# Stm1 regulates Ifh1 activity revealing crosstalk between ribosome biogenesis and ribosome dormancy

Eliana Bianco, Martina Bonassera, Federico Uliana, Janny Tilma, Martin Winkler, Sevil Zencir, Alvar Gossert, Michaela Oborská-Oplová, Reinhard Dechant, Jannik Hugener, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Eliana Bianco, Martina Bonassera, Federico Uliana, Janny Tilma, Martin Winkler, et al., Stm1 regulates Ifh1 activity revealing crosstalk between ribosome biogenesis and ribosome dormancy. Molecular Cell, 2025, 85 (9), pp.1806-1823.e17. 10.1016/j.molcel.2025.04.008. hal-05534775

HAL Id: hal-05534775

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Submitted on 3 Mar 2026

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# Molecular Cell

## Stm1 regulates Ifh1 activity revealing crosstalk between ribosome biogenesis and ribosome dormancy

### Graphical abstract

<figure><img src="image_3.png" /></figure>

### Authors

Eliana Bianco, Martina Bonassera, Federico Uliana, ..., Benjamin Albert, Philipp Kimmig, Matthias Peter

### Correspondence

eliana.bianco@fht.org (E.B.), matthias.peter@bc.biol.ethz.ch (M.P.)

### In brief

Ribosome abundance is balanced by production and degradation. Bianco et al. discover that the hibernation factor Stm1 has a dual role in ribosome homeostasis. Stm1 promotes ribosome biogenesis during growth conditions while protecting a subset of ribosomes from degradation during starvation. Stm1 thus coordinates ribosome homeostasis across cellular compartments involved in growth control.

### Highlights

* Stm1 balances ribosome storage and ribosome production through separable domains

* Stm1's C-terminal IDR promotes RPG transcription and ribosome biogenesis

* Stm1 directly binds Ifh1 and RNA through a conserved RGG/GAR motif

* Nutrient-dependent IDR phosphorylation regulates Stm1's interaction with Ifh1

<figure><img src="image_4.png" /></figure>
## Article

# Stm1 regulates Ifh1 activity revealing crosstalk between ribosome biogenesis and ribosome dormancy

Eliana Bianco,<fnref n="1" />,<fnref n="8" />,* Martina Bonassera,<fnref n="1" />,<fnref n="2" /> Federico Uliana,<fnref n="1" />,<fnref n="9" /> Janny Tilma,<fnref n="1" /> Martin Winkler,<fnref n="1" /> Sevil Zencir,<fnref n="3" />,<fnref n="10" />

Alvar Gossert,<fnref n="1" />,<fnref n="4" /> Michaela Oborská-Oplová,<fnref n="5" /> Reinhard Dechant,<fnref n="1" /> Jannik Hugener,<fnref n="2" /> Vikram Govind Panse,<fnref n="5" />,<fnref n="6" />

Martin Pilhofer,<fnref n="2" /> Benjamin Albert,<fnref n="7" /> Philipp Kimmig,<fnref n="1" /> and Matthias Peter<fnref n="1" />,<fnref n="11" />,*

## SUMMARY

Nutrient abundance boosts ribosome biogenesis, whereas ribosome dormancy factors limit ribosome degradation upon starvation. The equilibrium between the two pathways governs cell growth. In this study, we identified suppressor of Tom1 (Stm1) as a molecular link between ribosome protection and biogenesis in *Saccharomyces cerevisiae*. While Stm1 was previously described as a dormancy factor, we show that it activates Ifh1, a transcriptional activator of ribosomal protein genes. Stm1 transiently localizes to the nucleolus, where it interacts with pre-ribosomes and directly binds RNA and Ifh1 through its C-terminal intrinsically disordered region (IDR). Although the IDR is dispensable for ribosome protection, its loss compromises cell growth. The IDR is phosphorylated upon nutrient starvation, which disrupts its interaction with Ifh1. Our findings reveal a molecular pathway sensing and adjusting ribosome abundance in response to nutrient availability, reinforcing the relevance of regulated ribosome homeostasis in physiology and disease.

## INTRODUCTION

Ribosome biogenesis is the process through which ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) are synthesized and coordinately assembled into functional, cytoplasmic ribosomes.<fnref n="1" /> During ribosome biogenesis in yeast *Saccharomyces cerevisiae*, the primary 35S rRNA is transcribed by RNA polymerase I (RNA Pol I) in the nucleolus and processed into mature 18S, 25S, and 5.8S rRNAs. In the nucleus, 5S rRNA, tRNAs, and other short RNAs are transcribed by RNA polymerase III (RNA Pol III), while RP messenger RNA (mRNA), small nuclear RNAs (snRNA), and small nucleolar RNAs (snoRNA) are transcribed by RNA polymerase II (RNA Pol II). mRNAs are translated into proteins by ribosomes in the cytoplasm, and most RPs then return to the

nucleolus or nucleus to be assembled with rRNAs into new ribosomes aided by ribosome assembly factors (RAFs).<fnref n="2" />⁻<fnref n="5" /> Errors in ribosome biogenesis are linked to defects in cell growth, ribosomopathies, and cancer,<fnref n="6" />⁻<fnref n="8" /> and perturbing ribosome biogenesis causes proteotoxic stress and reduces cellular fitness.<fnref n="9" />,<fnref n="10" />

Molecular dissection reveals signaling crosstalk between nucleolar rRNA transcription and maturation, and nuclear RP gene (RPG) transcription involves the inhibitory function of the nucleolar CURI complex composed of Ckb2, Utp22, Rrp7, and Ifh1.<fnref n="11" />,¹² When RNA Pol I activity is downregulated, the small subunit processome (SSU) sub-complex UtpC subunits U three protein 22 (Utp22), and ribosomal RNA processing 7 (Rrp7), along with the casein kinase beta (CK2) subunit Ckb2, assemble into the CURI complex and sequester the RPG transcription

<footnote n="1">¹Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), 8093 Zürich, Switzerland</footnote>

<footnote n="2">²Department of Biology, Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), 8093 Zürich, Switzerland</footnote>

<footnote n="3">³Department of Cell Biology Sciences III, Université de Genève, 1211 Geneva, Switzerland</footnote>

<footnote n="4">⁴Biomolecular NMR Spectroscopy Platform (BNSP), Department of Biology, Eidgenössische Technische Hochschule (ETH), 8093 Zürich, Switzerland</footnote>

<footnote n="5">⁵Institute of Medical Microbiology, University of Zürich, 8006 Zürich, Switzerland</footnote>

<footnote n="6">⁶Faculty of Science, University of Zürich, Zürich, Switzerland</footnote>

<footnote n="7">⁷Molecular, Cellular and Developmental Biology Unit (MCD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 31062 Toulouse, France</footnote>

<footnote n="8">⁸Present address: Human Technopole, 20157 Milan, Italy</footnote>

<footnote n="9">⁹Present address: Johannes Gutenberg University of Mainz, 55122 Mainz, Germany</footnote>

<footnote n="10">¹⁰Present address: Department of Medical Biology, Faculty of Medicine, Pamukkale University, 20160 Denizli, Turkey</footnote>

<footnote n="11">¹¹Lead contact</footnote>

<footnote>*Correspondence: eliana.bianco@fht.org (E.B.), matthias.peter@bc.biol.ethz.ch (M.P.)</footnote>

<footnote>https://doi.org/10.1016/j.molcel.2025.04.008</footnote>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 1.** The ribosome hibernation factor *Stm1* promotes ribosome biogenesis</figcaption></figure>

(A) Drawing (left) of *Stm1* (orange) bound to 80S ribosome composed of 40S (light blue) and 60S (dark blue) subunits. Serial dilution spottings (right) on synthetic auxotrophic SD-URA media show wild-type (WT) and *stm1Δ* strains with control plasmid (empty) or overexpressing *Stm1* (2µ *Stm1*). Cells were untreated (UT) or rapamycin-treated (Rap) as indicated (1.75 and 2.0 nM) and incubated (3 days, 30°C). Representative image from *N* = 3 independent experiments.

(B) Representative cryo-tomograms of untreated WT and *stm1Δ* cells. Projections (10.7 nm thick) are shown. Regions (square dashed line) are magnified in the inset (solid black line). ER, endoplasmic reticulum; M, mitochondrion; PM, plasma membrane; V, vacuole; CW, cell wall. Scale bars: 100 nm. Additional cryo-tomograms are in Figure S1G.

(C) Quantification of ribosome concentration (µM) from cryo-tomograms of WT and *stm1Δ* cells, treated or not with rapamycin (Rap 1 µM, 2 h). Dots represent the mean concentration of five independent measurements within one tomogram (workflow, Figure S1F). Data values are mean with standard deviation. *N* = 10 tomograms per experimental condition. *p* values were calculated by one-way test for multiple comparison analysis (Holm-Sidák). Significance: **p* < 0.01; ***p* < 0.001.

(D) Bar plot quantifying 18S (orange), 25S (blue), and 35S (purple) rRNA levels in WT and *stm1Δ* cells, treated (Rap) or not (UT) with rapamycin (10 nM, 2 h). Black dots represent independent rRNA quantifications normalized to untreated WT controls. Mean values with standard deviation are shown. Representative gel image (*N* = 3 independent replicates) is shown in Figure S1J.

(E) Volcano plot of pulsed-SILAC MS measurements comparing newly synthesized proteins in WT and *stm1Δ* cells. Protein ratio incorporating heavy (H) lysine over total levels of light (L) and heavy lysine (log₂ (H/L + H)) with significance indicated by *p* values adjusted for multiple hypothesis testing (Benjamin-Hochberg). Orange: 40S subunit RPs; blue: 60S proteins; purple: proteins shared by both subunits (40S/60S). *N* = 4 independent replicates.

(legend continued on next page)
activator interaction with fork head 1 (Ifh1) in the nucleolus.¹¹
Therefore, Ifh1 promoter binding activity is inhibited, and RPG
transcription is downregulated.¹² In contrast, evidence is lacking
for cellular components and mechanisms that coordinate the
cytoplasmic translation rate of RPs and RAFs with nuclear Ifh1
activity and pre-ribosome assembly to ensure ribosome homeo-
stasis in response to changing growth conditions.

Molecular regulators governing ribosome biogenesis in
response to nutrient availability include protein kinase A (PKA)
and the target of rapamycin complex 1 (TORC1). These nutrient-
responsive kinases promote RAF and RP transcription by regu-
lating the activity of transcription repressors and activators.<sup>13–15</sup>
TORC1 and PKA also repress catabolic processes, such as
degradation of macromolecules or macro-autophagy.<sup>16,17</sup> Nitro-
gen starvation or treatment with the macrolide rapamycin is a
potent inhibitor of TORC1, thereby repressing RAF and RPG
mRNA transcription and translation,<sup>18</sup> and inducing autophagy
and ribophagy, which target ribosomes for degradation.<sup>17,19–22</sup>

When nutrients are replenished following starvation, cells
restart growth and resume translation. Growth restart requires
a specialized pool of protected ribosomes that are maintained
in an inactive state during starvation.²³,²⁴ Indeed, after rapamy-
cin treatment, cells preserve about 50%–60% of their ribosomes
compared with nutrient-rich conditions.²⁵,²⁶ Specific ribosome
dormancy factors have been identified in bacteria, which are up-
regulated upon adaptation to starvation and stress.²⁷,²⁸ Likewise,
factors protecting eukaryotic ribosomes from degradation
during starvation have been described, such as the budding
yeast suppressor of Tom1 (Stm1),²⁴,²⁹,³⁰ its zebrafish and Xeno-
pus homolog intracellular hyaluronan binding protein 4
(HABP4)³¹ or its human homolog SERPINE1 mRNA binding pro-
tein 1 (SERBP1).³² Stm1 occupies the mRNA tunnel and con-
tacts both 40S and 60S subunits of the 80S ribosome (Fig-
ure 1A).³⁰ Upon nutrient replenishment, target of rapamycin
complex 1 (TORC1) directly phosphorylates Stm1, which is
then released from dormant ribosomes, allowing translation
restart and resuming cell growth.²⁴ Deletion of Stm1 sensitizes
cells to rapamycin and compromises cell growth restart after ni-
trogen starvation,²³ which has been attributed to ribosome
depletion. To date, evidence is lacking for the functional coupling
of ribosome degradation/protection mechanisms to maintain
ribosome homeostasis. Moreover, factors regulating ribosome
abundance have yet to be described.

In this study, we report an unexpected nucleolar and nuclear
role of Stm1 during exponential growth, distinct from its cytosolic
dormancy function during starvation. Our results demonstrate
that in the absence of stress, Stm1 shuttles into the nucleolus/
nucleus and activates RPG production and ribosome biogen-
esis. Thus, Stm1 regulates ribosome homeostasis by coupling
ribosome biosynthesis and degradation in response to nutrient
availability.

RESULTS

Stm1 promotes ribosome biogenesis during exponential
growth

We used spotting assays to assess the rapamycin sensitivity of wild-type and *stm1Δ* cells with and without Stm1 overexpression (2µ Stm1). As expected,²⁹ *stm1Δ* cells were sensitive to rapamycin, while Stm1 overexpression confers resistance to higher rapamycin concentrations (Figure 1A). The sensitivity of wild-type and *stm1Δ* cells to the translation inhibitor cycloheximide (CHX)³³ was comparable,³⁴ whereas Stm1 overexpression showed increased toxicity (Figure S1A). Like rapamycin treatment, recovery of *stm1Δ* cells was compromised after 3 or 6 days of stationary phase compared with wild-type controls (Figure S1B), whereas their cell volume was equivalent (Figure S1C). We conclude that Stm1 is essential in cells with reduced TORC1 activity and this phenotype cannot be explained by a translation defect.

Surprisingly, *STM1* mRNA and protein levels were abundant in exponentially growing cells, with mRNA expression downregulated upon TORC1 inhibition or heat stress. Moreover, *Stm1* protein levels decreased during the stationary phase, like RAFs and RPs (Figures S1D and S1E). This expression pattern contrasts bacterial hibernation factors, which are upregulated during starvation.²⁷,²⁸ We next compared the 80S cytosolic ribosome concentration in wild-type and *stm1Δ* cells using an *in situ* cryoelectron tomography (cryo-ET) pipeline (Figure S1F). As expected,²⁵ the ribosome concentration in wild-type cells (~18 µM) dropped upon rapamycin treatment (~12 µM). In *stm1Δ* cells, the cytoplasmic ribosome concentration was significantly lower (~14 µM) than in wild-type cells, without further reduction upon rapamycin treatment (~13 µM) (Figures 1B, 1C, and S1G). Flow cytometry analysis confirmed a comparable percentage of viable cells and phophorylation levels of yeast Ser51-suppressor of

<footnote>(F) GO analysis (cellular component) calculated for downregulated proteins (*p* < 0.05) in *stm1Δ* cells. Enrichment significance is reported as −log₁₀ of *p* value.
(G) *In vivo* pulse-chase proximity biotin ribosome labeling system. During the pulse phase (on), the 60S subunit (blue) protein Rpl16a/b-hemagglutinin (HA)-Tobacco Etch virus protease site (TEV)-peptide tag for enzymatic biotinylation (AVI) is biotinylated (purple star) by the estradiol-inducible BirA ligase. BirA degradation by the auxin-inducible degron abolishes further biotin labeling (off). Biotinylated 60S ribosomes are chased (CHASE) over time and detected by streptavidin.</footnote>

<footnote>(H) Western blot of total cell lysates (TCL) and co-precipitated streptavidin-bound proteins (IP: Strep [biotin]) from WT and *stm1Δ* cells expressing endogenously tagged Rpl16a/b-HA-TEV-AVI. Samples were uninduced (—Estr) or with 5 nM estradiol for 2 h to induce BirA expression. After estradiol washout, samples were supplemented with biotin to induce labeling of Rpl16a/b-HA-TEV-AVI and 1 mM auxin (IAA) to initiate BirA degradation. Samples were harvested after the BirA biotinylation/degradation step (CHASE, 0 h) or 1 h after BirA shut-off and degradation (CHASE, 1 h). Blots were probed with HA antibodies (biotinylated Rpl16a/b-HA-TEV-AVI after TEV elution from Strep-beads), endogenous Rps5 and Rpl35, Stm1, and Pgk1 (loading control). N = 3 independent replicates. Blue boxes = Rpl16a/b and Rpl35. Orange boxes = Rps5.</footnote>

<footnote>(I) Bar plot quantifying total protein from TCLs (left) and affinity-purified biotinylated Rpl16a/b (right) (IP) in no estradiol (orange), 0 h pulse (blue), and 1 h pulse (purple). Black dots represent normalized protein quantifications, shown with standard deviations. Statistical significance was calculated with two-way ANOVA test for multiple comparison analysis (Sidák). N = 3 independent replicates. Significance: "ns" = not significant.</footnote>

<footnote>(J) Bar plot quantifying Rpl35 in TCL and IP as in (I).</footnote>

<footnote>(K) Bar plot quantifying Rps5 in TCL and IP as in (l).</footnote>
initiator codon 2 (Sui2)/human Ser52-alpha subunit of translation initiation factor (eIF2α)³⁵ (Figures S1H and S1I). Moreover, we quantified different rRNA species in wild-type and *stm1Δ* cells exposed or not to rapamycin. Indeed, 18S and 25S rRNA levels decreased by 10%–20% in unchallenged *stm1Δ* cells and were further reduced by 40% upon TORC1 inhibition (Figures 1D and S1J). Strikingly, the levels of unprocessed RNA Pol I 35S transcripts were markedly diminished by approximately 75% in untreated *stm1Δ* cells or cells exposed to low rapamycin concentrations (Figures 1D and S1J). Together, these results suggest that Stm1 is required for ribosome homeostasis and promotes ribosome biosynthesis in unstressed cells.

To assess the cause of decreased ribosomes in *stm1Δ* cells, we first quantified newly synthesized proteins in wild-type and *stm1Δ* cells by pulsed stable isotope labeling by amino acid in cell culture (SILAC) mass spectrometry (MS) experiments.³⁶ While the SILAC media did not affect doubling times (Figure S1K), Gene Ontology (GO) analysis showed a significant enrichment in *stm1Δ* cells for terms associated with ribosomal components (Figures 1E, 1F, and S1L; Table S1), suggesting that the reduced levels of newly synthesized proteins are a consequence of decreased RP expression. Although we noticed that CHX fails to pause ribosome elongation in *stm1Δ* cells, wild-type and *stm1Δ* cells incorporate puromycin at similar rates in untreated and TORC1-inhibition conditions (Figure S1M). Likewise, eIF2α phosphorylation is equal in both strains (Figure S1l), implying that growth and translation rates are comparable between *stm1Δ* and wild-type cells, despite reduced ribosome particles. Without Stm1, cells may compensate by engaging the excess of inactive 80S ribosomes in mRNA translation.³⁷ We conclude that the dormancy factor Stm1 has an unexpected function in ribosome homeostasis in exponentially growing cells.

We next developed a pulse-chase biotinylation proximity labeling system (Figure 1G) to assess ribosome turnover (Figure S1N). This system allows isolating biotinylated cytoplasmic ribosomes by affinity purification (AP) and thus directly comparing their turnover in wild-type and *stm1Δ* cells by immunoblotting and Coomassie staining. Interestingly, biotinylated ribosomal subunits Rpl16a/b, as well as associated Rpl35 and Rps5, were reduced to a similar extent in both strains after chase (1 h) in exponential growth, indicating that Stm1 does not affect ribosome stability in nutrient-rich conditions (Figures 1l–1K and S1O). These results imply that reduced RP expression and ribosome biogenesis explain the lower ribosome concentration of *stm1Δ* cells.

## Stm1 interacts with RAFs in the nucleus

To probe the Stm1-dependent ribosome biogenesis mechanism, we stably integrated a single copy of fully functional 3xMyc-tagged Stm1 into the genome of *stm1Δ* cells³⁸ and identified associated proteins through AP-MS. A control purification with cells expressing 3xMyc-tagged GFP and rigorous quality control criteria allowed filtering contaminants (Figures S2A–S2C). Stm1-3xMyc pulled down ribosomal subunits and ribosome-interacting proteins, consistent with its role as a cytoplasmic hibernation factor. However, GO enrichment analysis showed that Stm1 is associated with multiple RAFs, which predominantly reside in the nucleus and/or nucleus (Figures 2A–

2C and S2D; Table S2). Two-hybrid analysis corroborated that Stm1 binds several nucleolar RAFs, including Noc4 and SSU components Utp18 and Utp22 (Figure 2D). Moreover, endogenous Stm1 co-purified with Nop7- tandem affinity purification (TAP) and Noc4-TAP particles, which belong to the early steps of 60S (pre-66S) and 40S (SSU) biogenesis, respectively (Figure 2E). We noticed two upshifted Stm1 bands in Noc4-TAP but not Nop7-TAP particles, which may represent post-translational modifications.

As expected, N- and C-terminally GFP-tagged Stm1 was predominantly cytosolic (Figure S2E). However, we found two putative nuclear export sequences (NESs)³⁹: NES1 (amino acid [aa] 6–20) and NES2 (aa 160–174) (Figure S2F). Indeed, deleting NES2, but not NES1, leads to slight nuclear GFP-Stm1 retention (Figures S2G–S2I), consistent with nuclear shuttling. To corroborate, we implemented bimolecular fluorescence complementation (BiFC) assays.⁴⁰ Stm1 was fused to the C-terminal half of yellow fluorescent protein (YFP) (Vc), while the N-terminal half of YFP (Vn) was fused to the nucleolar protein Rrp7 (Rrp7-Vn), a component of the SSU sub-complex UtpC,⁴¹ which interacts with Stm1 in pull-down assays (Figure S4I). For the control, the Vn fragment was fused to the cytosolic ribosome subunit Rpl42a (Rpl42a-Vn), which, in the ribosome crystal structure,³⁰ binds adjacent to Stm1 (Figures 2F and 2G). As expected, Vc-Stm1::Rpl42a-Vn reconstituted YFP in the cytoplasm, with no signal observed during individual expression. Importantly, Vc-Stm1 readily interacted with Rrp7-Vn, as visualized by the nucleolar YFP fluorescence. Quantification of these BiFC assays revealed that over 60% of cells expressing both Stm1 and Rrp7 fusions gained nucleolar YFP, while over 90% stained positive for the Rpl42a cytoplasmic control (Figures 2F and 2G). Similar results were also obtained for the UtpC and SSU component Utp22⁴¹ (Figures S2J and S2K). Taken together, these results suggest that Stm1 interacts with multiple cellular components and partitions between mature ribosomes in the cytoplasm and RAFs and pre-ribosomes in the nucleus and nucleolus.

## Stm1 controls RPG transcription of lfh1 targets

To stage Stm1 function during early ribosome biogenesis, we analyzed *stm1Δ* negative genetic interactions available in the synthetic genetic analysis database.⁴² Interestingly, several components regulating the RNA Pol II machinery, such as Kin28 or subunits of the mediator complex (Med), are synthetically sick with *stm1Δ* (Figure S3A). Furthermore, the slow RNA Pol II mutants *rpo21/rpb1*<sup>N479S</sup> and *rpo21/rpb1*<sup>N1082S43</sup> exhibit negative genetic interactions (Figure S3B), implying that Stm1 may be essential in cells with reduced RNA Pol II activity. To confirm this hypothesis, we crossed *stm1Δ* with the slow RNA Pol II mutants *rpo21*<sup>N479S</sup>, *rpo21*<sup>N1082S</sup>, or *rpo21*<sup>H1085Q</sup>, integrated in a *rpo21Δ* background and observed sub-lethality in the double mutants (Figure S3C). Moreover, we engineered strains with the anchor-away system,⁴⁴ carrying two copies of the *RPO21* gene: an untagged copy of wild-type *RPO21* or the slow mutants *rpo21*<sup>N479S</sup>, *rpo21*<sup>N1082S</sup>, and *rpo21*<sup>H1085Q</sup>, as well as FRB-tagged *RPO21*, which is sequestered in the cytoplasm upon addition of the dimerization compound (+ anchor away). This genetic assay confirmed that the slow RNA Pol II mutants *rpo21*<sup>N479S</sup>, *rpo21*<sup>N1082S</sup>, and *rpo21*<sup>H1085Q</sup> exhibit a
<figure><img src="image_7.png" /><figcaption>Figure 2. Stm1 interacts with pre-ribosomal particles</figcaption></figure>

(A) Volcano plot showing the Stm1 interactome as log₂ fold protein enrichment co-purified with Stm1-3xMyc versus GFP-3xMyc. Statistical significance was calculated by two-sided unpaired Student's t tests. The *p* value was corrected for multiple hypothesis using the Benjamín-Hochberg method. *n* = 3 independent replicates. 40S (orange), 60S (blue) RPs, and RAF (purple) are depicted.

(B and C) GO enrichment for cellular components (B) and molecular function (C) of Stm1 interactors (defined as log₂ fold change [FC] > 2 and *p* < 0.05). Significance: –log₁₀ of *p* value.

(D) Two-hybrid analysis of Stm1 fused to the GAL4 transcription factor activation domain (AD) or AD control (–) with SSU components or various controls (–, lamin C, Cdc13) fused to the GAL4 DNA binding domain (DBD). Colonies were grown (2 days, 30°C) on permissive auxotrophic SD –Leu, –Trp (–LT), or selective –His, –Leu, –Trp agar media, supplemented with 2.5 mM 3-amino-1,2,4-triazole (–HLT +2.5 mM 3-AT). Representative image from *n* = 3 independent replicates. Linked to Figure S4A.

(E) Tandem affinity purification (TAP) of untagged, Nop7-TAP or Noc4-TAP pre-ribosomal particles. Silver-stained SDS-PAGE (left) and western blot (right) are shown. TCLs, total cell lysates; IP, TAP affinity purification. Blots were probed with antibodies to CBP (Nop7-TAP and Noc4-TAP), Stm1, Rps5, or Rpl35. Representative gel from *n* = 3 independent replicates.

(F and G) Drawing of the BiFC assay. The estradiol-inducible C-terminal yellow fluorescent protein (YFP) fragment Vc is fused to Stm1 (orange), and the constitutively expressed N-terminal YFP fragment Vn is cloned in frame with Rpl42a (F) or Rrp7 (G). Interaction of Stm1 with Rpl42a or Rrp7 reconstitutes YFP fluorescence (top). Representative images of estradiol-induced (10 nM, 2 h) cells expressing Vc-Stm1 (Stm1) or Rpl42a-Vn (Rpl42a) or Vc-Stm1 and Rpl24a-Vn (Stm1 + Rpl42a) are shown (middle). Scale bar: 5 µm. Merged YFP and bright-field images are shown (middle). BiFC quantification is shown as percentage of YFP positive cells (bottom graph) from at least 50 cells/sample and independent replicate (dots) (*n* = 3). Data values are shown as mean and standard deviation.

synthetically lethal interaction when combined with *stm1Δ* (Figure 3A), demonstrating that Stm1 is needed when RNA Pol II activity is perturbed.

While Stm1 associates with pre-ribosomal complexes, no RNA polymerase subunits co-immunoprecipitated with

Stm1-3xMyc in the MS analysis (Figure 2A). Likewise, Stm1 was absent in Rpb8-TAP⁴⁸ pull-downs (Figure S3D), excluding the possibility that Stm1 stably interacts with the transcription machinery. We next measured the occupancy of RNA Pol II Ser5 by chromatin immunoprecipitation sequencing (ChIP-seq)
<figure><img src="image_8.png" /><figcaption>**Figure 3.** *Stm1 promotes Ifh1 activity at RPGs*</figcaption></figure>

(A) Serial dilution spottings on yeast extract pentose dextrose (YPD) media of wild-type (WT) (–) or anchor-away strains.⁴⁴ WT or *stm1Δ* anchor-away strains carry an extra copy of genomically integrated RPO21 WT (*RPO21*) or different point mutations (*rpo21*^N479S, *rpo21*^N1082S, *rpo21*^N1085Q). Nuclear depletion of endogenous *RPO21* (anchor-away) was initiated by the addition of solvent control (– anchor-away) or 1 µM rapamycin (+ anchor-away), and cells were incubated (3 days, 30°C). Representative image of *n* = 3 independent replicates.

(B) Box plots showing RNA Pol II Ser5 occupancy. *stm1Δ* versus WT cells is shown for ribosomal protein genes (RP [*n* = 138]), ribosome biogenesis genes (RAF [*n* = 566]), Gcn4 targets genes (Gcn4 [*n* = 108]), heat-shocked genes (Hsf1 [*n* = 18]), or others (*n* = 4,311). Data are displayed as log₂ normalized fold change of RNA Pol II S5 occupancy in *stm1Δ* versus WT cells, for untreated conditions (UT, left) and 3 h after low-dose rapamycin treatment (Rap 6.5 nM, right). Independent replicate is shown in Figure S3E. The signal was quantified for each RNA Pol II transcribed gene between the transcription start (TSS) and transcription termination site (TTS). Gene categories are defined as: Zencir et al.⁴⁵ for RPG, RAF, Solís et al.⁴⁶ for Hsf1, and Srinivasan et al.⁴⁷ for Gcn4 targets. An independent replicate is shown in Figure S3E.

(C) Genome browser tracks showing RNA Pol II Ser 5 ChIP-seq read counts at open reading frames of *RPS18B* (left) and *RPL9A* (right) in WT (orange) and *stm1Δ* (blue) cells, treated (Rap 6.5 nM, 1 h) or not (UT) with rapamycin.

(D) Boxplots comparing differential RNA Pol II Ser5 occupancy of ribosome assembly factors (RAFs [*n* = 566]), ribosomal protein genes (RP [*n* = 138]) categories number 1 (Cat I), number 2 (Cat II), and number 3 (Cat III) in WT and *stm1Δ* strains. Data are displayed as the log₂ normalized fold change of RNA Pol II Ser5 occupancy in *stm1Δ* versus WT cells, treated or not (t = 0, left) with 6.5 nM rapamycin for 1 h (t = 1, center) or 3 h (t = 3, right). Gene categories according to literature.⁴⁵⁻⁴⁷ An independent replicate is shown in Figure S3J.

(E) Bar plot showing Ifh1-13xMyc occupancy fold enrichment at the RPGs *RPL28* (left) and *RPL37A* (right) analyzed by ChIP-qPCR and normalized to *ACT1*. WT and *stm1Δ* cells were treated (t = 1 h) or not (t = 0) with rapamycin (6.5 nM). An independent replicate is shown in Figure S3L.

(F) Schematic of Ifh1 and Ifh1-6 (top). Serial dilution spottings (bottom) of WT, *stm1Δ*, the Ifh1 truncation mutant *ifh1-6* (aa 645–1,085), and the *ifh1-6* *stm1Δ* double mutant on YPD media. Cells were either treated or not (UT) with rapamycin (Rap, 1.5 nM) and incubated for 3–4 days. Representative image from *n* = 3 independent experiments.
<figure><img src="image_9.png" /></figure>

Disordered
Ordered

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<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>Figure 4. Loss of *Stm1* *IDR* drives rapamycin sensitivity</figcaption></figure>

(A) *Stm1* drawing with aa numbers depicting the 60S and 40S binding domains separated by a linker region,³⁰ and a nuclear export signal (NES) (Figures S2F–S2I) (top). The disorder score (0–1) using PONDR VL-XT⁵⁹ and aa charge (−0.5 to +0.5) using Emboss Charge⁵⁰ (middle) is shown below. C-terminally tagged full-length *Stm1*-3xMyc (FL) and indicated mutants (Δ*N*, Δ*C*, Δ176–225, Δ*IDR*) were cloned into single integration pSIV vectors³⁸ and integrated at the *URA3* locus.

(B) Western blot of total lysates prepared from WT or *stm1Δ* cells treated or not with rapamycin (Rap 200 nM, 2 h). FL and *Stm1* mutants were integrated into *stm1Δ* cells and analyzed by immunoblotting with antibodies against phospho-eIF2alpha (Sui2Ser51), eIF2alpha, Myc, and Pgk1 (control). Representative image of *N* = 3 independent replicates.

(C) Serial dilution spottings on YPD media of WT, *stm1Δ*, and *stm1Δ* strains stably expressing 3xMyc-tagged FL or the indicated *Stm1* mutants (Δ*N*, Δ*C*, Δ176–225, Δ*IDR*). Cells were treated or not (UT) with rapamycin (Rap 3 nM) and incubated at 30°C for 2 days. Representative image of at least 3 independent experiments.

(D) Serial dilution spottings of WT and *stm1Δ* cells with empty control plasmid (vector), or plasmids overexpressing GFP-tagged *Stm1* (GFP-Stm1) or its C-terminal domain (GFP-C, aa 175–273), schematically depicted (top). Cells were treated or not (UT) with 2.5 rapamycin (Rap 2.5 nM) and incubated (30°C, 3 days). Representative image from *n* = 2 independent experiments.

(E) Western blot of control (vector), GFP-Stm1, and GFP-C in WT or *stm1Δ* cells, using GFP and Pgk1 antibodies. The asterisk (*) marks the signal from the GFP antibody migrating close to Pgk1. Representative image of *n* = 2 independent replicates.

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analysis in wild-type and *stm1Δ* cells (Figures 3B, 3C, and S3E-S3I; Table S3). Interestingly, *stm1Δ* upregulated Gcn4 targets in the absence and presence of low dose of rapamycin, while Hsf1 targets are increased only upon TORC1 inhibition, indicating a general level of cellular stress<sup>47,49</sup> (Figures 3B and S3E-S3I). However, Sui2 phosphorylation and GCN4-LacZ reporter analysis (Figures S1I and S3M)<sup>35,50</sup> showed that GCN4 mRNA translation is comparable in wild-type and *stm1Δ* cells, indicating that upregulation of Gcn4 targets might originate from alternative pathways that impact ribosome biogenesis.<sup>51-54</sup> Importantly, however, we found a profound decrease in occupancy of RNA Pol II at RPGs in *stm1Δ* cells, whereas the reduction was notably less pronounced at RAF genes. Transcription of RAF genes is regulated by Sfp1.<sup>55-57</sup> RPGs can be subclassified in three categories: class I and II comprise predominantly Ifh1 targets, while class III targets are mostly regulated by Sfp1.<sup>45</sup> Detailed analysis revealed that, in *stm1Δ* cells, the RNA Pol II occupancy of all three RPG categories is downregulated in the absence and presence of a low dose of rapamycin (Figures 3D and S3J; Table S3), with the largest effect observed for Ifh1 targets. We conclude that Stm1 predominantly promotes Ifh1 activity, thereby modulating ribosome homeostasis in exponentially growing cells.

Next, we measured the occupancy of Ifh1-13xMyc by ChIP-qPCR in wild-type and *stm1Δ* cells. Indeed, Stm1 promotes Ifh1 binding at RPG promoters of the class I gene *RPL37A* and the class II gene *RPL28* relative to *ACT1* in both untreated and rapamycin-treated conditions (Figures 3E and S3L). To confirm specificity, the endogenous promoter of the class II gene *RPL18A* was replaced by the doxycycline-repressible TetO7 promoter. The doxycycline-induced growth defect of *RPL18A* was comparable in the presence or absence of Stm1 (Figure S3K), suggesting that Stm1 specifically enhances the Ifh1-dependent transcription of RPGs. To genetically validate the Stm1-Ifh1 connection, we deleted *STM1* in the truncation mutant *ifh1-6*, encompassing the forkhead binding domain (FHB), activation domain (AD), and C terminus of Ifh1, which stably associates with RPG promoters.<sup>12</sup> Deletion of *STM1* and exposure to a low dose of rapamycin compromised viability of *ifh1-6* cells (Figure 3F), suggesting that Stm1 enhances Ifh1 activity in growing cells, thereby promoting RPG expression and ribosome assembly.

## Loss of Stm1 C-terminal IDR confers rapamycin sensitivity independent of its hibernation function

The structure of Stm1 bound to ribosomes indicates that its N terminus binds the 60S particle and the linker region bridges the 40S and 60S ribosomal subunits, while the C terminus is predicted to be unstructured.<sup>24,30</sup> To test domain function (Figure 4A), we expressed 3xMyc-tagged wild-type or truncation mutants, stably integrated as a single copy in *stm1Δ* strains, and confirmed expected expression by western blot (Figure 4B, lanes 1–6). Downregulation of mRNA translation was assessed by probing Sui2

phosphorylation,<sup>21,58</sup> which is induced, as expected, only when cells are treated with rapamycin. Surprisingly, growth assays revealed that rapamycin-treated cells tolerate short truncation of the Stm1 N terminus (*stm1ΔN*, Δ2–25), previously linked to the hibernation function.<sup>24</sup> In contrast, cells expressing Stm1 lacking the C terminus (*stm1ΔC*, Δ177–273) displayed reduced growth on rapamycin-containing media, like *stm1Δ* controls (Figure 4C). Overexpression of a GFP-tagged C-terminal Stm1 domain (GFP-C) rescued this growth defect (Figures 4D and 4E), suggesting that Stm1's C terminus is necessary and sufficient for its starvation function *in vivo*. We then constructed and stably integrated a single copy of 3xMyc-tagged Δ176–225 and Δ intrinsically disordered region (ΔIDR) (Δ226–270) C-terminal mutants into *stm1Δ* cells (Figure 4A). Immunoblotting confirmed expected Stm1 variant expression and Sui2 phosphorylation (Figure 4B, lanes 7 and 8). In contrast to *stm1Δ176–225*, cells expressing *stm1ΔIDR* were sensitive to rapamycin comparable with *stm1Δ* controls, suggesting that the C-terminal IDR domain is required for Stm1 function. To test its relevance for ribosome hibernation, we compared sucrose gradient profiles of wild-type, *stm1Δ*, and *stm1ΔIDR* cells in untreated and rapamycin-treated conditions. As expected, truncation of the C-terminal IDR did not impact the 80S or polysome levels (Figures 4F–4H), indicating the C-terminal IDR is irrelevant for ribosome protection. However, rapamycin sensitivity of the *stm1ΔIDR ifh1-6* double mutant was comparable to *stm1Δ* (Figure 5A), suggesting that the Stm1 C-terminal IDR is functionally relevant for RPG expression by promoting Ifh1 activity.

## Stm1 IDR drives the interaction with RAF and CURI complex and is regulated by phosphorylation

To test the Stm1 C terminus interactions, we used two-hybrid analysis with the *stm1ΔN* and *stm1ΔC* mutants. While some components, such as Krr1 or Enp2, preferentially bound *stm1ΔC*, most nuclear and nucleolar interactors require the C terminus (Figure S4A) and even show increased association with the *stm1ΔN* mutant (Figure S4A), as confirmed by AP-MS analysis (Figures S4B–S4G; Table S4). We conclude that *stm1ΔN* and *stm1ΔC* interact with distinct protein assemblies, highlighting their separate functions. Indeed, two-hybrid analysis showed that binding of Ifh1, Utp22, and Ckb2 requires the C-terminal IDR domain of Stm1 (Figure S4H), while deletion of other C-terminal domains had no effect.

We employed AP-MS analysis to systematically compare the interactomes of Stm1-3xMyc and Stm1ΔIDR-3xMyc. Following normalization for Stm1 abundance, we observed a reduction of interacting RPs, translation regulators, and RAFs (Figures 5B, 5C, and S2A–S2C; Table S2). Next, we used targeted parallel reaction monitoring (PRM)-MS analysis<sup>61,62</sup> to quantify the binding of Stm1 to Ifh1 and subunits of the CURI complex (Ckb2, Utp22, and Rrp7). With at least 5-fold enrichment compared with GFP-3xMyc controls (Figures S4I–S4K and S5A; Table S5), these

(F and G) Ribosome and polysome profiles recorded at $A_{260}$ absorbance of WT (blue), *stm1Δ* (orange), and *stm1ΔIDR-3xMyc* (purple) cells treated (G) or not (UT) (F) with 200 nM rapamycin (Rap 200 nM, 1 h). The position of free, 40S, 60S, 80S, and polysomes is indicated (top).

(H) Bar plot quantifying 80S (blue) and polysomes (orange) peaks from (F) and (G) as the area under the curve of WT, *stm1Δ* and Stm1ΔIDR-3xMyc cells, untreated (UT) or treated with rapamycin (200 nM, 1 h). Black dots represent independent quantifications. Mean values with standard deviations are shown from *n* = 3 independent replicates.
<figure><img src="image_17.png" /><figcaption>**Figure 5.** The Stm1-IDR drives interaction with CURI subunits and promotes Ifh1 activity</figcaption></figure>

(A) Representation of the *ifh1-6* mutant (aa 645–1,085) including its forkhead binding (FHB) and AD (top). Serial dilution spottings (bottom) of WT, *Stm1Δ, Stm1ΔIDR-3xMyc, ifh1-6 and ifh1-6 stm1Δ and ifh1-6 stm1ΔIDR-3xMyc* double mutants on YPD media. Cells were treated or not (UT) with rapamycin (Rap 1.5 nM) and incubated at 30°C for 2–3 days. *n* = 3 independent replicates.

(B) Volcano plot showing the log₂ fold change enrichment for proteins co-purified with 3xMyc-tagged Stm1ΔIDR in comparison with Stm1. Protein abundance was normalized to Stm1 levels after AP-MS. Significance calculated from two-sided unpaired Student's t test from *n* = 3 independent replicates. Proteins of the 40S (orange) and 60S (blue) ribosomal subunits and RAF (purple) are highlighted. Linked to Figures S2A–S2C.

(C) The GO for biological process was calculated for Stm1 interactors identified in Figure 5A (defined as log₂FC < −1 and *p* < 0.05). Statistical significance of the enrichment is reported as the −log₁₀ of *p* value.

(D) Targeted PRM measurement of Ifh1 and CURI complex members co-precipitating with Stm1-3xMyc or Stm1ΔIDR-3xMyc, reporting the log₂FC enrichment for the indicated peptides (Figures 5C and S4I). Data values are shown as log₂ ratio of light (endogenous peptide) to heavy (stable heavy isotope-labeled peptide) (l/h), normalized to Stm1-3xMyc data. Each dot is a technical replicate (*n* = 3), with the box plot boundaries indicating the quantiles Q1 (25%) and Q3 (75%). The average value is denoted by a line (*n* = 3).

(E) Representation of Stm1 phosphorylation sites during exponential growth (red or orange) or 5 days of stationary phase (blue and dark blue) (*n* = 2 biological replicates). Numbers: relevant aa; S, serine; T, threonine. Circles: phosphosites with a localization probability higher than 0.8 with colored classification: red: unique during growth; dark blue: unique in stationary phase; orange: more abundant during growth; blue: more abundant during stationary phase. Gray boxes: 60S and 40S binding regions and the NES; purple box: IDR.

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measurements confirmed that the Stm1 IDR domain is neces-
sary to efficiently interact with Ifh1 and the CURI subunits
(Figures 5D and S4I–S4K; Table S5), in contrast to aa 176–225
(Figure S4H).

To examine how the two Stm1 functions are coordinated,
we hypothesized that the interaction of Stm1 with Ifh1 and the
CURI components may be regulated by phosphorylation. Phos-
pho-enrichment and MS analysis of exponential growth or station-
ary phase cells detected many phosphorylated serine and threo-
nine residues throughout the Stm1 protein in at least one of the
experimental conditions (Figures 5E and S5B), indicating that
phosphorylation may differentially regulate Stm1 activity. Four
previously uncharacterized phosphorylation sites—S229, T248,
S267, and S271—map to the C-terminal IDR domain and are
more abundant or specific to the stationary phase. To test their bio-
logical relevance, we overexpressed phospho-mimicking aspartic
acid mutants (Stm1S229D, T248D, S267D, S271D, Stm1S-T/D) or non-
phosphorylatable alanine mutants (Stm1S229A, T248A, S267A, S271A;
Stm1S-T/A) in stm1Δ cells from a 2-μm plasmid. Interestingly, while
stm1S-T/A and stm1S-T/D did not affect cell growth in unperturbed
conditions, both increased rapamycin sensitivity compared with
wild-type controls, with a stronger effect of the stm1S-T/D mutant
(Figure 5F). Two-hybrid analysis revealed that both Stm1S-T/D
and Stm1S-T/A abolish the binding of Stm1 and Ifh1 (Figure 5G).
Taken together, we conclude that the C-terminal IDR domain of
Stm1 is phosphorylated upon starvation, regulating its interaction
with Ifh1.

Stm1-IDR and Ifh1-AD directly interact at the RGG/
GAR motif

We next analyzed the binding of Ifh1 truncation mutants¹² to Stm1
in two-hybrid assays. While the N-terminal domain (aa 1–702,
ifh1-2) was unable to bind, a fragment encompassing the AD
and parts of the C-terminal FHB domain (ifh-7) was sufficient to
interact with full-length Stm1 (Figure 6A). We used fluorescent po-
larization (FP) of purified GST-tagged Ifh1 AD-domain (glutathione
S-transferase [GST]-Ifh1-AD, aa 703–811) to examine direct bind-
ing to 5-carboxytetramethylrhodamine (TAMRA)-labeled Stm1-
IDR peptide and measured a K<sub>d</sub> of approximately 1.4 µM (Fig-
ure 6B). To precisely map the interaction site, we <sup>15</sup>N-labeled the
Stm1-IDR fragment (aa 227–273) and recorded 2D [15N, 1H]
NMR spectra with or without purified GST-Ifh1-AD. All aa could
be assigned (Figure 6C), and chemical shifts were observed for
G238, G241, K244, and G245 residues of Stm1 (Figures 6D and
6E), indicative of binding to GST-Ifh1-AD. Surprisingly, the first
two residues are located within a glycine/arginine-rich RGG/
GAR motif,<sup>63,64</sup> while the other two flank this putative RNA-binding
motif (Figure 6F). To demonstrate RNA binding, we performed pel-
leting assays with purified Stm1-IDR (aa 226–273) and total yeast
RNA. While the Stm1-IDR fragment alone remains soluble, it pre-

cipitates into the pellet fraction with RNA (Figure 6G), dependent
on an intact RGG/GAR motif (Stm1-IDR-8A) (Figure S6). Finally,
FP assays confirmed the binding of total RNA to TAMRA-labeled
Stm1-IDR but not the Stm1-IDR-8A control (Figure 6H), demon-
strating that Stm1 binds RNA through its C-terminal RGG/GAR
motif. Taken together, we conclude that Stm1's C-terminal
RGG/GAR motif not only binds RNA but also associates with
Ifh1, regulated by starvation-induced phosphorylation.

DISCUSSION

The conundrum of unlinking ribosome concentration
from cell viability

Despite the low number of mature ribosomes, *stm1Δ* cells do not show a significantly reduced growth rate in nutrient-rich conditions (Figures 1A, S1A, and S1B). This was surprising at first, since the cell proliferation rate is directly linked to ribosome content.¹,³,⁶⁵ However, like bacteria, yeast cells store excess inactive ribosomes in rich conditions, along with elongating ribosomes for rapid adaptation to changes.³⁷,⁵⁸ Since Stm1 is expressed (Figures S1D and S1E) and bound to inactive 80S ribosomes in exponential growth,⁶⁶ empty 80S ribosomes may compensate for the lower translation capacity in *stm1Δ* cells. Additionally, Stm1 may shorten individual ribosome re-engagement time so that the translating ribosome fraction remains unchanged even though total ribosome particles are decreased. This adaptation mechanism is consistent with the puromycin-incorporation assays (Figure S1M) and polysome profiles (Figures 4F–4H), showing that Stm1 absence does not impact mRNA translation rates. While bacteria have developed dedicated systems to adapt cell growth and translation, such as the alarmone guanosine tetra- or pentaphoshate ((p) ppGpp),⁶⁷,⁶⁸ Stm1 may be part of a eukaryotic sensing mechanism linking inactive ribosome reserves to ribosome production. Alternatively, the translational activity of individual ribosomes may be increased in the absence of Stm1. Indeed, Stm1 may repress translation by preventing the association of the translation elongation factor eEF3 with ribosomes.⁶⁹ Further work measuring translational output and the activity of single ribosomes will be required to distinguish between these possibilities.

Stm1 promotes RAF transcription by regulating Ifh1 activity

Previous studies described Stm1 as a ribosome dormancy
factor and TORC1-regulated translation inhibitor. These func-
tions involve Stm1's N-terminal domain intercalating in the
ribosome mRNA tunnel, with its linker region (aa 40-60)
bridging the 40S and 60S binding domains (Figure 4A). Indeed,
stm1Δ or N-terminal truncation (Δ2-40 or Δ41-80) shows

<footnote>(F) Illustration of the phospho-mimicking Stm1S229D, T248, S267D, S271D (Stm1S-T/D) and non-phosphorylatable Stm1S229A, T248A, S267A, S271A (Stm1S-T/A) (top) muta-
tants. Serial dilution spottings (bottom) of stm1Δ cells with empty control plasmid or plasmids overexpressing Stm1 (WT), Stm1S-T/D or Stm1S-T/A on YPD media.
Cells were treated or not (UT) with 2.0 or 2.5 nM rapamycin (Rap) and incubated (3 days, 30°C). Representative image from *n* = 3 independent experiments.
(G) Two-hybrid analysis of Stm1, Stm1S-T/A, and Stm1S-T/A fused to the GAL4 AD with Ifh1, the CURI components Utp22, Ckb2, and Rrp7, or the lamin C control
fused to the GAL4 DNA binding domain (DBD). Colonies were grown for 3 days at 30°C on permissive auxotrophic SD –Leu, –Trp (–LT), or selective –His,
–Leu, –Trp agar media, supplemented with 2.5 or 5 mM 3-amino-1,2,4-triazole (–HLT +2.5 mM 3-AT or +5 mM 3-AT). Representative image from *n* = 3 in-
dependent replicates.</footnote>
A

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C

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B

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E

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F

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G

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H

<figure><img src="image_24.png" /><figcaption>Figure 6. Stm1 protects 80S ribosomes upon starvation but promotes RPG transcription by activating Ifh1 during exponential growth</figcaption></figure>

(A) Two-hybrid assay of Stm1 fused to the GAL4 AD (AD Stm1) and Ifh1 truncation mutants¹² fused to the GAL4 DNA binding domain (DBD). Lamin C and untagged Ifh1 were used as controls. Representative image acquired after 19 h 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL) incubation from n = 3 independent replicates.

(B) Fluorescent polarization (FP) assay quantifying binding of purified GST-tagged Ifh1 AD (aa 703–811) (GST-Ifh1-AD) and TAMRA-labeled Stm1-IDR peptide (aa 226–273) (Stm1 IDR-TAMRA), normalized to GST. The dissociation constant (K_d) is indicated. n = 3 independent replicates shown as mean (dot) and standard deviation (error bar).

(C) 2D [¹⁵N,¹H]-heteronuclear single quantum coherence (HSQC) of ¹⁵N-labeled Stm1-IDR (aa 227–273) with resonance assignments indicated.

(D) Overlay of the Stm1 spectrum in the presence of 260 µM Ifh1 (purple) on top of the apo spectrum (black; same spectrum as in C). Resonances with significant chemical shift perturbations are identified. Magnification of the glycine region (black box) is shown.

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reduced 80S ribosomes after starvation.²⁴ In this study, we used a shorter N-terminal truncation (stm1ΔN: Δ2–25) that does not affect its linker region and binds RPs and RAFs. Importantly, *stm1ΔN* cells are not sensitive to rapamycin, indicating Stm1 ribosome protection cannot fully explain the sensitivity of *stm1Δ* cells to TORC1 inhibition. Instead, our results demonstrate that Stm1 plays a second, unlinked function by regulating ribosome number in nutrient-rich conditions. We speculate that the reduced ribosome number explains the rapamycin sensitivity of *stm1ΔIDR* cells, because they cannot recycle excess ribosomes into building blocks during prolonged starvation conditions. Several lines of evidence suggest that Stm1 regulates ribosome biogenesis by increasing nuclear Ifh1 activity. First, while the concentration of cytoplasmic ribosomes is decreased in *stm1Δ* cells, ribosome degradation was comparable to wild-type controls (Figures 1B–1K and S1F–S10). Second, 35S rRNA levels drop in *stm1Δ* cells (Figures 1D and S1J), and Stm1 is essential for cell growth when RNA Pol II (Figures 3A and S3C) or Ifh1 activity is reduced (Figure 3F). Finally, Stm1 enhances the binding of RNA Pol II and Ifh1 to RPG promoters (Figures 3B, 3D, S3E, and S3J). The exact mechanism by which Stm1 modulates Ifh1 activity remains elusive. Stm1 binding to the AD of Ifh1 may increase its ability to recruit Pol II, concomitant with rRNA synthesis and processing.¹¹,¹² This interaction may be transient, and structural work will be required to unravel the function of Stm1 when bound to Ifh1. Alternatively, Stm1 and Ifh1 may compete for binding to the CURI complex, thereby releasing Ifh1 to allow its binding to RPG promoters. Finally, Stm1 was identified as a suppressor of Tom1,⁷⁰ which prevents aggregation of RPs.⁹,⁷¹,⁷² Stm1 may thus impact Ifh1 solubility in the crowded nuclear/nucleolar compartments. Thus, further studies are needed to decipher the detailed molecular mechanisms of how the C-terminal Stm1 IDR motif promotes Ifh1 activity and RPG expression.

## Stm1 may promote ribosome biogenesis independently of rRNA and RAF transcription

While Stm1 enhances Ifh1 activity, it also promotes ribosome biogenesis in the nucleolus downstream of rRNA transcription. Indeed, Stm1 co-purifies with pre-ribosomal particles (Figures 2A–2E), with enhanced association of Stm1 N-terminal mutants (*stm1ΔN*), in part because of its nuclear export signal (Figures S4A–S4G). Stm1 interacts with several components of the SSU processome⁷³ (Figures 2D, 2E, and S4A), which associates with the nascent pre-rRNA and orchestrates RNA folding/cleavage and pre-ribosomal rRNA degradation by the RNA exo-

some. We speculate that Stm1 may alter rRNA processing by regulating SSU activity. Stm1 regulates multiple RNA-mediated processes and binds G4 quadruplex and purine triplex motifs,⁷⁴,⁷⁵ RNA folds present in rRNA and telomeric structure. Moreover, Stm1 was identified as an RNA-binding protein in response to stress⁷⁶ and as a component of the cytoplasmic mRNA degradation machinery,⁷⁷ connecting mRNA decapping to ribosome function. RNA binding triggers phase transition *in vitro* and promotes mRNA de-adenylation and decay of a subclass of mRNAs, including the Dhh1 mRNA targets *EDC1* and *COX17*.⁷⁷ Our studies demonstrate that Stm1 binds RNA through a conserved RGG/GAR-motif, which surprisingly also directly interacts with Ifh1. Future studies will examine whether other RGG/GAR motifs or distinct RNA-binding regions may similarly bind specific proteins in addition to RNA. While we cannot exclude the possibility that RNA and Ifh1 bind Stm1 in a tertiary complex, the overlapping residues mapped by NMR point toward a mutually exclusive structure. If so, the binding of RNA and Ifh1 may compete for Stm1 binding, possibly as part of a regulatory mechanism.

## Differential phosphorylation of Stm1 may switch its growth-promoting and dormancy functions

Our results demonstrate that separable Stm1 domains control ribosome dormancy in response to nutrient starvation²⁴,²⁹ and ribosome production during exponential growth (Figure 7). In nutrient-rich conditions, Stm1 regulates ribosome levels by binding Ifh1 and other CURI subunits, thereby increasing RPG expression and ribosome biogenesis. In the cytoplasm, Stm1 binds 80S ribosomes and polysomes²⁴,⁷⁸ and may delay re-engagement of excess 80S ribosomes in translation.³⁴,⁶⁹ Nutrient starvation switches Stm1 from ribosome production to ribosome hibernation, protecting a subset of mature 80S ribosomes from degradation.²⁴,²⁹ Other ribosome hibernation factors such as yeast late-annotated small open reading frame 2 (Lso2) and its human homolog coiled-coil domain-containing protein 124 (CCDC124)⁷⁹,⁸⁰ may support this process. Starvation triggers phosphorylation of four sites in the C-terminal IDR domain of Stm1, which reduces Ifh1 interaction and contributes to RPG repression. Conversely, TORC1 phosphorylates the N-terminal domain of Stm1 during exponential growth,²⁴ while nutrient starvation leads to Stm1 dephosphorylation to clamp the two ribosomal subunits, preventing their dissociation and subsequent degradation.

## SERBP1 and its homolog HABP4 may similarly regulate ribosome homeostasis in mammalian cells

Mammalian cells express the Stm1 homolog SERBP1, an onco-gene associated with poor prognosis in glioblastoma (GBM).⁸¹

(E) Chemical shift perturbations (CSPs) are plotted against the Stm1 sequence. Gray horizontal lines show one and two standard deviations of the CSP data. P indicates proline 270, which is not visible in the spectrum.

(F) Representation of Stm1 and its C-terminal IDR domain (aa 226–273) (purple box): RGG/GAR RNA-binding motif: orange; phosphorylation sites: blue; Ifh1-interacting residues: purple.

(G) Representation of the Stm1 IDR (aa 226–273), with the RGG/GAR binding motif highlighted in orange (top). Coomassie blue-stained gel of the Stm1 IDR peptide (aa 226–273) incubated with or without yeast total RNA (± tot. RNA) and separated in supernatant (SUP) and pellet (PELLET) (bottom). M, molecular weight (kDa) marker. n = 3 independent replicates.

(H) FP assay quantifying binding of yeast total RNA (total RNA [ng/µL]) to TAMRA-labeled Stm1-IDR WT peptide (aa 226–273) (Stm1 IDR-TAMRA) or a mutant (Stm1 IDR (8A)-TAMRA) with arginine and lysine residues in the RNA-binding RGG/GAR motifs changed to alanine (aa 236–243 ->8A). TAMRA-maleimide controls for non-specific binding. n = 3 independent replicates shown as mean (dot) and standard deviation (error bar).
<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /><figcaption>**Figure 7.** *Stm1 regulates ribosome homeostasis during exponential growth and starvation*</figcaption></figure>

(A and B) Models depicting Stm1 functions during exponential growth (A) and nutrient starvation (B). During nutrient-rich conditions, Stm1 localizes to the nucleolus and interacts with CURI components and the transcription factor Ifh1. Stm1 directly binds RNA and Ifh1 through its RGG/GAR motif and promotes RPG transcription. Stm1 also interacts with pre-ribosomes and may promote ribosome maturation (A). During starvation, ribosome biogenesis is downregulated and cytoplasmic Stm1 inactivates and protects a subset of ribosomes (B).

Like Stm1, SERBP1 binds 80S ribosomes in the cytosol, inhibiting subunit splitting and translation activity.<sup>32,82</sup> SERBP1 also shows nucleolus accumulation in multiple stress conditions,<sup>83</sup> where it interacts with RAF and may function in ribosome biogenesis and rRNA modification.<sup>84</sup> Interestingly, mammalian cells also express HABP4, which shares many sequence motifs with SERBP1 and may thus perform redundant functions.<sup>85</sup> Together with the Dap1/Dap-elf5a module, HABP4 inactivates ribosomes by interacting with EEF2 at the A-site. Indeed, HABP4 plays a critical role in ribosome dormancy within mature oocytes, ensuring ribosome stability.<sup>31</sup> Given the conserved functions of Stm1 and SERBP1/HABP4, our findings may be relevant for future studies to unravel mechanisms of ribosome homeostasis and ribosome crosstalk across multiple cellular compartments.

## Limitations of the study

While our results demonstrate that Stm1 regulates ribosome abundance during exponential growth, multiple activities may contribute to *stm1Δ* rapamycin sensitivity. Stm1 promotes ribosome biogenesis and enhances Ifh1 activity, but the detailed mechanisms remain unclear. Stm1 is required for efficient recruitment of Ifh1 to RPG promoters but may also enhance the transcriptional activity of Ifh1 by directly binding its AD. Further work is needed to understand how Stm1 coordinates RPG expression and ribosome biogenesis, as well as how Stm1 may alter the ribosome pool available for translation. Moreover, open questions persist regarding how the two opposing functions of Stm1 are temporally and spatially regulated by phosphorylation. During growth, the hernation function of Stm1 is prevented by TORC1 phosphorylation,<sup>24</sup> while Stm1 binding to Ifh1 is diminished upon starvation by phosphorylation of the C-terminal IDR. Since both

non-phosphorylatable and phospho-mimicking Stm1-IDR mutants interfere with Ifh1 binding, further work will be required to understand this regulatory mechanism. Surprisingly, the RGG/GAR motif of Stm1 binds both RNA and Ifh1, and it remains to be examined whether this dual binding mode also applies to other RNA-binding motifs. Finally, further work is warranted to understand the conservation of Stm1 functions in mammalian cells.

## RESOURCE AVAILABILITY

### Lead contact

The lead contact is Prof. Matthias Peter (matthias.peter@bc.biol.ethz.ch).

### Materials availability

Find reagents and materials in the key resources table. Materials from this study are available upon request from the lead contact.

### Data and code availability

* All fluorescence microscopy data have been deposited in the ETH Research Collection: https://doi.org/10.3929/ethz-b-000727429 for the BiFC assay and the NES1-2 GFP analysis. All electron microscopy raw data files were deposited in the Electron Microscopy Data Bank (EMDB); EMDB: EMD-52589, EMD-52590, EMD-52591, EMD-52592. All ChIP-seq FASTQ raw files were deposited in the European Nucleotide Archive (ENA): ENA: PRJEB72705. All MS raw and analyzed data files, as well as scripts, were deposited in the Proteomics IDEntifications Database (PRIDE): PRIDE: PXD046775 and PXD046821 for AP-MS of Stm1ΔIDR and Stm1ΔN, respectively; PRIDE: PXD046821 for targeted analysis of CURI complex components with Stm1ΔIDR; PRIDE: PXD046820 for the pulsed-SILAC dataset); and PRIDE: PXD059338 for the phosphorylation analysis of Stm1. All magnetic resonance raw data files were deposited in the ETH repository server and Biological Magnetic Resonance Bank (BMRB): BMRB: 52971. These data are publicly available as of the date of publication.

* The cryo-ET script for ribosome analysis has been deposited on Zenodo at https://doi.org/10.5281/zenodo.15129772.
* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank Tom Dever, Jingwei Xu, Ludovic Gillet, Lennart Opitz, Marina Kunzi, and Anastasia Timofiva for their support with reagents, analysis and experiments; David Shore, Prashant Rawat, Caroline Wilson-Zbinden, and members of the Peter, Neurohr, and Weis laboratories for valuable scientific input; and the ETH ScopeM, Light Imaging Toulouse CBI, ETH FCGZ, ETH Flow Cytometry Core Facility, and the D-BIOL Biomolecular NMR Spectroscopy Platform for essential support. BioRender and Adobe Illustrator were used. M. Pilhofer is supported by the NOMIS Foundation, B.A. by the French National Research Agency (ANR-21-CE12-0008-01), and the Peter laboratory by the Swiss National Science Foundation (SNSF) and ETH Zürich.

## AUTHOR CONTRIBUTIONS

Conceptualization: E.B. and M. Peter; formal analysis: E.B; investigation: E.B., M.B., F.U., M.W., J.T., S.Z., B.A., M.O.-O., R.D., J.H., A.G., and P.K.; writing – original draft: E.B. and M. Peter; writing – review and editing: E.B. and M. Peter, with input from all authors; visualization: E.B. and M. Peter; supervision: V.G.P., M. Peter, and M. Pilhofer; funding acquisition: V.G.P., M. Pilhofer, and M. Peter.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* METHOD DETAILS

* Strains and plasmids

* Spotting assay

* Growth curves and doubling times

* Cell size measurement

* Cell viability assay

* Two-hybrid assay

* β-Galactosidase assay for the yeast two hybrid

* β-Galactosidase assay for the 4uORF GCN4-lacZ reporter

* Protein sample preparation and western blotting

* Ribosome polysome analysis by sucrose gradients

* Chromatin-immunoprecipitation, sequencing analysis, and qPCR

* Protein immunoprecipitation

* Proteomics sample preparation

* Phosphorylation enrichment

* Proteomics sample preparation for pulsed-SILAC experiment

* Mass spectrometry data acquisition

* Mass spectrometry data analysis

* Phosphorylation dataset

* Tandem affinity purification

* In vivo pulse chase biotinylation assay

* Puromycin incorporation assay

* Plunge-freezing

* Cryo-FIB milling and CryoET analysis

* Template matching and concentration estimation for ribosomes

* Live cell microscopy, BiFC measurements, and image analysis

* RNA extraction and GelRed staining

* Protein purification

* Fluorescence polarization assays

* NMR data acquisition and resonance assignment

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.molcel.2025.04.008.

Received: April 2, 2024

Revised: January 26, 2025

Accepted: April 4, 2025

Published: May 1, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Mouse monoclonal anti-Pgk1 (clone 22C5D8) 1:3000</td><td>Life technologies</td><td>Cat# 459250; RRID: AB_2532235</td></tr><tr><td>Mouse monoclonal anti-c-Myc (clone 9E510) 1:3000</td><td>Thermo Fisher Scientific</td><td>Cat# MA1-980; RRID: AB_558470</td></tr><tr><td>Rabbit polyclonal anti-calmodulin binding protein (CBP) 1:2000</td><td>Millipore</td><td>Cat# 07-482; RRID: AB_11212268</td></tr><tr><td>Mouse monoclonal anti-GFP (clones 7.1 and 13.1) 1:3000</td><td>Roche Diagnostics</td><td>Cat# 11814460001; RRID: AB_390913</td></tr><tr><td>Rabbit anti-eIF2α (yeast Sui2) 1:1000</td><td>Generous gift from T. Dever (NIH)</td><td>N/A</td></tr><tr><td>Rabbit polyclonal anti-pSer<sup>52</sup>-eIF2α 1:3000</td><td>Enzo Life Sciences</td><td>Cat# BML-SA405-0020; RRID: AB_2050517</td></tr><tr><td>Mouse monoclonal anti-puromycin (clone 12D10) 1:5000</td><td>Millipore</td><td>Cat# MABE343; RRID: AB_2566826</td></tr><tr><td>Mouse monoclonal anti-Rpb1 1:3000</td><td>Biolegend</td><td>Cat# 664906; RRID: AB_2565554</td></tr><tr><td>Rabbit anti-Rps5 1:3000</td><td>Generous gift from V.G. Panse (UZH)</td><td>N/A</td></tr><tr><td>Rabbit anti-Rpl35 1:3000</td><td>Generous gift from V.G. Panse (UZH)</td><td>N/A</td></tr><tr><td>Rabbit anti-Stm1 (clone 1740) 1:2000</td><td>This study, developed with Eurogentec</td><td>N/A</td></tr><tr><td>Mouse Anti-HA.11 epitope tag antibody</td><td>Biolegend</td><td>Cat# 901515; RRID: AB_2565334</td></tr><tr><td>Goat Anti-Rabbit IgG (H + L)-HRP Conjugate 1:3000</td><td>Biorad</td><td>Cat# 1706515; RRID: AB_11125142</td></tr><tr><td>Goat Anti-Mouse IgG (H + L)-HRP Conjugate 1:3000</td><td>Biorad</td><td>Cat# 1706516; RRID: AB_11125547</td></tr><tr><td>Rabbit polyclonal anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ChIP)</td><td>Abcam</td><td>Cat# ab5131; RRID: AB_449369</td></tr><tr><td>Mouse monoclonal anti-Myc tag antibody [9E10] (ChIP grade)</td><td>Abcam</td><td>Cat# ab32; RRID: AB_303599</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>Rosetta2(DE3) pLysS</td><td>Novagen</td><td>Cat# 71403</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Peptide Stm1 wild type (amino acids 226-273) with N-terminal cysteine for TAMRA labelling-malemide</td><td>GL Biochem (Shanghai) Ltd</td><td>N/A</td></tr><tr><td>Peptide Stm1 8A (amino acids 226-273 with amino acids 236-243 mutated to alanine) with N-terminal cysteine for TAMRA labelling-malemide</td><td>GL Biochem (Shanghai) Ltd</td><td>N/A</td></tr><tr><td>GST-tagged Ifh1 activation domain (amino acids 703-811)(GST-Ifh1-AD)</td><td>This study</td><td>N/A</td></tr><tr><td><sup>15</sup>N-labelled Stm1 IDR (amino acids 227-273)</td><td>This study</td><td>N/A</td></tr><tr><td>Clarity Western ECL substrate</td><td>Biorad</td><td>Cat# 1705061</td></tr><tr><td>SuperSignal™ West Femto Maximun Sensitivity Substrate</td><td>ThermoFisher</td><td>Cat# 34094</td></tr><tr><td>Cycloheximide (CHX)</td><td>Sigma</td><td>Cat# C7698</td></tr><tr><td>Rapamycin</td><td>LC-Laboratories</td><td>Cat# R-5000</td></tr><tr><td>L-Lysine-2HCl (<sup>13</sup>C<sub>6</sub>, 99%; <sup>15</sup>N<sub>2</sub>, 99%)</td><td>Cambridge Isotope Laboratories</td><td>Cat# CNLM-291-H</td></tr><tr><td>3-Amino-1,2,4-triazole</td><td>Merck</td><td>Cat# A8056</td></tr><tr><td>X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)</td><td>Merck</td><td>Cat# 3117073001</td></tr><tr><td>Concanavalin A</td><td>Sigma</td><td>Cat# 11028-71-0</td></tr><tr><td>NuPAGE™ LDS Sample Buffer (4X)</td><td>Invitrogen</td><td>Cat# NP0007</td></tr></tbody></table>

(Continued on next page)
**Continued**

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>HRP-conjugated streptavidin 1:10'000</td><td>ThermoFisher</td><td>Cat# N100</td></tr></tbody></table>

**Critical commercial assays**

<table><tbody><tr><td>14x89mM polypropylene tubes</td><td>Beckman</td><td>Cat# 331372</td></tr><tr><td>Dynabeads™ M-280 Sheep Anti-Rabbit IgG</td><td>Invitrogen</td><td>Cat# 11203D</td></tr><tr><td>Dynabeads™ M-280 Sheep Anti-Mouse IgG</td><td>Invitrogen</td><td>Cat# 11202D</td></tr><tr><td>MinElute PCR Purification Kit</td><td>Qiagen</td><td>Cat# 28004</td></tr><tr><td>NEBNext® Ultra™ DNA Library Prep Kit for Illumina®</td><td>NEB</td><td>Cat# E7370L</td></tr><tr><td>cOmplete™ EDTA free protease inhibitor</td><td>Roche</td><td>Cat# 11836170001</td></tr><tr><td>Streptavidin-conjugated magnetic beads</td><td>Invitrogen</td><td>Cat# 65605D</td></tr><tr><td>Anti-c-Myc magnetic beads</td><td>Thermo Fisher Scientific</td><td>Cat# 88843</td></tr><tr><td>IgG Sepharose™ 6 Fast Flow</td><td>Merck</td><td>Cat# GE17-0969-01</td></tr><tr><td>Calmodulin-Sepharose™ 4B</td><td>Merck</td><td>Cat# GE17-0529-01</td></tr><tr><td>Yeast nitrogen base without amino acids and biotin (YNB-Biotin)</td><td>Sunrise Science Products</td><td>Cat# 1523-100</td></tr><tr><td>Vivacon® 500, 10,000 MWCO Hydrosart</td><td>Sartorius</td><td>Cat# VN01H01</td></tr><tr><td>GelRed® nucleic acid gel stain</td><td>Botium</td><td>Cat# 41003</td></tr><tr><td>ROTI® Aqua-Phenol, 250 ml</td><td>Roth</td><td>Cat# A980.1</td></tr><tr><td>BD Pharmigen™ PI/RNase Staining Buffer For Flow Cytometry</td><td>BD Biosciences</td><td>Cat# 550825</td></tr></tbody></table>

**Deposited data**

<table><tbody><tr><td>Cryo-tomogram of a FIB-milled wild-type untreated yeast cell</td><td>This study</td><td>EMDB: EMD-52589</td></tr><tr><td>Cryo-tomogram of a FIB-milled STM1-deletion untreated yeast cell</td><td>This study</td><td>EMDB: EMD-52590</td></tr><tr><td>Cryo-tomogram of a FIB-milled wild-type rapamycin treated yeast cell</td><td>This study</td><td>EMDB: EMD-52591</td></tr><tr><td>Cryo-tomogram of a FIB-milled STM1-deletion rapamycin treated yeast cell</td><td>This study</td><td>EMDB: EMD-52592</td></tr><tr><td>Pulsed SILAC experiment</td><td>This study</td><td>Table S1; PRIDE: PXD046820</td></tr><tr><td>Characterization STM1 pulldown (C terminal) DDA</td><td>This study</td><td>Table S2;PRIDE: PXD046775</td></tr><tr><td>RNA Pol II Ser5 ChIP-Seq</td><td>This study</td><td>Table S3; ENA: PRJEB72705</td></tr><tr><td>Characterization STM1 pulldown (N terminal)"</td><td>This study</td><td>Table S4; PRIDE: PXD046774</td></tr><tr><td>Characterization STM1 pulldown (C terminal)" PRM</td><td>This study</td><td>Table S5; PRIDE: PXD046821</td></tr><tr><td>Phosphoenrichment Stm1</td><td>This study</td><td>Table S6;PRIDE: PXD059338</td></tr><tr><td>BiFC assay</td><td>This study</td><td>ETH Research Collection: 10.3929/ethz-b-000727429</td></tr><tr><td>NES1-2 GFP analysis</td><td>This study</td><td>ETH Research Collection: 10.3929/ethz-b-000727429</td></tr><tr><td>Stm1 IDR NMR assignments</td><td>This study</td><td>BMRB: 52971</td></tr></tbody></table>

**Experimental models: Organisms/strains**

<table><tbody><tr><td>BY4741: MAT<sub>a</sub> his3Δ1 leu2Δ0 met15Δ0 ura3Δ0</td><td>OpenBiosystems</td><td>N/A</td></tr><tr><td>BY4742: MAT<sub>α</sub> his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0</td><td>Karsten Weis</td><td>N/A</td></tr><tr><td>W303: MAT<sub>a</sub> leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15</td><td>OpenBiosystems</td><td>N/A</td></tr><tr><td>NMY32: MAT<sub>a</sub> trp1 leu2 his3 ade2 LYS2::lexA-HIS3 ade2::lexA-ADE2 URA3::lexA-lacZ</td><td>Vikram G. Panse</td><td>N/A</td></tr><tr><td>yJW1779: BY4741 sporulated, rpl16a::RPL16a-HA-TEV-AVI rpl16b::RPL16b-HA-TEV-AVI ura3Δ0 met15Δ0 his3Δ1 leu2Δ0</td><td>Jan et al.<sup>86</sup></td><td>N/A</td></tr></tbody></table>

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<figure><img src="image_28.png" /></figure>

Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>YEB5: yJW1779, pADH1-OsTir1-9xMyc::HIS3<br>GEV-RFP::LEU2 pGAL-HA-2xIAA-BirA::URA3</td><td>This study</td><td>N/A</td></tr><tr><td>YJT474: yJW1779 pADH1-OsTir1-9xMyc::HIS3<br>GEV-RFP::LEU2 pGAL-HA-2xIAA-BirA::URA3<br>stm1::NATMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YJT370: BY4741, pRS426 empty (pMJ364)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT371: BY4741 WT, pRS426 STM1 (pJT35)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT372: BY4741, STM1::NATMX6, pRS426<br>empty (pMJ364)</td><td>This study</td><td>N/A</td></tr><tr><td>YEB134: BY4741, STM1::NATMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YEB687: BY4742, STM1::NATMX6</td><td>This study</td><td>N/A</td></tr><tr><td>BY4741, noc4::Noc4-TAP::HIS3</td><td>Horizon Discovery</td><td>N/A</td></tr><tr><td>BY4741, nop7::Nop7-TAP::HIS3</td><td>Horizon Discovery</td><td>N/A</td></tr><tr><td>BY4741, rpb8::Rpb8-TAP::HIS3</td><td>Horizon Discovery</td><td>N/A</td></tr><tr><td>BY4741 rpl24::Rpl24-TAP::HIS3</td><td>Horizon Discovery</td><td>N/A</td></tr><tr><td>YEB23: BY4741 GEV-RFP::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT428: BY4741 pGAL-Vc-STM1::HIS3 GEV-RFP::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT429: BY4742 utp22::UTP22-Vn::KANMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YJT430: BY4742 rpl42a::RPL42a-Vn::KANMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YJT444: BY4741 STM1::pGAL-Vc-STM1::HIS<br>GEV-RFP::LEU2 rrp7::Utp22-Vn::KANMX6 (clone 1)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT445: BY4742 STM1::pGAL-Vc-STM1::HIS<br>GEV-RFP::LEU2 rrp7::RRP7-Vn::KANMX6 (clone 2)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT453: BY4741 STM1::pGAL-Vc-STM1::HIS<br>GEV-RFP::LEU2 rrp7::RRP7-Vn::KANMX6 (clone 1)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT454: BY4742 STM1::pGAL-Vc-STM1::HIS<br>GEV-RFP::LEU2 rrp7::RRP7-Vn::KANMX6 (clone 2)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT455: BY4742 rrp7::RRP7-Vn::KANMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YJT458: BY4742 STM1::pGAL-Vc-STM1::HIS3<br>GEV-RFP::LEU2 rpl42a::RPL42a-Vn::KANMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YPK148: W303, STM1::NATMX6</td><td>This study</td><td>N/A</td></tr><tr><td>HHY170: W303 tor1-1 fpr1::NATMX6,<br>trp1::RPL13A-2×FKBP12::TRP1,<br>kan::RPO21-FRB::KANMX6</td><td>Haruki et al.<sup>44</sup></td><td>N/A</td></tr><tr><td>HHY170 STM1::URA3</td><td>This study</td><td>N/A</td></tr><tr><td>YJT466: HHY170 rpo21(H1085Q)::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT467: HHY170 rpo21<sup>N1082S</sup>::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT468: HHY170 rpo21<sup>N479S</sup>::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT469: HHY170 RPO21 WT::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT470: HHY170 STM1::URA3 RPO21WT::LEU2</td><td>Haruki et al.<sup>44</sup></td><td>N/A</td></tr><tr><td>YJT471: HHY170 STM1::URA3 rpo21<sup>N479S</sup>::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT472: HHY170 STM1::URA3 rpo21<sup>N1082S</sup>::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>YJT473: HHY170 STM1::URA3 rpo21<sup>H1085Q</sup>::LEU2</td><td>This study</td><td>N/A</td></tr><tr><td>BEN22 (YDS2): W303</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>BEN24: W303 ifh1-6(Δ0-644)-16xMyc::HIS3</td><td>Benjamin Albert</td><td>N/A</td></tr><tr><td>BEN392: W303 GEM(Gal4-ER-Msn2)::HIS3<br>Nop56-3xmCherry::KANMX6</td><td>Benjamin Albert</td><td>N/A</td></tr><tr><td>YJT411: BEN22 STM1::NATMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YJT427: BEN24 STM1::NATMX6</td><td>This study</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>YJT476: BEN24 stm1::NATMX6 STM1ΔIDR (226-270Δ)-6xGLY-3xMYC::URA3 (pJT46)</td><td>This study</td><td>N/A</td></tr><tr><td>BY4741 TetO7-RPL18A::KANMX6, CMV-tTA::URA3</td><td>OpenBiosystems</td><td>N/A</td></tr><tr><td>YJT400: BY4741 TetO7-RPL18A::KANMX6 CMV-tTA::URA3 stm1::NATMX6</td><td>This study</td><td>N/A</td></tr><tr><td>YEB676:BY4741 GFP-3xMyc::URA3 (pEB169)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT416: BY4741 stm1::NatMX6NATMX6 STM1ΔIDR (226-270Δ)-6xGLY-3xMYC::URA3 (pJT46)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT417: BY4741 stm1::NatMX6, STM1(Δ176-225)-6xGLY-3xMYC::URA3 (pJT47)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT418: BY4741 stm1::NatMX6, STM1ΔN(25-273)-6xGLY-3xMYC::URA3 (pJT44)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT419: BY4741 stm1::NatMX6, STM1ΔC(176-270Δ)-6xGLY-3xMYC::URA3 (pJT43)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT421: BY4741 stm1::NatMX6, STM1-6xGLY-3xMYC::URA3 (pJT41)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT432: BY4741 WT, pSIVura</td><td>This study</td><td>N/A</td></tr><tr><td>YJT433: BY4741 stm1::NatMX6, pSIVura</td><td>This study</td><td>N/A</td></tr><tr><td>YJT438: BY4741, pRS426-yEGFP-7aa-Stm1 (pEB64)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT439: BY4741 stm1::NatMX6, pRS426-yEGFP-7aa-STM1 (pEB64)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT440: BY4741 stm1::NatMX6, pRS426-yEGFP-7aa linker-STM1(175-273)</td><td>This study</td><td>N/A</td></tr><tr><td>YJT441: BY4741, pRS426-yEGFP-7aa linker-STM1(175-273)</td><td>This study</td><td>N/A</td></tr><tr><td>YEBJT148: BY4741 stm1::Stm1-GFP::HIS3</td><td>This study</td><td>N/A</td></tr><tr><td>YEB155: BY4741 stm1::yEGFP-Stm1</td><td>Thermo Fischer Scientific</td><td>N/A</td></tr><tr><td>yJT373: BY4741, stm1::NATMX6, pRS426 STM1 (pJT35)</td><td>This study</td><td>N/A</td></tr><tr><td>gcn2::KANMX6/GCN2</td><td>Euroscarf</td><td>N/A</td></tr><tr><td>yJT505: gcn2::KANMX6, p180 (4 uORF GCN4 lacz wt)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT512: gcn2::KANMX6; stm1::NAT, p180 (4 uORF GCN4 lacz wt)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT513: BY4741, p180 (4 uORF GCN4 lacz wt)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT514: stm1::NATMX6, p180 (4 uORF GCN4 lacz wt)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT552: stm1::NATMX6, pRS426-pSTM1-STM1<sup>S229A, T248A, S267A, S271A</sup>-tSTM1 (pJT82)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT558: stm1::NATMX6, pRS426-pSTM1-STM1<sup>S229D, T248D, S267D, S271D</sup>-tSTM1 (pJT83)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT578: rpo21::KANMX6, stm1::NATMX6, rpo21<sup>N479S</sup>::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT579: rpo21::KANMX6, stm1::NATMX6, rpo21<sup>H1085Q</sup>::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT580: rpo21::KANMX6, rpo21<sup>N479S</sup>::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT581: rpo21::KANMX6, stm1::NATMX6, rpo21::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT582: rpo21::KANMX6, RPO21::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT583: rpo21::KANMX6, stm1::NATMX6, rpo21<sup>N1082S</sup>::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT584: rpo21::KANMX6, rpo21<sup>N1082S</sup>::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT585: rpo21::KANMX6, rpo21<sup>H1085Q</sup>::URA</td><td>This study</td><td>N/A</td></tr><tr><td>yJT586: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-Stm1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT587: NMY32 pLexA-DBD(N)-Ifh1, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT588: NMY32 pLexA-DBD(N)-Rrp7, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>yJT589: NMY32 pLexA-DBD(N)-Utp22, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT590: NMY32 pLexA-DBD(N)-Ckb2, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT591: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-STM1<sup>S229A, T248A, S267A, S271A</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT592: NMY32 pLexA-DBD(N)-Ifh1, pACT2.2-AD(N)-STM1<sup>S229A, T248A, S267A, S271A</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT593: NMY32 pLexA-DBD(N)-Rrp7, pACT2.2-AD(N)-STM1<sup>S229A, T248A, S267A, S271A</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT594: NMY32 pLexA-DBD(N)-Utp22, pACT2.2-AD(N)-STM1<sup>S229A, T248A, S267A, S271A</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT595: NMY32 pLexA-DBD(N)-Ckb2, pACT2.2-AD(N)-STM1<sup>S229A, T248A, S267A, S271A</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT596: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-STM1<sup>S229D, T248D, S267D, S271D</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT597: NMY32 pLexA-DBD(N)-Ifh1, pACT2.2-AD(N)-STM1<sup>S229D, T248D, S267D, S271D</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT598: NMY32 pLexA-DBD(N)-Rrp7, pACT2.2-AD(N)-STM1<sup>S229D, T248D, S267D, S271D</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT599: NMY32 pLexA-DBD(N)-Utp22, pACT2.2-AD(N)-STM1<sup>S229D, T248D, S267D, S271D</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT600: NMY32 pLexA-DBD(N)-Ckb2, pACT2.2-AD(N)-STM1<sup>S229D, T248D, S267D, S271D</sup></td><td>This study</td><td>N/A</td></tr><tr><td>yJT586: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yEB505: NMY32 pLexA-DBD(N)-IFH1, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT446: NMY32 pLexA-DBD(N)-IFH1(1-811)-13xMYC, pACT2.2-AD(N) STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT447: NMY32 pLexA-DBD(N)-IFH1(1-702)-13xMYC, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT448: NMY32 pLexA-DBD(N)-IFH1(0-188del)-13xMYC, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT449: NMY32 pLexA-DBD(N)-IFH1(0-555del)-13xMYC, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT450: NMY32 pLexA-DBD(N)-IFH1(0-644del)-13xMYC, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT451: NMY32 pLexA-DBD(N)-IFH1(0-695del)-13xMYC, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT452: NMY32 pLexA-DBD(N)-IFH1-13xMYC, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT464: NMY32 pLexA-DBD(N)-IFH1(1-561)-13xMYC, pACT2.2 STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT589: NMY32 pLexA-DBD(N)-UTP22, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT590: NMY32 pLexA-DBD(N)-CKB2, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJT588: NMY32 pLexA-DBD(N)-RRP7, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yJ587: NMY32 pLexA-DBD(N)-IFH1, pACT2.2-AD(N)-STM1 FL</td><td>This study</td><td>N/A</td></tr><tr><td>yEB537: NMY32 pLexA-DBD(N)-UTP22, pACT2.2-AD(N)-STM1ΔC(1-175)</td><td>This study</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
**Continued**

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>yEB606: NMY32 pLexA-DBD(N)-IFH1, pACT2.2-AD(N)-STM1ΔC(1-175)</td><td>This study</td><td>N/A</td></tr><tr><td>yEB607: NMY32 pLexA-DBD(N)-CKB2, pACT2.2-AD(N)-STM1ΔC(1-175)</td><td>This study</td><td>N/A</td></tr><tr><td>yEB608: NMY32 pLexA-DBD(N)-RRP7, pACT2.2-AD(N)-STM1ΔC(1-175)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT385: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-STM1ΔC(1-175)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT406: NMY32 pLexA-DBD(N)-RRP7, pACT2.2-AD(N)-STM1-IDRΔ(226-270Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT407: NMY32 pLexA-DBD(N)-CKB2, pACT2.2-AD(N)-STM1-IDRΔ (226-270Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT408: NMY32 pLexA-DBD(N)-IFH1, pACT2.2-AD(N)-STM1-IDRΔ (226-270Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT409: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-STM1-IDRΔ (226-270Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT410: NMY32 pLexA-DBD(N)-UTP22, pACT2.2-AD(N)-STM1-IDRΔ (226-270Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT422: NMY32 pLexA-DBD(N)-CKB2, pACT2.2-AD(N)-STM1(176-225Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT423: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-STM1(176-225Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT424: NMY32 pLexA-DBD(N)-IFH1, pACT2.2-AD(N)-STM1(176-225Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT425: NMY32 pLexA-DBD(N)-RRP7, pACT2.2 Stm1(N)-STM1(176-225Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT426: NMY32 pLexA-DBD(N)-UTP22, pACT2.2-AD(N)-STM1(176-225Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT386: NMY32 pLexA-DBD(N)-LaminC, pACT2.2-AD(N)-STM1 C-ONLY(176-273)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT394: NMY32 pLexA-DBD(N)-CKB2, pACT2.2-AD(N)-STM1 C-ONLY(176-273)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT395: NMY32 pLexA-DBD(N)-IFH1, pACT2.2-AD(N)-STM1 C-ONLY(176-273)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT397: NMY32 pLexA-DBD(N)-RRP7, pACT2.2-AD(N)-STM1 C-ONLY(176-273)</td><td>This study</td><td>N/A</td></tr><tr><td>yJT399: NMY32 pLexA-DBD(N)-UTP22, pACT2.2-AD(N)-STM1 C-ONLY(176273)</td><td>This study</td><td>N/A</td></tr></tbody></table>

**Recombinant DNA**

<table><tr><td>pFA6a-NATMX6 (AMP)</td><td>Euroscarf</td><td>Cat# 30437</td></tr><tr><td>pFA6a-KANMX6 (AMP)</td><td>Euroscarf</td><td>Cat# 39296</td></tr><tr><td>pSIVura (AMP)</td><td>Addgene</td><td>Cat# 81089</td></tr><tr><td>pSIVleu (AMP)</td><td>Addgene</td><td>Cat# 81090</td></tr><tr><td>pCZ65:pFA6a-VN173-KANMX6 (AMP)</td><td>Bioneer</td><td>N/A</td></tr><tr><td>pCZ68: pFA6a-HIS3MX6-PGAL1-VC155 (AMP)</td><td>Bioneer</td><td>N/A</td></tr><tr><td>pJT35: pRS426 pStm1-Stm1-tStm1 (URA)</td><td>This study</td><td>N/A</td></tr><tr><td>pMJ364: pRS426 empty plasmid (URA)</td><td>This study</td><td>N/A</td></tr><tr><td>pJT40:pSIVura pStm1-STM1-tStm1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT41: pSIVura pStm1-STM1-6xGLY-3xMYC-tStm1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT42: pSIVura pStm1-STM1(RRG/GAR to 8Ala) (AA236-243)-6xGLY-3xMYC-tStm1</td><td>This study</td><td>N/A</td></tr></table>

(Continued on next page)
<figure><img src="image_29.png" /></figure>

Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>pJT43: pSIVura pStm1-STM1ΔC(176-270Δ)-6xGLY-3xMYC-tStm1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT44: pSIVura pStm1-STM1ΔN(25-273)-6xGLY-3xMYC-tStm1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT45: pSIVura pStm1-STM1ΔRGG/GAR (236-243Δ)-6xGLY-3xMYC-tStm1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT46: pSIVura pStm1-STM1ΔIDR (226-270Δ)-6xGLY-3xMYC-tStm1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT47: pSIVura pStm1-STM1(Δ176-225)-6xGLY-3xMYC-tStm1</td><td>This study</td><td>N/A</td></tr><tr><td>pEB169: pSIVu-pGAP-GFP-3xMyc-tCyc</td><td>This study</td><td>N/A</td></tr><tr><td>pLexA-DBD(N) LexA DNA Binding Domain (N terminal on backbone)</td><td>Vikram G. Panse</td><td>N/A</td></tr><tr><td>pLexA-DBD(N)-LaminC</td><td>Vikram G. Panse</td><td>N/A</td></tr><tr><td>pEB135: pLexA-DBD(N)-NOP56</td><td>Vikram G. Panse</td><td>N/A</td></tr><tr><td>pJT65: pLexA-DBD(N)-Spel-empty with single base pair insertion in MCS</td><td>This study</td><td>N/A</td></tr><tr><td>pJT57: pLexA-DBD(N) with additional restriction site in MCS (Spel)</td><td>This study</td><td>N/A</td></tr><tr><td>pEB100: pACT2.2-AD(N)-STM1</td><td>This study</td><td>N/A</td></tr><tr><td>pACT2.2-AD(N)-STM1ΔC(1-175)</td><td>This study</td><td>N/A</td></tr><tr><td>pEB162: pACT2.2-AD(N)-STM1 C-ONLY(176-273)</td><td>This study</td><td>N/A</td></tr><tr><td>pJT39: pACT2.2-AD(N)STM1-ΔIDR(226-270Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>pJT48: pACT2.2-AD(N)STM1(176-225Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>pEB114: pLexA-DBD(N)-Cdc13</td><td>This study</td><td>N/A</td></tr><tr><td>pEB127: pLexA-DBD(N)-MPP10</td><td>This study</td><td>N/A</td></tr><tr><td>pLexA-DBD(N)-KRR1</td><td>This study</td><td>N/A</td></tr><tr><td>pEB95: pLexA-DBD(N)-SAS10</td><td>This study</td><td>N/A</td></tr><tr><td>pEB129: pLexA-DBD(N)-LCP5</td><td>This study</td><td>N/A</td></tr><tr><td>pEB96: pLexA-DBD(N)-UTP18</td><td>This study</td><td>N/A</td></tr><tr><td>pLexA-DBD(N)-Noc4</td><td>This study</td><td>N/A</td></tr><tr><td>pEB122: pLexA-DBD(N)-UTP22</td><td>This study</td><td>N/A</td></tr><tr><td>pEB8: pLexA-DBD(N)-ENP2</td><td>This study</td><td>N/A</td></tr><tr><td>pEB124: pLexA-DBD(N)-RPA49</td><td>This study</td><td>N/A</td></tr><tr><td>pEB128: pLexA-DBD(N)-RPA190</td><td>This study</td><td>N/A</td></tr><tr><td>pLexA-DBD(N)-RRP7</td><td>This study</td><td>N/A</td></tr><tr><td>pLexA-DBD(N)-CKB2</td><td>This study</td><td>N/A</td></tr><tr><td>pLexA-DBD(N)-IFH1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT58: pLexA-DBD(N)-IFH1(1-811)-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pJT59: pLexA-DBD(N)-IFH1(1-702)-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pJT60: pLexA-DBD(N)-IFH1(1-561)-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pJT61: pLexA-DBD(N)-IFH1(0-188del)-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pJT62: pLexA-DBD(N)-IFH1(0-555del)-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pJT63: pLexA-DBD(N)-IFH1(0-644del)-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pJT64: pLexA-DBD(N)-IFH1(0-695del)-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pJT66: pLexA-DBD(N)-IFH1-13xMYC</td><td>This study</td><td>N/A</td></tr><tr><td>pVM49: 2μ URA3 GAD-ifh1-1(1-811)</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>pVM50: 2μ URA3 GAD-ifh1-2(1-702)</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>pVM52: 2μ URA3 GAD-ifh1-4(Δ0-188)</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>pVM53: 2μ URA3 GAD-ifh1-5(Δ0-555)</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>pVM54: 2μ URA3 GAD-ifh1-6(Δ0-644)</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>pVM55: 2μ URA3 GAD-ifh1-7(Δ0-695)</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>pVM75: pRS313 IFH1-13MYC mutated at ATG to contain Smal site</td><td>Albert et al.<sup>12</sup></td><td>N/A</td></tr><tr><td>pJT51: pRS426-pSTM1-yEGFP-7AA linker-STM1(175-273)-tSTM1</td><td>This study</td><td>N/A</td></tr><tr><td>pEB64: pRS426-pSTM1-yEGFP-7AA linker-STM1-tSTM1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT52: pRS426-pSTM1-STM1-6xGLY-yEGFP-tSTM1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT53: pRS426-pSTM1-STM1(175-273)-6xGLY-yEGFP-tSTM1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT67: pSIVleu-Sacl-pRPO21-RPO21-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pJT68: pSIVleu-Sacl-pRpO21-rpo21<sup>N479S</sup>-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pJT69: pSIVleu-Sacl-pRPO21-rpo21<sup>N1082</sup>-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pJT70: pSIVleu-Sacl-pRPO21-rpo21<sup>H1085Q</sup>-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pMW_079:pGEX-5x-1-Cys-IFH1_AD</td><td>This study</td><td>N/A</td></tr><tr><td>pK27-Smt3</td><td>Dirk Görlich</td><td>N/A</td></tr><tr><td>pMW_093: pK27 Twin Strep Twin-Strep-Smt3-Stm1_IDR (amino acid 226-273)</td><td>This study</td><td>N/A</td></tr><tr><td>pPGK-BirA (cytosolic)</td><td>Jan et al.<sup>86</sup></td><td>N/A</td></tr><tr><td>pEB3: pRS305-pPGK1-BirA</td><td>This study</td><td>N/A</td></tr><tr><td>pEB5: pRS306 pGal HA-2xIAA-BirA</td><td>This study</td><td>N/A</td></tr><tr><td>pEB10: pRS305-pcyc-GEV-RFP</td><td>This study</td><td>N/A</td></tr><tr><td>pEB11: pRS303-pADH1-OsTIR1-9xMyc-HIS</td><td>This study</td><td>N/A</td></tr><tr><td>pJT82: pRS426-pSTM1-STM1<sup>S229A, T248A, S267A, S271A</sup>-tSTM1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT83: pRS426-pSTM1-STM1<sup>S229D, T248D, S267D, S271D</sup>-tSTM1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT89: pSIVura-Sacl-pRPO21-RPO21-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pJT90: pSIVura-Sacl-pRPO21-rpo21<sup>N479S</sup>-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pJT91: pSIVura-Sacl-pRPO21-rpo21<sup>N1082S</sup>-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pJT92: pSIVura-Sacl-pRPO21-rpo21<sup>H1085Q</sup>-tRPO21-BamHI</td><td>This study</td><td>N/A</td></tr><tr><td>pJT93: pRS416 pGAL1-GFP-STM1 FL-tCYC1</td><td>This study</td><td>N/A</td></tr><tr><td>pJT97: pRS416 pGAL1-GFP-STM1ΔNES1-tCYC1 (6-20Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>pJT98: pRS416 pGAL1-GFP-STM1ΔNES2_tCYC1 (160-174Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>pJT99: pRS416 pGAL1-GFP-STM1ΔΔNES-tCYC1 (6-20Δ, 160-174Δ)</td><td>This study</td><td>N/A</td></tr><tr><td>pJT103: pACT2.2-STM1<sup>S229A, T248A, S267A, S271A</sup></td><td>This study</td><td>N/A</td></tr><tr><td>pJT109: pACT2.2-STM1<sup>S229D, T248D, S267D, S271D</sup></td><td>This study</td><td>N/A</td></tr><tr><td>p180: URA3, ARS1, CEN4 plasmid containing a GCN4::lacZ fusion with lacZ fused in-frame at the 55th codon of GCN4</td><td>Hinnebusch<sup>50</sup></td><td>N/A</td></tr></tbody></table>

Software and algorithms

<table><tr><td>Fiji (v. 2.14.0/1.54f)</td><td>Schindelin et al.<sup>87</sup></td><td>https://imagej.net/software/fiji/</td></tr><tr><td>Fastp (v0.20)</td><td>Chen et al.<sup>88</sup></td><td>https://github.com/Openene/fastp</td></tr><tr><td>Bowtie2 (v2.4.1)</td><td>Langmead and Salzberg<sup>89</sup></td><td>https://bowtiebio.sourceforge.net/bowtie2/index.shtml</td></tr><tr><td>Rsubread (v2.4)</td><td>Liao et al.<sup>90</sup></td><td>https://bioconductor.org/packages/release/bioc/html/Rsubread.html</td></tr><tr><td>edgeR (v3.30)</td><td>Robinson et al.<sup>91</sup></td><td>https://bioconductor.org/packages/release/bioc/html/edgeR.html</td></tr><tr><td>ShinyGO (0.76)</td><td>Ge et al.<sup>92</sup></td><td>https://bioinformatics.sdstate.edu/go/</td></tr><tr><td>SnapGene software (v57.2.21)</td><td>N/A</td><td>https://www.snapgene.com/</td></tr><tr><td>GraphPad Prism software (v10.2.1-)</td><td>N/A</td><td>https://www graphpad.com/</td></tr><tr><td>Microsoft Office Excel (v16.93)</td><td>Microsoft</td><td>https://www.microsoft.com/</td></tr><tr><td>MaxQuant software (v2.4.13)</td><td>Cox and Mann<sup>93</sup></td><td>https://www.maxquant.org/</td></tr><tr><td>Micro-manager (v.1.4.23)</td><td>Edelstein et al.<sup>94</sup></td><td>https://micro-manager.org/</td></tr></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Topspin (v4.0.9)</td><td>Bruker</td><td>https://www.bruker.com/en/products-and-solutions/mr/nmr-software/topspin/</td></tr><tr><td>Computer Aided Resonance Assignment (CARA)</td><td>N/A</td><td>http://www.cara.nmr.ch/doku.php/Home</td></tr><tr><td>FlowJo (v10)</td><td>BD Bioscience</td><td>https://www.bdbiosciences.com/en-ch/products/software/flowjo-v10-software</td></tr><tr><td>SerialEM</td><td>Mastronarde<sup>95</sup></td><td>https://bio3d.colorado.edu/SerialEM/</td></tr><tr><td>IMOD</td><td>Kremer et al.<sup>96</sup></td><td>https://bio3d.colorado.edu/imod/</td></tr><tr><td>IsoNet</td><td>Liu et al.<sup>97</sup></td><td>https://github.com/IsoNet-cryoET/IsoNet</td></tr><tr><td>BD CSampler Plus Software (v1.0.34.1)</td><td>BD Biosciences</td><td>Cat# 661310</td></tr><tr><td>Dynamo</td><td>Castaño-Díez<sup>98</sup></td><td>https://www.dynamo-em.org/w/index.php?title=Main_Page</td></tr><tr><td>Matlab R2023a</td><td>MathWorks</td><td>https://www.mathworks.com/products/new_products/release2023a.html</td></tr><tr><td>PACE-TOMO</td><td>Eisenstein et al.<sup>99</sup></td><td>https://github.com/eisfabian/PACETomo</td></tr><tr><td>UCSF ChimeraX</td><td>Pettersen et al.<sup>100</sup></td><td>https://www.rbvi.ucsf.edu/chimerax/</td></tr><tr><td>TomoSampling.py</td><td>This study</td><td>https://github.com/mbonassera/ribo_cryoET</td></tr><tr><td>Spectrostar plate reader</td><td>Spectrostar BMG Labtech</td><td>N/A</td></tr><tr><td>VersaMax microplate reader</td><td>Molecular Devices</td><td>N/A</td></tr><tr><td>Coulter Counter Multisizer</td><td>Beckman</td><td>N/A</td></tr><tr><td>Polyvinylidene (PVDF) membranes</td><td>Sigma</td><td>Cat# P2938</td></tr><tr><td>Amersham Protran 0.45 NC nitrocellulose Western blotting membranes</td><td>Amersham</td><td>Cat# 10600002</td></tr><tr><td>NuPAGE™ Bis-Tris Mini Protein Gels, 4–12%, 1.0–1.5 mmp</td><td>Invitrogen</td><td>Cat# NP0321</td></tr><tr><td>Fusion Fx instrument</td><td>Vilber Lourmat</td><td>N/A</td></tr><tr><td>Quantifoil copper grids (R2/2 200 mesh)</td><td>Quantifoil</td><td>https://www.quantifoil.com/</td></tr><tr><td>Vitrobot Mark IV</td><td>Thermo Fisher Scientific</td><td>N/A</td></tr><tr><td>Cryo-transmission electron microscope Titan Krios (300kV)</td><td>Thermo Fisher Scientific</td><td>N/A</td></tr><tr><td>Crossbeam 550 FIB-SEM</td><td>Zeiss</td><td>N/A</td></tr><tr><td>BD Accuri C6 Plus flow cytometer</td><td>BD Biosciences</td><td>Cat# 661310</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Budding yeast *Saccharomyces cerevisiae* (RRID: SCR_007282) was used as experimental model.

## METHOD DETAILS

### Strains and plasmids

All plasmids and strains used in this study are listed in **key resources table**. Plasmids were generated using standard molecular biology methods and checked by sequencing. Yeast strains are derivatives of BY4741, BY4742, W303 and NMY32 backgrounds. Gene replacement and tagging at the endogenous locus were obtained by PCR-based homology recombination or by crossing and sporulation. DNA oligonucleotide sequences used for cloning are available upon request to the Lead contact. Cells were grown
at 30°C in yeast extract pentose dextrose (YPD) (1% yeast extract, 2% peptone and 2% dextrose) or in synthetic defined (SD) medium (0.17% Yeast nitrogen base without amino acids, 2% carbon source, 0.5% NH₄ sulfate and 1x amino acids mix) and harvested in mid-log phase (OD₆₀₀ = 0.6), unless differently stated.

### Spotting assay

Cells were inoculated overnight. The next day, cultures were diluted to OD₆₀₀ = 0.1-0.15. After two generations cells were normalized to OD₆₀₀ = 0.3. Five 1:5 serial dilutions were prepared in a 96-well round bottom plate (50µL cells suspension in 250µL total volume). Dilutions were spotted on YPD or SD agar plates containing rapamycin (dissolved in 90% EtOH and 10% Tween) or cycloheximide (dissolved in DMSO) at the indicated concentrations. Pictures were taken after growth for 2-3 days at 30°C.

### Growth curves and doubling times

For stationary phase experiments, cells were inoculated overnight. The next day they were diluted in fresh media at OD₆₀₀= 0.1. Cells were incubated for 3 or 6days. Then, cells were normalized to OD₆₀₀= 0.1 and diluted in fresh media in a flat bottom plate. The optical density was recorded over time using a Spectrostar plate reader. Measurements of 3 biological independent replicates are plotted as mean and standard deviation in GraphPad Prism (v10.2.1).

For pulse SILAC experiments, cells were inoculated in SD without L-Lysine (SD - Lys) media but supplemented with light L-Lysine (25mg/L) and diluted after 8h into exponential phase. After 20 hours, OD₆₀₀ was measured and normalized to 0.1. Cells to be grown in SD media containing 25mg/mL heavy Lysine (¹³C₆, ¹⁵N₂) (Cambridge Isotope Laboratories) were filter washed using a 0.45µM pore size nitrocellulose membrane, washed twice with SD + heavy Lysine and resuspended in SD + heavy Lysine. The same procedure was executed with SD + light Lysine for cells to be grown in light Lysine. The optical density was recorded over time using a Spectrostar plate reader. Measurements of 3 biological independent replicates are plotted as mean and standard deviation in GraphPad Prism (v10.2.1). Doubling times were extracted from exponential growth phase, and calculated using the GraphPad Prism in-built nonlinear regression equation for exponential growth:

$$Y = Y0 * \exp(k * X)$$

$$Doubling time = \frac{\ln(2)}{k}$$

Y0 is the initial Y value, X the time expressed in hours and k the rate constant. Y is expressed as hours⁻¹.

### Cell size measurement

Cells were grown overnight at 30°C in YPD. In the morning, cells were diluted to OD₆₀₀ = 0.05 in fresh medium. At OD₆₀₀ = 0.5, cells were treated with 200nM of rapamycin (dissolved in 90% EtOH and 10% Tween) for 1h. Yeast cells were then subjected to three sonication pulses of 30s medium intensity using a Bioruptor sonicator (Diagenode). Cell volume (fl) of yeast cells was directly measured on a Beckman Coulter Counter Multisizer 4e using Isotone II (Beckman) as a diluent and a 100µm aperture. Cell volume distributions were analyzed using a custom R script.

### Cell viability assay

Cells were grown overnight at 30°C in YPD medium. In the morning, cells were diluted to OD₆₀₀ = 0.05 in fresh medium. Around OD₆₀₀ = 0.5, cells were treated with 1µM rapamycin (LC laboratories, dissolved in 90% EtOH and 10% Tween) for 2h, as previously described in Delarue et al.²⁵ For each sample, 0.25 OD of exponentially growing cells was then harvested and resuspended in 500µl of 1x PBS with 4% Propidium Iodide (BD Pharmigen PI/RNase Staining Buffer For Flow Cytometry). Samples were acquired on a BD Accuri C6 Plus flow cytometer (BD Biosciences), equipped with 2 lasers (488nm and 633nm), 2 light scatter detectors and 4 fluorescence detectors. Flow cytometer data was measured using a BD CSampler Plus Software (v1.0.34.1) and analysed using FlowJo (v10) software (BD Biosciences). Specifically, cells were gated for singlets and the percentage of propidium iodide positive (dead) and negative (viable) cells was measured.

### Two-hybrid assay

Full-length ORFs were amplified by PCR from genomic DNA and cloned into pLexA (encoding for the DNA binding domain) or pACT2.2 plasmids (encoding for the activation domain) (Dual System AG) using restriction enzymes, mutagenesis or Gibson assembly.¹⁰¹ The correct integration into the multiple cloning site was verified by sequencing (Microsynth AG) and SnapGene software (v57.2.21). Plasmids were co-transformed into NMY32 yeast cells. Clones originating from single cells were selected and grown overnight in SD media with auxotrophic selection (-Leu, -Trp). Cells suspensions were then diluted into fresh media and allowed to reach exponential phase. A single dilution of cells (OD₆₀₀ = 0.3) was spotted on SC -Leu, -Trp (-LT) or SC -His, -Leu, -Trp (-HLT) agar plates containing 3-Amino-1,2,4-triazole (3-AT)) at the indicated dilutions. Pictures were taken after growth at 30°C for 2-4 days.
## β-Galactosidase assay for the yeast two hybrid

β-galactosidase expression was monitored using the Pellet-X-G assay.<sup>102</sup> Briefly, yeast cells were inoculated in selective medium (SD-Leu-Trp) and grown overnight at 30°C. Cultures were then diluted to OD<sub>600</sub> = 0.2 and grown for approximately 5 hours to reach OD<sub>600</sub> = 0.6-0.8. 1.4 OD of cells were pelleted by centrifugation at 2500 g for 3 min and resuspended in 130 µL X-gal reagent (0.1% (w/v) X-Gal, 0.05% (v/v) β-mercaptoethanol and 0.1% (w/v) SDS in 1x PBS). 125 µL was transferred to a transparent flat bottom 96-well microplate and incubated at room temperature for several hours. Color changes were scanned at different time points using a flatbed scanner.

## β-Galactosidase assay for the 4uORF GCN4-lacZ reporter

β-galactosidase activity was determined quantitatively using the Yeast β-Galactosidase Assay Kit, according to the stopped microplate plate assay protocol (Thermo Fisher Scientific).

Yeast strains carrying 4uORF GCN4-lacZ reporter plasmids were inoculated in SD medium in the morning and diluted in the evening to reach exponential phase next morning. Then, cultures were diluted to OD<sub>600</sub> 0.1 - 0.2 in 9 ml of medium, divided over three tubes and treated with either 0, 10 or 200 nM rapamycin. Cells were grown for an additional 4-5 hours until start of the experiment. Absorbance at 420 nm was measured using a VersaMax microplate reader (Molecular Devices) after 60 minutes of incubation in working solution. Stop solution was not used in this assay.

## Protein sample preparation and western blotting

Cells corresponding to 1 OD were resuspended in 1mL of ice-cold water, lysed on ice in 150 µL 2M NaOH with 0.7% (v/v) β-mercaptoethanol and precipitated with TCA solution (55% w/v). Protein samples were centrifugated at 16,000 g for 15 min at 4°C and resuspended in 33 µL of HU Buffer (120mM Tris pH 6.8, 5% glycerol, 8M urea, 0.1% bromophenol blue, 3% (v/v) β-mercaptoethanol freshly added) and boiled at 65°C for 15 minutes.

Protein samples were loaded on polyacrylamide 4-12%, Bis-Tris gradient gels and separated at 120-150V in 1X MOPS or MES buffer. Proteins were then transferred on polyvinylidene (PVDF) or nitrocellulose membranes by semi-dry electro-transfer. Protein transfer was confirmed using Ponceau Red staining (0.5% (w/v), Ponceau S dissolved in 1% (v/v) acetic acid). Membranes were blocked in 1x PBS-Tween20 (0.05%) containing 5% milk powder or 1x TBS-Tween20 (0.05%) with 5% BSA. Primary antibodies are listed in the key resources table. Secondary horseradish peroxidase-coupled antibodies were applied prior to ECL-based chemiluminescence detected using a Fusion Fx instrument (Vilber Lourmat).

## Ribosome polysome analysis by sucrose gradients

Sucrose gradient analysis of ribosome polysomes was performed adapting experimental conditions from Shetty et al.<sup>24</sup> Briefly, 200mL of exponentially growing cells were treated with CHX 100µg/mL freshly dissolved in DMSO and cooled on ice for 2 minutes. Cells were harvested by centrifugation in conical 500mL bottles (2 min 2000 g), washed once with 20mL ice cold 1X PBS containing CHX 100µg/mL and once with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM MgCl<sub>2</sub>, 1% Triton X100, 1 mM DTT, 100 µg/ml CHX, 0.1 mM PMSF) and lysed in 1mL lysis buffer and 0.2g (400-600µm) glass beads by vortexing (Disruptor Genie, Scientific Industries) for 15min. After recovering the lysates in a fresh tube, they were clarified by centrifugation at 4°C for 10 minutes, top speed. RNA concentration at 260nm was measured and aliquots of 4 units of A<sub>260</sub> were supplemented with 5% glycerol and flash-frozen in liquid nitrogen. Each aliquot was layered on a 10-50% sucrose gradient in 20 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM MgCl<sub>2</sub>, 1 mM DTT, 100 µg/ml CHX, 0.1 mM PMSF buffer and subjected to ultracentrifugation at 36,000 rpm at 4°C for 2h using a SW41 rotor (Beckman) and 14 x 89 mm polypropylene tubes (Beckman). Profiles were recorded at 260nm with a fractionator device (Biocomp). RNA absorbance A<sub>260</sub> was recorded and plotted with Microsoft Office Excel. The area under the curve of 80S and polysomes peaks were quantified using a custom R script.

## Chromatin-immunoprecipitation, sequencing analysis, and qPCR

Yeast cells were grown overnight in YPD medium at 30°C before diluting the cultures to OD<sub>600</sub> = 0.1. The cells were grown to log-phase (OD<sub>600</sub> = 0.3-0.4) and treated with 6.5 nM rapamycin final concentration (10 µM of stock solution resuspended in 90% ethanol, 10% Tween-20) for 60 min before crosslinking. ChIP experiments were performed with exponential phase cells harvested around OD<sub>600</sub> ≈ 0.6-0.8. Yeast cultures of 100 mL in complete medium were collected and then crosslinked with 1% (v/v) formaldehyde for 15 min and quenched by adding 125 mM glycine for 5 min at room temperature. Cells were then washed with ice-cold HBS (50 mM HEPES-Na pH7.5, 140 mM NaCl) and resuspended in 0.6 mL of ChIP lysis buffer (50 mM HEPES-Na pH7.5, 140 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate) supplemented with 1 mM PMSF and 1x protease inhibitor cocktail (Roche). The cells were broken using Zirconia/Silica beads (BioSpec) and lysates centrifuged at 13,000 rpm for 30 min at 4°C. Pellets were resuspended in 300 µL ChIP lysis buffer containing 1 mM PMSF and sonicated for 15 min (30s ON - 60s OFF) in a Bioruptor sonicator (Diagenode). The lysates were then centrifuged at 7000 rpm for 15 min at 4°C and soluble fractions were transferred to new tubes. A defined amount (5% of total chromatin) of crosslinked and sonicated chromatin from S. pombe was added to S. cerevisiae chromatin prior to the immunoprecipitation step. ChIP was performed by using RNAPII and Myc antibodies (see key resources table) and incubated overnight at 4°C with rotation. Magnetic beads coupled to IgG against rabbit or mouse (Invitrogen, Dynabeads™ M-280 Sheep Anti-Rabbit or Anti-Mouse IgG) were washed three times with 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na₂HPO₄, 1.8 mM KH₂PO₄) containing 0.5% BSA and added to the lysates (30 µL of beads/300 µL of cell lysate). The samples were incubated for 2 hr at 4°C with rotation. The beads were washed twice with AT1 buffer (50 mM HEPES-Na pH7.5, 140 mM NaCl, 1 mM EDTA, 0.03% SDS), once with AT2 buffer (50 mM HEPES-Na pH7.5, 1 M NaCl, 1 mM EDTA), once with AT3 buffer (20 mM Tris-Cl pH7.5, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) and twice with TE buffer (10 mM Tris, 1 mM EDTA). Chromatin was eluted from the beads by resuspension in TE containing 1% SDS and incubation at 65°C for 10 min. The eluate was transferred to new eppendorf tubes and incubated overnight at 65°C to reverse the crosslinks. The DNAs were purified using the MinElute PCR Purification Kit (Qiagen).

DNA libraries were prepared using NEBnext Ultra according to manufacturer's specifications. The libraries were sequenced with Illumina's NovaSeq6000 at FGCZ (Zürich) in single read mode 100nt. The data analysis was performed as follows: the raw reads were first cleaned by removing adapter sequences, trimming low quality ends, and filtering reads with low quality (phred quality <20) using Fastp (v0.20).⁸⁸ Read alignment was done with Bowtie2 (v2.4.1).⁸⁹ As reference we used the Ensembl genome build R64 with the gene annotations downloaded on 2019-12-03 from Ensembl (release 98). Count values were computed with the featureCounts-function from the R package Rsubread (v2.4).⁹⁰ The options for featureCounts were: - min mapping quality 10 - min feature overlap 10bp - count multi-mapping reads - count only primary alignments - count reads also if they overlap multiple genes. Differentially expressed (DE) regions were identified using the R package edgeR (v3.30)⁹¹ applying a generalised linear model (glm) regression, a quasi-likelihood (QL) differential expression test and the trimmed means of M-values (TMM) normalisation. The entire dataset is available in the ENA repository (ENA: PRJEB72705).

Gene categories of Gcn4 targets, ribosomal protein genes category 1 to 3, RAF and Hsf1 targets were defined according to previous studies.⁴⁵⁻⁴⁷ FASTQ files were mapped to the sacCer3 and the 972-h pombe genome assemblies using the Galaxy server (https://usegalaxy.org). For each sample, reads mapped uniquely to the sacCer3 genome were used for further analysis, while reads that mapped to both genomes were discarded. The signal was quantified for each gene between the transcription start site (TSS) and transcription termination site (TTS) for RNAPII. The gene list is reported in Table S3. For Ifh1-qPCR, after cross-linking, quantification of immunoprecipitated DNA was performed as described in Ribaud et al.¹⁰³ by real-time qPCR (Roche LightCycler 480). Ifh1 occupancy at the RPL28 and RPL37A promoters was calculated as a fold enrichment relative to ACT1 controls in *stm1Δ* and wild-type strains. Primer sequences for ChIP-qPCR assay are available upon request.

## Protein immunoprecipitation

For Streptavidin pull-down of biotinylated protein, a volume of cells corresponding to 80 OD e.g. 100mL of OD₆₀₀ = 0.8 cells in mid-log phase, were treated with 100µg/mL CHX and cooled on ice for 5 minutes. Cells were harvested by centrifugation in conical bottles (3500 g 4°C), washed and disrupted with glass beads in 800µL lysis buffer (50mM Tris-HCl pH7.5, 100mM NaCl, 1mM EDTA pH 8.0, 20mM MgCl₂, 100µg/mL CHX, 1mM PMSF, cOmplete™ EDTA free protease inhibitor, NP40 0.1%). Lysates were spun at 20'000 g for 20min at 4°C and the recovered supernatant was transferred to a new tube. After measuring protein concentration, lysates were normalized and 20µL input sample was taken. Lysates were incubated for 1 hour with 50µL of slurry Streptavidin-conjugated magnetic beads rotating at 4°C. Beads were recovered with a magnet and washed 5 times in lysis buffer with 100µg/mL CHX, 1mM PMSF (without NP40 and cOmplete™ EDTA free protease inhibitor). Beads were then resuspended in 40µL of lysis buffer with CHX and PMSF (without NP40 and cOmplete™ EDTA free protease inhibitor) before adding TEV protease. Samples were rotated overnight at 4°C or at 16°C for 1h. Beads were recovered with the magnet, the supernatant transferred to a fresh tube and mixed with 40µL urea loading buffer (120mM Tris pH 6.8, 5% glycerol, 8M urea, 0.1% bromophenol blue, 3% (v/v) β-mercaptoethanol). Samples were heated at 65°C for 15 minutes before Western blotting analysis or SDS-PAGE and Coomassie staining.

Extracts for immunoprecipitation with c-Myc magnetic beads were prepared from 100mL of OD₆₀₀ = 0.6-0.8 cells expressing Myc-tagged proteins, which were treated with 100µg/mL CHX and cooled on ice for 5 minutes. Cells were harvested by centrifugation in conical bottles (3500 g, 4°C), washed and disrupted with glass beads in lysis buffer (50mM Tris-HCl pH7.5, 100mM NaCl, 1mM EDTA pH 8.0, 20mM MgCl₂, 100µg/mL CHX, 1mM PMSF, cOmplete™ EDTA free) in a 1.5mL tube. After addition of NP40 0.5%, lysates were spun on a tabletop centrifuge (20'000 g for 15min at 4°C) and cleared with GST agarose beads for 20 minutes. Anti-c-Myc magnetic beads were washed in lysis buffer and added to the lysates for 1h 30' at 4°C with rotation. Beads were washed five times with lysis buffer supplemented with CHX and PMSF (without NP40), boiled in 50µL 1X SDS loading buffer and analyzed by Western blotting.

## Proteomics sample preparation

After Myc-affinity purification, samples were digested in-gel (AP-MS comparison Stm1-3xMyc and Stm1ΔN-3xMyc) or on-beads (AP-MS comparison Stm1-3xMyc and Stm1ΔIDR-3xMyc). For in-gel digestion, proteins were separated/concentrated by SDS-PAGE, reduced (6.5mM DTT, 1 hour at 60°C), alkylated (54mM Iodoacetamide, 30min, RT) and digested with 1µg of Trypsin (Promega).¹⁰⁴ Before MS injection, peptides were subjected to C18 7-70µg cleanup (The Nest group) following manufacture instructions, dried and re-suspend in 2% acetonitrile 0.1% formic acid.

For on-bead digestion, proteins were digested after purification using Filter Assisted Spin Proteolysis (FASP) as previously described.¹⁰⁵,¹⁰⁶ Briefly, proteins were loaded in 10 kDa molecular weight cutoff spin column (Vivacon 500, Sartorious) and centrifuged at 9000 g for 15min until dryness. Samples were dissolved in urea 8M, reduced (5mM) and alkylated (10mM), washed with ammonium bicarbonate (0.1M) and proteolyzed with 0.4µg of LysC (Wako) for 3 hours and 1.2µg of Trypsin (Promega) overnight. Peptides were collected in a tube by centrifugation (8000 g, 20min) and the spin column was additionally washed with MS water.
Proteolysis reaction was quenched with 5% formic acid. Before MS injection, peptides were subjected to C18 cleanup (C18 7-70µg, The Nest Group) following manufacture instructions, dried and re-suspend in 2% acetonitrile 0.1% formic acid. For targeted analysis, isotope-labeled heavy peptides corresponding to the selected proteotypic peptides, containing either heavy lysine (¹³C₆ ¹⁵N₂) or arginine (¹³C₆ ¹⁵N₄) were spiked with RT peptides (Biognosys) to each sample before LC-MS/MS analysis. The inclusion list with analyzed target peptides is reported in Table S5.

## Phosphorylation enrichment

50mL of cell culture (BY4741) were quenched with 6.25% (v:v) of 100% trichloroacetic acid purum (TCA) on ice for 10min. Cell pellets (1500g, 5min at 4°C) were lysed in 0.4 ml of 8M Urea, 100mM ammonium bicarbonate, 5mM EDTA by glass bead beating (using beads with 500µm diameter in an amount equal to the volume of the lysate). Mechanical lysis was carried out at 4°C under the following conditions: 6 m/s for 20 seconds across 5 cycles, using fresh lysis buffer and retaining the supernatant after each cycle. Protein concentration was quantified using the BCA Protein Assay (total 3mg of protein extract). Proteins were reduced (5 mM TCEP, 30min at 37°C), alkylated (12mM IAA, 30 min), diluted to 1M Urea with ammonium bicarbonate (50mM) and digested overnight at 37°C and 300 rpm using sequencing-grade Trypsin (150µg Promega). Proteolysis was quenched (1% formic acid) and peptide mixture were subjected to C18 phase separation clean up (3cc SepPak C18 cartridge) following the manufacture procedure. After elution, peptides were resuspended in 350 µl of phospho-enrichment loading buffer (50% acetonitrile, 29.2% lactic acid and 0.1% trifluoroacetic) and incubated with 6mg of TiO₂ resin (GL Science) equilibrated with phospho-enrichment loading buffer (1 hour). Subsequently, the resin was washed twice with 500 µl of loading buffer, 500 µl of 50% acetonitrile, 0.1% TFA and finally 500 µl of 0.1% TFA. Phosphopeptides were eluted in basic condition in two steps with 150µl of 50mM ammonium hydroxide (at pH 10.8). The elution solution was quenched immediately with 4.4µl of 50% TFA. Enriched peptides were desalted using ultra-micro spin columns (The Nest Group) and subsequently dried using a speedvac. For MS analysis, peptides were reconstituted to a concentration of 1 µg/µl in 0.1% formic acid, with retention time standard peptides (1:50 iRT, Biognosys) spiked into the samples prior to LC-MS analysis.

## Proteomics sample preparation for pulsed-SILAC experiment

Yeast cultures were grown at 30°C in SD with 25mg/mL light L-lysine in exponential phase for 24h. At OD₆₀₀ = 0.4, cells were pulsed with heavy media. Cells were vacuum filtered on 0.45µm nitrocellulose membrane, washed with warm SD media containing heavy ¹³C₆ ¹⁵N₂ L-lysine 25mg/mL (Cambridge Isotope Laboratories), and resuspended in warm SD heavy L-lysine. After 2h of pulse labeling, samples equivalent to 50mL OD₆₀₀ = 0.8 were harvested by vacuum filtering on nitrocellulose membrane, washed with distilled water, scraped and snap frozen in liquid nitrogen. The samples where then processed for protein extraction and mass spectrometry analysis. Briefly, samples were resuspended in 100µL lysis buffer (8M urea, 100 mM Ammonium Bicarbonate, 5 mM EDTA, pH 8) and bead beaten with 200µL acid-washed glass beads. The samples were spun for 5 min with 500 g at 4°C and the supernatant transferred to a new 1.5mL tube. These steps were repeated 4 times. The collected supernatant was spun at top speed for 20 min at 4°C and subsequently, protein concentration was measured by BCA assay. 100µg of proteins in 50µL lysate were first incubated with TCEP 5mM for 30 min at 37°C (500rpm) and then with iodoacetamide 10mM in the same conditions and in the dark. 50µL ammonium bicarbonate 100mM was added to dilute urea to 4M before starting proteolysis by LysC (Wako, 0.4µg) at 37°C for 3h at 500rpm. Additional 100µL ammonium bicarbonate 100mM was then added to further dilute urea to 2M. Trypsin (Promega, 1.2µg) was added for proteolysis at 37°C overnight. The reaction was quenched with formic acid 5% and the C18 clean-up (C18 17-170µg, The Nest Group) was performed as described above.

## Mass spectrometry data acquisition

### Untargeted differential interactome analysis of WT Stm1 and the ΔN terminal truncation

LC-MS/MS analysis was performed on an Orbitrap QExactive+ mass spectrometer (Thermo Fisher) coupled to an EASY-nLC-1000 liquid chromatography system (Thermo Fisher). Peptides were separated using a reverse phase column (75 µm ID x 400 mm New Objective, in-house packed with ReproSil Gold 120 C18, 1.9 µm, Dr. Maisch GmbH) across a gradient from 5 to 30% in 90 minutes (buffer A: 0.1% (v/v) formic acid; buffer B: 0.1% (v/v) formic acid, 95% (v/v) acetonitrile). The DDA data acquisition mode was set to perform one MS1 scan followed by a maximum of 20 scans for the top 20 most intense peptides with MS1 scans (R=70'000 at 400 m/z, AGC=3e6 and maximum IT=64 ms), HCD fragmentation (NCE=30%), isolation windows (1.4 m/z) and MS2 scans (R=17'500 at 400 m/z, AGC=1e5 and maximum IT=55ms). A dynamic exclusion of 30s was applied and charge states lower than two and higher than seven were rejected for the isolation.

### Untargeted differential interactome analysis of WT Stm1 and the Stm1ΔIDR mutant

LC-MS/MS analysis was performed on an Orbitrap Exploris480 mass spectrometer (Thermo Fisher) coupled to an Vanquish Neo liquid chromatography system (Thermo Fisher). Peptides were separated using a reverse phase column (75 µm ID x 400 mm New Objective, in-house packed with ReproSil Gold 120 C18, 1.9 µm, Dr. Maisch GmbH) across a gradient from 7 to 35% in 120 minutes (buffer A: 0.1% (v/v) formic acid; buffer B: 0.1% (v/v) formic acid, 80% (v/v) acetonitrile). The DDA data acquisition mode was set to perform one MS1 scan followed by a maximum of 20 scans for the top 20 most intense peptides with MS1 scans (R=60'000 at 400 m/z, normalized AGC (%)=100 and max. IT set to auto), HCD fragmentation (NCE=28%), isolation windows (1.4 m/z) and
MS2 scans (R=15'000 at 400 m/z, normalized AGC (%)=200 and max. IT set to auto). A dynamic exclusion of 25s was applied and charge states lower than two and higher than seven were rejected for the isolation.

**Targeted differential interactome analysis of Stm1 WT and the Stm1ΔIDR mutant**

LC-MS/MS analysis was performed on an Orbitrap Exploris480 mass spectrometer (Thermo Fisher) coupled to an Vanquish Neo liquid chromatography system (Thermo Fisher). Peptides were separated using a reverse phase column (75 µm ID x 400 mm New Objective, in-house packed with ReproSil Gold 120 C18, 1.9 µm, Dr. Maisch GmbH) across a gradient from 7 to 35% in 60 minutes (buffer A: 0.1% (v/v) formic acid; buffer B: 0.1% (v/v) formic acid, 80% (v/v) acetonitrile). A targeted scheduled method was set up with one MS1 scan (R=120'000 at 400 m/z, normalized AGC (%)=100 and max. IT set to auto) and a cycle time of 3s to monitor 53 peptides (variable normalized AGC target and max IT) using an isolation window of 1.5m/z and normalized collision energy 27%.

**Phosphorylation analysis of cells in starvation condition**

Phospho enriched peptides were analyzed in Orbitrap Fusion Lumos (Thermo) equipped with a nanoelectrospray ion source coupled to ACQUITY UPLC system M-Class (Waters). Peptides were separated on a 75µm-diameter capillary column packed with ReproSil-Pur C18-AQ resin (1.9µm, Dr. Maish) at 50°C. Peptides were eluted at 300nl/min over a linear gradient of 2 hours (5-35% of Buffer A; Buffer A composition: 0.1% formic acid, Buffer B composition: 100% acetonitrile, 0.1% formic acid).

The data acquisition mode was set to perform one MS1 scan followed by a MS2 scans for a cycle time of 3sec; MS1 scans (R=120'000 at 400 m/z, AGC=400'000 and maximum IT= 10ms), HCD fragmentation (NCE=28%), isolation windows (1.6m/z) and MS2 scans (R=30'000 at 400 m/z, AGC=100'000 and maximum IT=54ms). A dynamic exclusion of 30s was applied and charge states lower than two and higher than seven were rejected for the isolation.

**Pulsed-SILAC data acquisition**

LC-MS/MS analysis was performed on an Orbitrap Exploris480 mass spectrometer (Thermo Fisher) coupled to an Vanquish Neo liquid chromatography system (Thermo Fisher). Peptides were separated using a reverse phase column (75 µm ID x 400 mm New Objective, in-house packed with ReproSil Gold 120 C18, 1.9 µm, Dr. Maisch GmbH) across a linear gradient from 7 to 35% in 120 min (buffer A: 0.1% (v/v) formic acid; buffer B: 0.1% (v/v) formic acid, 80% (v/v) acetonitrile). The DDA data acquisition mode was set to perform one MS1 scan followed by a maximum of 20 dependent scans with MS1 scans (R=60'000 at 400 m/z, normalized AGC (%)=100 and max. IT set to auto), HCD fragmentation (NCE=28%), isolation windows (1.4 m/z) and MS2 scans (R=15'000 at 400 m/z, normalized AGC (%)=200 and max. IT set to auto). A dynamic exclusion of 25s was applied and charge states lower than two and higher than seven were rejected for the isolation.

**Mass spectrometry data analysis**

**Untargeted analysis**

For untargeted analysis, acquired spectra were searched using the MaxQuant software package version 1.5.2.8 embedded with the Andromeda search engine⁹³ against the *Saccharomyces cerevisiae* reference dataset (http://www.uniprot.org/, downloaded on 14.05.2020, 6'052 proteins) extended with reverse decoy sequences. The search parameters were set to include only full tryptic peptides, maximum two missed cleavage, carbamidomethyl as static peptide modification, oxidation (M) and deamidation (N-ter) as variable modification and “match between runs” option. The MS and MS/MS mass tolerance was set to 10 ppm, and a False discovery rate of < 1% was used protein identification. Protein abundance was determined from the intensity of top two peptides. Intensity values were median normalized and imputed using random sampling from a normal distribution generated 1% less intense values. ANOVA statistical analysis was employed to compare multiple condition. The entire dataset, including raw data, generated Tables and scripts used for the data analysis are available in the PRIDE repository (PRIDE: PXD046775 and PRIDE: PXD046821 for Stm1 delta IDR and Stm1 delta N terminal, respectively).

**Targeted analysis**

For targeted analysis, peptides were analyzed manually using Skyline daily,¹⁰⁷ and correct identification with six fragment ions per peptide was assigned based on the coelution of light and heavy peptide and matching peak shape for precursor and product ions from light and heavy peptides. Peptide abundance was analyzed by summing the integrated areas of three fragment ions per peptide. Significance of change in intensity was estimated with *p*-values using two-sided, not paired t-tests. The entire dataset, including raw data, generated tables and scripts for the data analysis are available in the PRIDE repository (PRIDE: PXD046821).

**Phosphorylation dataset**

**Pulsed-SILAC dataset**

Acquired spectra were searched using the MaxQuant software package version 1.5.2.8 embedded with the Andromeda search engine⁹³ against the *Saccharomyces cerevisiae* reference dataset (http://www.uniprot.org/, downloaded on 17.05.2021, 6'057 proteins) extended with reverse decoy sequences. The search parameters were set to include a double multiplicity with heavy Lys (Lys8) and a maximum of three labeled amino acids. Semi-tryptic (free N terminal) with a maximum of two missed cleavage were considered. Carbamidomethyl was set as static peptide modification, while oxidation (M) and deamidation (N-ter) as variable modification. The “match between runs” option was set up for the search, and the MS and MS/MS mass tolerance set to 10 ppm. A false discovery rate of < 1% was used for protein identification. The experiment was performed with four independent replicates and only proteins identified in the light and heavy form in at least 2 replicates per condition were considered for the analysis. Protein labeling was then computed as log₂(H/(H+L)) where H and L are respectively the intensities of new synthesized proteins and protein intensity
before the pulsed. Statistical analysis was performed using unpaired two-sided t-test. The entire dataset, including raw data, generated tables and scripts used for the data analysis are available in the PRIDE repository (PRIDE: PXD046820).

## Tandem affinity purification

Tandem affinity purification (TAP) of pre-ribosomal particles and untagged control cells was performed as described in Altvater et al.<sup>108</sup> Briefly, 2 liters of OD<sub>600</sub> = 3 were harvested and lysed with glass beads and lysis buffer (50mM Tris-HCl pH 7.5, 75mM NaCl, 1.5mM MgCl<sub>2</sub>, 0.15% NP40) in a Planetary mill PULVERISETTE. Lysates were briefly clarified by centrifugation at 4°C to remove debris and further cleared at 18'000 rpm using a SS-34 rotor at 4°C for 15 min. Lysates were incubated with IgG Sepharose beads (Merck), before eluting the TAP particles by TEV protease. Eluates were incubated with calmodulin CaM-Sepharose beads (Merck), and the TAP particles eluted with elution buffer (10mM Tris-HCl pH 8, 50mM NaCl, 5mM EGTA). Proteins were then precipitated with TCA, resuspended in 1x NuPAGE LDS buffer (Invitrogen), separated on NuPAGE 4-12% Bis-Tris gradient gels (Invitrogen) and analyzed by silver staining or Western blotting.

## In vivo pulse chase biotinylation assay

Cells were grown overnight in 20mL SD complete media without biotin (0.17% (w/v) yeast nitrogen base without amino acids and biotin (Sunrise Science Products), 2% (v/v) glucose, 0.5% (w/v) NH<sub>4</sub> sulfate and amino acids mix) supplemented with low biotin (5nM). Cells were then diluted to OD<sub>600</sub>= 0.3, and estradiol was added to a final concentration of 5nM for 2 hours. Subsequently, cells were filter washed twice on a 0.45µM pore size nitrocellulose membrane, with an appropriate amount of SD complete low biotin to remove excess estradiol. Cells were resuspended in a flask containing fresh SD media containing 1mM auxin (IAA) and high biotin (10nM) to induce BirA degradation and biotinylation of AVI-tagged proteins. After 1 hour of biotin pulse and BirA degradation, cells were harvested for immunoblotting or immunoprecipitation with Streptavidin magnetic beads (Invitrogen). Filter washed cells were transferred to fresh SD complete media with low biotin and samples were collected immediately or after 1 hour (chase step) and further processed for pull-down with Streptavidin conjugated magnetic beads.

## Puromycin incorporation assay

The assay was performed as in Shetty et al.<sup>24</sup> Briefly, exponentially growing cells were incubated with puromycin 100µg/mL for 15 minutes at 30°C. TCA was added to stop the reaction at 10% (v/v) final concentration and samples were incubated for 20 minutes on ice. Cells were washed twice with ice-cold acetone and resuspended in 0.2M NaOH PMSF 0.1mM for 10 min on ice. Then 2X Laemml buffer (100mM Tris-HCl pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, fresh 200mM DTT, 0.2% (w/v) Bromophenol Blue) was added and samples were boiled for 15 min before Western blotting analysis.

## Plunge-freezing

Yeast cell cultures were grown overnight in YPD medium on a shaker (180 rpm) at 30°C. Cultures were diluted to OD<sub>600</sub> = ~0.05 in fresh YPD medium, grown until OD<sub>600</sub> = ~0.3 and then divided. One half was treated with 1 µM of rapamycin (LC laboratories, dissolved in 90% EtOH and 10% Tween) for 2 hours, and the other half with the same volume of EtOH as a negative control.<sup>25</sup> Samples were harvested at OD<sub>600</sub> = ~0.6 by spinning down for 2 min at 800 g at room temperature and concentrated to OD<sub>600</sub> = ~3 with conditioned media. Yeast cells were vitrified by plunge freezing using a Vitrobot Mark IV (Thermo Fisher Scientific).<sup>109</sup> 4 µL of cell suspension was applied onto negatively glow-discharged (two times, 25mA for 45 sec) copper EM grids (R2/2, 200 mesh, Quantifoil). Grids were blotted 5-6 seconds from the backside (using a Teflon sheet on one side) at 25 °C, 95% humidity, before plunging into the liquid ethane-propane mixture.<sup>110</sup>

## Cryo-FIB milling and CryoET analysis

Vitrified cells on EM grids were subjected to automated sequential FIB milling using a Focused Ion Beam - Scanning Electron Microscope (FIB-SEM; Crossbeam 550, Zeiss) as previously described.<sup>111</sup> Grids were loaded onto a pre-tilted Autogrid holder<sup>110</sup> (Leica Microsystems) using a VCM loading station and transferred using a VCT500 shuttle (Leica Microsystems) to an ACE500 (Leica Microsystems) for tungsten sputter coating (4nm thick) under cryogenic conditions.<sup>111,112</sup> The grids were then transferred using the VCT500 to the FIB-SEM instrument, where an organometallic platinum precursor was applied onto each grid via a gas injection system (GIS). Subsequently, automated FIB-milling was set up targeting for a lamella thickness of 250 nm by sequentially applying currents of 700 pA, 300 pA, and 100 pA for rough milling, and 50 pA for polishing.

The cryo-FIB milled samples were imaged in a Titan Krios 3 (Thermo Fischer Scientific) at an acceleration voltage of 300 kV and equipped with a BioContinuum imaging filter (slit width used: 10 eV) (Gatan) and a K3 direct electron detector (Gatan). Tilt series were collected at 33,000x magnification (pixel size of 2.68 Å) with a defocus value ranging from -3 µm to -8 µm in SerialEM<sup>113</sup> using PACE-TOMO.<sup>99</sup> The tilt series were collected in a dose symmetric scheme, with an angular range between -51° and +69° and 3° increment. The cumulative dose for a tilt series for samples was between 141 and 150 e/Å<sup>2</sup>. Frames were aligned using 'align-frames' and tomograms were manually reconstructed in IMOD<sup>96</sup> at a binning factor of 4. The contrast of the reconstructed tomograms (pixel size of 10.71 Å) was further improved by filtering via IsoNet package applying defocus 5; snrfalloff 1.2; deconvolution strength 1.<sup>97</sup>
## Template matching and concentration estimation for ribosomes

Template matching was performed using Dynamo in MATLAB⁹⁸ on contrast transfer function (CTF) deconvolved tomograms obtained via IsoNet.⁹⁷ Firstly, ~2,000 particles from 20 tomograms of the unstressed yeast WT cells were manually picked and sub-tomogram averaging was performed at a binning factor of 4. The averaged ribosome structure was then visualised with UCSF ChimeraX¹⁰⁰ and used as a reference in template matching for the reconstructed tomograms from different conditions. The general workflow of template matching was followed based on the details in dynamo wiki webpage (https://wiki.dynamo.biozentrum.unibas.ch/w/index.php/Walkthrough_for_template_matchin), where the threshold ('mcc') value applied was 0.4. For each condition, ten tomograms with similar thickness (~220 nm) were selected for analysis. To estimate the ribosome concentration in tomograms, a custom python script (TomoSampling.py, see key resources table) was written to generate serials of boxes with a defined volume and randomly placed inside the cellular cytoplasm. The centers of each box were manually checked to avoid that they overlap with one another, other organelles, cellular structures or were placed outside the cell (including the plasma membrane). For each tomogram, the script performed five independent measurements reflecting how many template matching detected ribosomes were contained in a box of defined volume of 200 nm × 200 nm × 200 nm. Then the concentration of ribosomes per tomogram was calculated by averaging the five measurements and applying the following formula:

$$ \text{ribosome concentration} = \frac{\text{number of ribosome hits}}{\text{Avogadro's number} \times \text{box volume}} $$

## Live cell microscopy, BiFC measurements, and image analysis

Cells were grown overnight at 30°C in SD media, diluted into fresh growth medium and incubated for ~5h at 30°C to resume exponential phase. Cells were then diluted to OD₆₀₀= 0.05 and spotted on a glass bottom plate functionalized with Concanavalin A (Sigma). Live-cell microscopy was performed using a Nikon Eclipse Ti-E microscope equipped with a Hamamatsu camera, a motorized XY stage and a piezo Z drive and using Micro-Manager (v.1.4.23) software. The incubation chamber of the device was set at 30°C and images were acquired with a 100X oil objectives (NA = 1.45 100x Apo), under non-saturating conditions.

For BiFC assays, a spinning disk confocal fluorescence microscope (Visitron system) equipped with an inverted microscope (Nikon Eclipse TiE), an EM-CCD camera (Andor iXon Ultra, Andor), a motorized XY stage, and a piezo Z drive was used. The system is controlled by VisiVIEW software 5.0.0 (Visitron system). Cells were imaged at 30°C in a temperature-controlled incubator with a ×100 objective lens (NA = 1.4, Nikon CFI Plan Apo).

For quantification, images were processed by Fiji (v. 2.14.0/1.54f) software using custom made macros for maximum intensity projections and composites. BiFC quantification of YFP positive cells were carried out using the Fiji plug-in Cell Counter. Minimum and maximum values were adjusted equally for all images within the same experiment by Fiji (v. 2.14.0/1.54f) software for the preparation or representative images.

All the fluorescence microscopy data are available at ETH Repository collection: https://doi.org/10.3929/ethz-b-000727429.

## RNA extraction and GelRed staining

Total RNA was extracted with the hot acid phenol method.¹¹⁴ Briefly, 5-10 OD of cells OD₆₀₀= 0.5-0.6 were harvested by centrifugation, washed in RNAse-free water and resuspended in 200µL AE buffer (50mM NaAc, 10mM EDTA), 20µL 10% SDS and 250µL of acid saturated phenol pH 4.5-5 (Roth). RNA was extracted at 65°C with shaking, precipitated with ethanol and resuspended in RNAse-free water. 5µg of RNA were mixed with 2.5X sample denaturation mix (65% formamide, 22% formaldehyde, 13% 10X E-buffer), denaturated at 55°C for 15°C, mixed with loading dye (50% Glycerol, 0.3% Xylene cyanol, 0.3% bromophenol blue, 1mM EDTA) and loaded on a 1.2% agarose- 6% formaldeyde gel in 1X E-buffer (20mM MOPS, 5mM NaAc, 0.5mM EDTA pH 7.0). RNAs were visualized with GelRed® nucleic acid gel stain (Botium) and photographed with a Quantum imager (Vilber). rRNA bands were quantified using Fiji (v. 2.14.0/1.54f) software.

## Protein purification

GST-tagged Ifh1-AD was purified from Rosetta2(DE3) pLysS cells grown in TB medium. Cells were grown to OD₆₀₀= 2.5, cold-shocked for 20 min in ice-water and induced with 0.5 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 16 hours at 30°C. Cells were harvested by centrifugation, resuspended in lysis buffer (GST-buffer containing DNAsel, PMSF, Pepstatin A, Leupeptin and Lysozyme) and lysed by high-pressure homogenization (Emulsiflex). Cleared supernatant was loaded onto a GSTprep 16/60 column (Cytiva), washed with GST-buffer (25 mM MOPS, pH 7.6; 150 mM NaCl; 1 mM DTT; 5 % (v/v) GlyOH) containing 1 M NaCl and eluted with 10 mM GSH in GST-buffer. GST-tagged Ifh1-AD was fractionated by gel filtration (S75 16/600 pg, Cytiva), analyzed by SDS-PAGE, and pure protein was pooled and flash-frozen in liquid nitrogen.

¹³C, ¹⁵N-labelled Stm1 IDR (227-273) was expressed and purified as a Twin-Strep-Smt3 fusion protein. In brief, transformed Rosetta2(DE3) pLysS cells were grown in M9 minimal medium supplemented with 4.0g/L ¹³C₆ D-glucose and 1.0 g/L ¹⁵NH₄Cl until OD₆₀₀ reached 0.6. Protein expression, cell harvesting and lysis were carried out as described for GST-tagged Ifh1-AD. Twin-Strep-Smt3-Stm1 IDR (227-273) was purified over a StrepTactin Superflow Plus column (Qiagen), followed by tag removal with 14xHis-Ulp1. Purification until protease cleavage was carried out in 25 mM MOPS, pH 7.6, 100 mM NaCl, 1 mM DTT, 5 % (v/v) GlyOH.
Finally, Stm1 IDR (227-273) was purified and buffer exchanged into 40 mM MMT, pH 6.14, 25 mM D-Trehalose via gel filtration (S75 16/600 pg, Cytiva). To remove uncleaved fusion protein, Twin-Strep-Smt3 and 14xHis-Ulp1 appropriate columns were connected during gel filtration.

## Fluorescence polarization assays

Fluorescence polarization assays were performed in 20mM MOPS, pH 7.6; 100 mM NaCl; 25 mM D-Trehalose; 1 mM DTT; 1%(v/v) GlyOH; 0.01 % (v/v) Triton X-100; 0.1 g/L BSA. Briefly, 25 µL of 40 nM TAMRA-labeled Stm1 IDR-peptide was mixed 1:1 with increasing amounts of GST-lfh1-AD and incubated for 20 min at room temperature. Fluorescence polarization was measured using a CLARIOStar micro-plate reader (BMG Labtech). The target value of free fluorescent peptide was set to 20 mP. Triplicates were measured with different protein preparations of GST-lfh1-AD and baseline corrected against a GST-only control. To obtain K<sub>d</sub> value(s) baseline corrected data were then fit to a one site specific binding model in GraphPad Prism (v10.2.1).

## NMR data acquisition and resonance assignment

For NMR resonance assignments, samples consisting of approximately 0.4 mM uniformly ¹³C, ¹⁵N-labelled Stm1 IDR (227-273) in 40 mM MMT, pH 6.14, 25 mM D-trehalose, 1 mM DTT and 10% D₂O were used. Spectra were recorded at 25 °C in 3 mm diameter NMR tubes (Bruker). 3D HNCACB, CBCACONH, HN(CA)CO and HNCO spectra¹¹⁵⁻¹¹⁸ were recorded on a 600 MHz AVNEO spectrometer equipped with a TCI cryo-probe (Bruker). The CACB correlation spectra consisted of 2048×64×200 complex points in the ¹H, ¹⁵N, and ¹³C dimensions with respective spectral widths of 16, 26, and 64 ppm and were recorded with 8 scans per increment. The CO correlation spectra consisted of 2048×64×176 complex points in the ¹H, ¹⁵N, and ¹³C dimensions with respective spectral widths of 16, 26, and 14 ppm and were recorded with 8 scans per increment. Sample degradation was evident in the latter spectra. Binding experiments were performed on approximately 40 µM ¹⁵N-labeled Stm1-IDR in absence and presence of 260 µM of GST-lfh1. 2D [¹⁵N, ¹H]-SOFAST HSQC experiments¹¹⁹ were recorded using 512 scans and an interscan delay of 0.2 s. 310 increments were recorded in the indirect dimension of 24 ppm spectral width. Chemical shift perturbations were analyzed with combined chemical shifts CSP = $\sqrt{((\Delta\delta(¹H))^2 + (\Delta\delta(¹⁵N)/7))^2}$.

Spectra were processed with the program Topspin (v4.0.9) (Bruker) and analyzed with the program CARA (www.cara.nmr.ch). Stm1 IDR NMR assignments have been deposited on Biological Magnetic Resonance Bank (BMB): BMRB: 52971.

## QUANTIFICATION AND STATISTICAL ANALYSIS

All data are represented as mean ± standard deviation over at least three independent experiments, unless otherwise stated. For all comparisons *P*-values are displayed on Figure panels, with the statistical method specified in the Figure legends. Statistical significance is represented in the Figures by asterisks (*), with the corresponding *P*-values specified in the Figure legends.
Supplemental information

Stm1 regulates Ifh1 activity revealing crosstalk
between ribosome biogenesis and ribosome dormancy

Eliana Bianco, Martina Bonassera, Federico Uliana, Janny Tilma, Martin Winkler, Sevil Zencir, Alvar Gossert, Michaela Oborská-Oplová, Reinhard Dechant, Jannik Hugener, Vikram Govind Panse, Martin Pilhofer, Benjamin Albert, Philipp Kimmig, and Matthias Peter
A

WT + empty
WT + 2µ Stm1
*stm1Δ + empty
stm1Δ + 2µ Stm1

<figure><img src="image_30.png" /></figure>

CHX 0.1µg/mL

B

Growth into stationary phase

<figure><img src="image_31.png" /></figure>

Growth restart after
3 day stationary phase

<figure><img src="image_32.png" /></figure>

Growth restart after
6 days stationary phase

<figure><img src="image_33.png" /></figure>

C

<figure><img src="image_34.png" /></figure>

D

<figure><img src="image_35.png" /></figure>

E

Exp or Stat.
phase (days):

<figure><img src="image_36.png" /></figure>

F

Tomogram
filtering
(IsoNet)

<figure><img src="image_37.png" /></figure>

Ribosome
average
(Dynamo)

<figure><img src="image_38.png" /></figure>

Template
Matching
(Dynamo)

Ribosome concentration
(N° ribosomes/volume)
calculation

<figure><img src="image_39.png" /></figure>

G

<figure><img src="image_40.png" /></figure>

1µM Rap

H

<figure><img src="image_41.png" /></figure>

I

<figure><img src="image_42.png" /></figure>

M

<figure><img src="image_43.png" /></figure>

J

<figure><img src="image_44.png" /></figure>

GelRed

K

<figure><img src="image_45.png" /></figure>

L

<figure><img src="image_46.png" /></figure>

N

<figure><img src="image_47.png" /></figure>

O

<figure><img src="image_48.png" /></figure>

*TEV
Figure S1. The ribosome hibernation factor Stm1 promotes ribosome biogenesis.

Related to Figure 1

A) Serial dilution spotting assay monitoring growth of wild-type (WT) and *stm1Δ* cells carrying an empty control plasmid (+ empty) or a 2µ plasmid overexpressing Stm1 (+2µ Stm1) on synthetic auxotrophic SD-URA media. Cells were treated or not (UT) with cycloheximide (CHX 0.1µg/mL) and incubated for 3 days at 30°C. A representative image from *n* =3 independent experiments is shown.

B) Growth curves of wild-type (WT) (orange) and *stm1Δ* (blue) cells. OD<sub>600</sub> measurements for 24 hours of cells in early exponential phase (left panel) or upon addition of fresh media to cells kept for 3 or 6 days in stationary phase (center and right panel). Mean and standard deviation of 3 independent replicates is shown.

C) Coulter counter-based cell volume mean measurements (fl) for wild-type (WT) and *stm1Δ* cells treated or not with 200nM rapamycin for 1 hour. Mean of *n* = 2 independent replicates is shown.

D) Distribution of ChIP-Seq reads for RNA Pol II Ser5 occupancy. The abundance of the *STM1* gene was compared to other Chromosome XII genes (*PCD1*, *YLR152C*, *MRPL4*, *SEC39*), before (Veh), after 20 min of rapamycin treatment (200nM) (Rap), and after heat shock for 5 min at 40°C. Data are re-analyzed from the ChIP-Seq RNA Pol II dataset (GSE125226)[S1].

E) Western blot of total cell lysates prepared from wild-type (WT) or *stm1Δ* cells carrying an empty control plasmid (+ empty) or a 2µ plasmid overexpressing Stm1 (+2µ Stm1). Samples were collected from exponential growth phase (exp) or after 1, 2 or 3 days of stationary phase (Stat. phase (days)). Blots were probed with antibodies against Stm1 and Pgk1 to control equal loading. *n* = 2 independent replicates.

F) Schematic representation of the workflow to calculate ribosome concentration in cryo-tomograms. Briefly, tomograms were Contrast Transfer Function (CTF) deconvolved with Isonet[S2] and used for template matching analysis with Dynamo[S3]. The average structure of a cytoplasmic ribosome was used as template reference. To calculate the ribosome concentration, five boxes of equal volume were randomly placed in the cytoplasm of each tomogram and ribosomes in the box were counted. The number of coordinate points within each box was then used to calculate
the molar concentration of cytosolic ribosomes. For more details, please refer to STAR
Methods.

**G)** Representative cryo-tomograms of rapamycin (Rap 1µM for 2 hours) treated wild-type (WT) (left panel) or *stm1Δ* (right panel) cells, which were subjected to plunge freezing and focused ion beam (FIB)-milling, prior to cryo-ET. The region of interest (dashed box) is shown magnified as an inset (solid black box) in the upper left corner. ER: endoplasmic reticulum, V: vacuole. Scale bars: 100 nm. Quantification of these images is shown in **Fig 1C**.

**H)** Bar plot quantifying viability of wild-type (WT) and *stm1Δ* cells treated or not with rapamycin 1µM for 2h (Rap 1µM), stained with propidium iodide and analyzed by flow cytometry. Black dots represent independent replicates (n = 3). Data are shown as mean and standard deviation (error bar).

**I)** Western blot analysis of total cell lysates of wild-type (WT) and *stm1Δ* cells, untreated (UT) and treated with 1µM rapamycin for 2 hours (Rap 1µM) as in **Fig 1B**, S1F-G. Blots were probed with antibodies against phosphorylated pSer<sup>51</sup>-Sui2, Sui2, Stm1 and Pgk1 to control equal loading. n = 3 independent replicates.

**J)** GelRed®-stained agarose gel analysis of total RNA samples (tot. RNA) from exponentially growing wild-type (WT) and *stm1Δ* cells. The cell number was normalized by OD<sub>600</sub>, and equivalent sample volume was loaded (4 µL). Cells were untreated (-) or treated (+) with rapamycin (Rap 10nM) for 2 hours. As standards, increasing amounts of Luciferase mRNA (Luciferase RNA (µg)), and increasing volume of total yeast RNA (yeast tot. RNA (µL)) were loaded on the agarose gel (left). The positions of the 35S, 25S, 18S and Luciferase RNA are marked. Quantification of n = 3 independent experiments is shown in **Fig 1D**.

**K)** Bar plot showing the doubling time extracted from growth curves of wild-type (WT) and *stm1Δ* cells grown in SD media containing light lysine (orange) or grown in light lysine and then switched to SD media containing heavy lysine (blue). Data values are shown as dot as well as the mean and the standard deviation. n = 3 independent replicates.

**L)** Violin plot showing the ratio of proteins incorporating heavy and light lysine in wild-type (WT) and *stm1Δ* cells (n = 4). Linked to **Fig 1E**.
**M)** Western blot of total cell lysates prepared from wild-type (WT) and *stm1Δ* cells treated with cycloheximide 200µg/ml for 15' (CHX 15'), rapamycin 10nM for 1 or 2 hours (Rap 1h, Rap 2h) or left untreated (UT) prior to exposure to puromycin for the last 15' of treatment. Blots were probed with antibodies against puromycin, *Stm1* and *Pgk1* (loading control). Bar plot quantification of puromycin incorporation normalized to the *Pgk1* loading control is shown (lower panel). Data values are shown as dots as well as the mean and standard deviation. Representative blot of *n* = 2 independent replicates.

**N)** Schematic representation of the ribosome biotinylation assay (upper panel). Cells were grown to exponential phase in low-biotin media and then supplemented with 5nM estradiol for 2 hours to induce *BirA* expression. Cells were filter-washed and split in three flasks. One flask was treated with auxin 1mM for 30' (+IAA 30') and another one for 60' (+IAA 60') to promote *BirA* degradation, while the last one was left untreated (-IAA). Each flask was then supplemented with biotin for 30' (biotin 30') to induce biotinylation or left untreated for control. Protein samples were collected before estradiol induction (lane 1) and after each auxin and/or biotin treatment (lanes 2-7). Western blot of total cell lysates prepared from cells expressing *BirA* tagged at its N-terminus with HA-2xIAA, and Rpl16a/b C-terminally tagged at its endogenous locus with HA-TEV-AVI (lower panel). Blots were probed with HA-antibodies to detect HA-2xIAA-BirA and total Rpl16a/b-HA-TEV-AVI. HRP-conjugated streptavidin was used to visualize biotinylated Rpl16a/b-HA-TEV-AVI (Biotinylated Rpl16a/b-HA-TEV-AVI), and *Pgk1* controls equal loading. Representative blot of *n* = 3 independent replicates.

**O)** Coomassie stain of protein samples used in **Fig 1H**. **M:** protein ladder. **\*TEV:** TEV protease treatment, **RPs:** ribosomal proteins. Representative gel from *n* = 3 independent replicates.
<figure><img src="image_49.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_52.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_57.png" /><figcaption>J</figcaption></figure>

+Estradiol

<figure><img src="image_58.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_59.png" /></figure>
**Figure S2. Stm1 interacts with pre-ribosomal particles. Related to Figure 2**

A) Bar plot displaying the number of protein identified across all replicates for AP-MS analysis of GFP-3xMyc, Stm1-3xMyc and Stm1ΔIDR-3xMyc as in **Fig 2A**. *n* = 3 independent replicates.

B) Box plot showing the log₂ intensity across all replicates for AP-MS analysis of GFP-3xMyc, Stm1-3xMyc and Stm1ΔIDR-3xMyc as in **Fig 2A**. *n* = 3 independent replicates.

C) Bar plot displaying the spectra counts for AP-MS of GFP and Stm1 across the following conditions: GFP-3xMyc, Stm1-3xMyc and Stm1ΔIDR-3xMyc as in **Fig 2A**. *n* = 3 independent replicates.

D) Gene ontology for Biological Process calculated for Stm1-3xMyc protein interactors identified in **Fig 2A** (defined as log₂FC> 2 and *p*-value< 0.05). The statistical significance of the enrichment is reported as the -log₁₀ of *p*-value.

E) Schematic representation of Stm1 with GFP inserted at either the N- (GFP-Stm1) or C-terminus (Stm1-GFP) (top panel). Representative wide-field light microscopy images of live cells stably expressing GFP-Stm1 (top row) or Stm1-GFP (bottom row). Nuclei were stained with Hoechst. Scale bar: 5µm. *n* = 2 independent replicates.

F) Schematic representation of the Stm1 primary sequence displaying 60S and 40S ribosome binding regions connected by a linker region. Blue boxes depict the predicted nuclear export sequences (NES)[S4] (top). Schematic representation of Stm1 full length (FL), Stm1ΔNES1 (amino acids 1-5, 21-273), Stm1ΔNES2 (amino acids 1-159, 176-273) and Stm1ΔΔNES (amino acids 1-5, 21-159, 176-273) fused to N-terminal GFP (bottom).

G) Analysis of the GFP signal was performed by quantifying the ratio between the mean signal in a nuclear region of interest (ROI) (yellow circle) adjacent to nucleolus visualized by Nop56-mCherry and the mean signal in a corresponding cytoplasmic ROI.

H) Representative fluorescence microscopy images of exponentially growing cells expressing Nop56-mCherry and GFP-Stm1 FL or NES mutants (GFP-X). Scale bar: 2µm.

I) Quantification of the ratio between nuclear and cytoplasmic GFP signal as in **Fig S2G**. Data are presented as mean values ± standard deviation. An unpaired two-tailed *t*-test was used for statistics analysis. Significant differences (compared to FL condition)
are indicated as *p*-values on the graph. n>25 cells were pooled from 2 independent experiments.

J) BiFC assay to monitor interaction between Stm1 and Rpl42a, Rrp7 and Utp22 as in **Fig 2F and G**. Strains were all treated with estradiol 10nM for 2h to induce Vc-Stm1 expression. Representative images of estradiol induced (10nM for 2h) cells expressing Vc-Stm1 (Stm1), Rpl42a-Vn (Rpl42a), Rrp7-Vn (Rrp7), Utp22 (Utp22-Vn), Vc-Stm1 and Rpl24a-Vn (Stm1 + Rpl42a), Vc-Stm1 and Rrp7-Vn (Stm1 + Rrp7) and Vc-Stm1 and Utp22-Vn (Stm1 + Utp22) are shown. The reconstituted YFP (YFP), the chimeric transcription factor for Vc-Stm1 induction (GEV-RFP) and composites (merge) are shown. YFP and GEV-RFP intensities were equally adjusted with Fiji. Adjusted intensities are shown for Stm1 + Rrp7 and for Stm1 + Utp22 (adj. int.). Scale bar: 5µm.

K) Bar plot showing quantification of BiFC assays in Fig S2J as percentage of YFP positive cells. At least 20 cells per sample were analyzed from each independent replicate (n = 2). Data are shown as mean and standard deviation are shown, dots represent independent measurements.
<figure><img src="image_60.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_61.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_62.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_63.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_64.png" /><figcaption>Rap 6.5nM</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_68.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_69.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_70.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>M</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>L</figcaption></figure>

<figure><img src="image_73.png" /></figure>
<caption>**Figure S3. Stm1 promotes Ifh1 activity at RPGs. Related to Figure 3**</caption>

A) Distribution of the negative genetic interaction score of *stm1Δ* cells analyzed from the DRYGIN repository[S5]. *stm1Δ* was combined with genome-wide gene deletions and the viability of the double mutants was measured. The DRYGIN default cut off for intermediate genetic interaction strength is highlighted by the dashed line at y= 0.08. Components of the RNA Pol II machinery are labelled.

B) Correlation plot of *rpo21<sup>N479S</sup>* and *rpo21<sup>N1082S</sup>* genetic interaction maps analyzed from[S6]. *STM1* deletion (*stm1Δ*) is highlighted in orange.

C) Serial dilution spotting assay monitoring growth of wild-type and *stm1Δ* cells carrying a copy of integrated plasmid encoding wild-type (RPO21) or mutant RPO21 (*rpo21<sup>N479S</sup>, *rpo21<sup>N1082S</sup>*, *rpo21<sup>H1085Q</sup>*) on rich YPD media. Since *rpo21Δ* cells are inviable, the diploid strain *rpo21Δ/RPO21 STM1Δ* was complemented with the RPO21 integrative plasmids (wild-type or mutants), sporulated and dissected. Haploid strains were selected based on auxotrophic and antibiotic markers. Cells were incubated at 30°C for 2 days. A representative image from *n* = 3 independent experiments is shown.

D) Tandem affinity purification of the RNA Pol II subunits Rpb8-TAP and 60S ribosomal protein Rpl24-TAP. Silver-stained gel (left panel) and Western blots (right panel) are shown. Blots were probed with antibodies against CBP (Rpb8-TAP and Rpl24-TAP), Stm1 and Rpb1/Rpo21. Total cell lysates (TCL) *n* = 1, affinity purification (IP) *n* = 2 independent replicates.

E) Box plots showing RNA Pol II Ser 5 occupancy. The differential analysis of *stm1Δ* and wild-type (WT) cells is shown for ribosomal protein genes (RP (n=138)), ribosome assembly factors (RAF (n=566)), Gcn4 targets genes (Gcn4 (n=108)), heat shock genes (Hsf1 (n=18)) and others (n=4311). Data are displayed as the log₂ normalized fold change of RNA Pol II S5 occupancy in *stm1Δ* versus WT cells, either using untreated conditions (UT, left panel) or after 3 hours exposure to low rapamycin concentration (Rap 6.5nM, right panel). The signal was quantified for each RNA Pol II transcribed gene between the transcription start (TSS) and termination site (TTS). The gene categories have been defined according to scientific literature [S7]·[S8]·[S9]. An independent replicate is shown in **Fig 3B**.
**F)** and **G)** Gene ontology enrichment for biological process calculated for genes with downregulated (**F**) and upregulated (**G**) RNA Pol II occupancy in *stm1Δ* versus wild-type (WT) cells in untreated conditions defined as log₂FC< -0.5 and false discovery rate FDR< 0.001. The x-axis shows the enrichment compared to the whole yeast genome, the size of the circle represents the number of genes, and the color is indicative for the significance of the enrichment expressed as -log₁₀(FDR). The analysis was performed with ShinyGO [S10] from data linked to **Fig 3B** and **Fig S3E**.

**H)** and **I)** Gene ontology enrichment for Biological process calculated for genes with downregulated (**H**) upregulated (**I**) RNA Pol II occupancy in *stm1Δ* versus wild-type (WT) cells in rapamycin conditions (Rap 6.5nM for 1h) defined as log₂FC< -0.5 and false discovery rate FDR< 0.001. The x-axis shows the enrichment compared to the whole yeast genome, the size of the circle represents the number of genes, and the color is indicative for the significance of the enrichment expressed as -log₁₀(FDR). The analysis was performed with ShinyGO [S10] from data linked to **Fig 3B** and **S3E**.

**J)** Box plots showing RNA Pol II Ser 5 occupancy change in *stm1Δ* versus wild-type (WT) cells for ribosome assembly factors (RAF (n= 566)), ribosomal protein genes (RP (n=138)) categories number 1 (Cat 1), number 2 (Cat 2) and number 3 (Cat 3) [S7]. Data from biological replicate *n* = 2 are displayed as the log₂ normalized fold change of RNA Pol II occupancy in *stm1Δ* versus wild-type cells after no treatment (t=0, left panel) or treatment with 6.5nM rapamycin for 1h (t=1, central panel) or 3h (t=3, right panel). An independent replicate is shown in **Fig 3D**.

**K)** Serial dilution spotting assay to monitor growth on YPD media of wild-type (WT) and *stm1Δ* cells, and WT or *stm1Δ* cells where the endogenous promoter of *RPL18A* was replaced by a doxycycline repressible promoter with seven *TetO* operator repeats (*TetO7-Rpl18a*). Cells were treated or not (UT) with 1µg/mL doxycycline (Dox) and imaged after 3 days. A representative picture of *n* = 3 independent experiments is shown.

**L)** Bar plot showing Ifh1-13xMyc occupancy change analyzed by ChIP-qPCR in *stm1Δ* versus wild-type (WT) cells for the RPGs *RPL28* and *RPL37A* in untreated (t=0) and treatment with 6.5nM rapamycin for 1 h (t=1h) normalized on *ACT1*. *n* = 1; an independent replicate is shown in **Fig 3E**.
M) Bar plot quantifying the β-galactosidase activity of wild-type (WT), *stm1Δ*, *gcn2Δ*, *gcn2Δ STM1Δ* strains carrying 4uORF GCN4-lacZ reporter plasmids untreated (UT) or exposed to rapamycin 10 or 200 nM rapamycin for 4-5 hours. Data values are shown as mean with standard deviation. *n* = 3 independent replicates. The indicated *p*-values were calculated by a two-way ANOVA test for multiple comparison analysis (Tukey). Significance: * *p*-value < 0.05. Note that there is no significant difference (ns) of the β-galactosidase activity observed in WT + Rap, *STM1Δ* and *STM1Δ + Rap*.
<figure><img src="image_74.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_77.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_80.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_82.png" /><figcaption>I</figcaption></figure>

CURI

<figure><img src="image_83.png" /></figure>

<figure><img src="image_84.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_85.png" /></figure>
<caption>**Figure S4. Stm1-IDR interacts with RAF and CURI members. Related to Figure 4 and 5**</caption>

A) Two-hybrid analysis of full length Stm1 (Stm1) and N- (stm1ΔN) or C-terminally (stm1ΔC) truncated Stm1 fused to the GAL4 activation domain (AD) with components of the SSU fused to the GAL4 DNA binding domain (DBD). For control, unfused AD or DBD domains (-), or DBD-fusions to Lamin C and Cdc13 were included. Colonies were grown at 30°C for 3 days on permissive auxotrophic SD -Leu, -Trp (-LT), or selective -His, -Leu, -Trp agar media, supplemented with 2.5mM or 10mM 3-amino-1,2,4-triazole (-HLT +3-AT). A representative picture of n = 3 independent replicates is shown. Extension of Fig 2D.

B) Bar plot displaying the number of protein identifications across all conditions for AP-MS of GFP-3xMyc, Stm1-3xMyc and Stm1ΔN-3xMyc. Linked to **Fig S4E**.

C) Box plot showing the distribution of protein intensities across all conditions for AP-MS of GFP-3xMyc, Stm1-3xMyc and Stm1ΔN-3xMyc. Linked to **Fig S4E**.

D) Bar plot displaying the spectra counts of GFP and Stm1 for the AP-MS of GFP-3xMyc, Stm1-3xMyc and Stm1ΔN-3xMyc. Data values for each biological replicate are shown (n = 3). Linked to **Fig S4E**.

E) Volcano plot showing the log₂ fold change enrichment of proteins co-purified with Stm1ΔN-3xMyc compared to Stm1-3xMyc. The protein abundance was normalized for Stm1 levels after AP-MS. The significance is calculated using a two-sided unpaired Student's t-test. n = 3 independent replicates. Components of the 40S (orange) and 60S (blue) ribosomal subunits and RAF (purple) are highlighted.

F) Gene ontology for Biological Process was calculated for the Stm1 interactors identified in **Fig S4E** defined as log₂FC > 1 and p-value< 0.05. The significance of the enrichment is reported as the -log₁₀ of p-value.

G) Box plot of the log₂ fold change enrichment of RAF identified in the AP-MS analysis of the Stm1 N- (Stm1ΔN-3xMyc) and C-terminal (Stm1ΔIDR-3xMyc) truncation mutants, relative to full-length Stm1-3xMyc. Linked to **Fig S4E** and **Fig 5A**.

H) Two-hybrid analysis of full length Stm1 (FL), Stm1ΔC, Stm1ΔIDR, Stm1Δ176-225, and the Stm1 C-terminus (amino acids 176-273) fused to the GAL4 activation domain (AD) with components of the CURI complex fused to the GAL4 DNA binding domain (DBD). The DBD-LaminC fusion was included for negative control. Cells were grown at 30°C on permissive auxotrophic SD -Leu, -Trp (-LT), or selective -His, -Leu, -Trp agar media,
supplemented with 1mM or 2.5mM 3-amino-1,2,4-triazole (-HLT +3-AT). Cells were imaged after 3 days of incubation at 30°C. Representative image of n =3 independent replicates.

I) Targeted PRM measurement of Ifh1 and CURI subunits (schematic, upper left) co-purified with Stm1. Box plots showing the log₂ fold change enrichment of Ckb2, Ifh1, Rrp7 and Utp22 in Stm1ΔIDR-3xMyc compared to wild-type Stm1 (Stm1-3xMyc). Data values (normalized for Stm1 abundance) are shown as log₂ ratio of light (endogenous peptide) to heavy (stable heavy isotope-labeled peptide) (l/h). Protein abundance is calculated from the average intensity of the measured peptides (all measured peptides are reported in Fig S5A). The number of peptides used for each protein is indicated below the protein name. Independent replicates are represented by a dot (n = 3), with the box plot boundaries indicating the quantiles Q1 (25%) and Q3 (75%). The average value is indicated by a line across the box. Statistical analysis was performed with Student t-test's, two sided unpaired.

J) Quality control for targeted analysis reporting the retention time (RT) of iRT peptides (internal standards for targeted analysis). Linked to Fig S4I.

K) Quality control for targeted analysis reporting intensity of iRT peptides (internal standards for targeted analysis). The intensity of the peptides is obtained from the sum of all peptide transitions. Linked to Fig S4I.
A

log2(Iih)

<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>

B

<figure><img src="image_89.png" /></figure>
**Figure S5.** The *Stm1-IDR* interacts with *CURI* subunits and is phosphorylated upon nutrient starvation. Related to Figure 5

A) Targeted measurement of Ifh1 and CURI complex members co-purified with *Stm1-3xMyc*. This panel reports the log₂FC enrichment for all measured peptides in AP-MS measurements of *Stm1-3xMyc* compared to the *GFP-3xMyc* control (Fig 5C, S4I). Data values are shown as log₂ ratio of light (endogenous peptide) to heavy (stable heavy isotope-labeled peptide) (l/h). Each dot represents the results for a technical replicate (*n* = 3), with the box plot boundaries indicating the quantiles Q1 (25%) and Q3 (75%) and the average value denoted by a line across the box (*n* = 3). Linked to Fig S4I.

B) Intensity of the indicated *Stm1* phosphorylation sites identified in exponentially growing or stationary phase cells (5 days after exponential growth) (*n* = 2 biological replicates). Intensities are represented as grey dots, with orange triangles indicating the average intensity. Phosphorylation sites that were not detected in both biological replicates are annotated as 'NA'.
Stm1 IDR wt
RGG/GAR

226 | 273

Stm1 IDR 8A
8xAla

226 | 273
236 243

SUP.

PELLET

<figure><img src="image_90.png" /></figure>

tot. RNA
**Figure S6. The RGG/GAR motif within the Stm1 IDR domain drives RNA interaction. Related to Figure 6**

Schematic representation of the Stm1 IDR (amino acids 226-273), with the RGG/GAR
binding motif highlighted in orange (top panel). Coomassie blue stained gel of the wild-
type Stm1 IDR peptide (amino acids 226-273) and the corresponding Stm1 IDR 8A mutant
with amino acids 236-243 changed to alanine residues. The peptides were incubated with
deionized water (- tot. RNA) or with yeast total RNA (+ tot. RNA)) and separated
in supernatant (SUP.) and pellet (PELLET) fractions. M: molecular weight (kDa) marker. *n*
= 1 independent replicate.
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