EMBO/soda-nel huggingface dataset:
Genome-wide RNAi screen reveals a role for multipass membrane proteins in endosome-to-golgi retrieval. (2014)
Figure 6

ZDHHC5, SFT2D2, or GRINA Depletion Affect Levels of Endosome-to-Golgi Cargo Proteins

(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown.

(B–E) Quantitative analysis of SFT2D2, ZDHHC5, and GRINA KD cells immunofluorescence using automated microscopy (see Experimental Procedures). (B) Representative images showing VPS35, TGN46, and CIMPR staining. Scale bar, 50 μm. (C) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2, ZDHHC5, or GRINA KD cells. (D) SFT2D2, ZDHHC5, and GRINA KD increase the Pearson’s correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130. In some cases, identical correlation coefficients were measured in the replicate experiments. (E) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells.

In (C)–(E), error bars indicate the SD of the replicate measurements. ∗p < 0.05; ∗∗p < 0.01.
Phospholipid imbalance impairs autophagosome completion (2022)
Figure 1

Figure 1: Loss of PC biosynthesis via the CDP-DAG pathway impairs autophagic flux

A. Scheme of PC biosynthesis pathways in the yeast saccharomyces cerevisiae

B, C, D. WT, ∆cpt1∆ept1 (B), Δcho2 (C) or Δopi3 (D) cells expressing GFP-Atg8 were grown to logarithmic phase in SD-URA, and shifted to SD-N for 4 h of starvation. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was used as a loading control. Middle panel- Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by student's t-test (paired, two tailed) (ns- not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount, during SD and SD-N. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns- not significant, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.

E. WT, Δopi3, and Δcho2 as well as Δatg1 (negative autophagy control) cells expressing chromosomally-tagged Fba1-GFP were grown to log phase in SD, and starved in SD-N for 4 hours. Cells were harvested at indicated starvation time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Right panel- Autophagic activity during starvation was quantified, by calculating the ratio of free GFP to total GFP (Fba1-GFP + free GFP). Statistical analysis was done by Anova, Dunnet's multiple comparisons test (*, p≤0.05, ***, p≤0.0005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.

F. WT, Δopi3, Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain, were grown to log phase in SD, and starved in SD-N for 4 hours. Pho8 activity was measured by the alkaline phosphatase assay. Statistical analysis was done by Anova, Dunnet's multiple comparisons test (ns- not significant, *, p≤0.05, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.
Phospholipid imbalance impairs autophagosome completion (2022)
Figure 2

Figure 2: PC deficiency specifically compromises the autophagic pathway

A. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD-N) (left panel). Scale bar 1 µm. Right panel- Quantification of of cells with or without GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001) (ns- not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n=446), Δcho2 (n=272), Δopi3 (n=272)), SD-N (WT (n=398), Δcho2 (n=224), Δopi3 (n=213)).

B. CPY maturation assay: precursor CPY (pCPY) is transported to the vacuole and processed into a mature form (mCPY) in the vacuolar lumen. WT, Δopi3 and Δcho2 cells, as well as Δpep4 cells as negative control for CPY maturation, all expressing GFP-Atg8, were grown to log phase in SD-URA, and shifted to SD-N for 3 h. Cells were harvested at the indicated time points and subjected to western blotting (see Fig. EV2B). Quantification of CPY maturation (mCPY/(mCPY+pCPY)) is shown for samples in log phase (SD) and starvation (3h SD-N). Statistical analysis was done by Anova multiple comparison test-Dunnett's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of at least 3 independent experiments.

C. WT, Δopi3 and Δcho2 cells were grown to log phase in SD medium, stained for 30 min on ice with FM4-64, washed 3 times with cold SD, and observed at indicated time points after wash by widefield microscopy (see Fig. EV2C). Quantification of cells with stained vacuoles (%), statistical analysis was done by Sidak's multiple comparison test compared to WT (ns-not significant), error bars represent SEM of two independent exp. Number of cells analyzed for each strain and time points; WT: 0 min (n=170), 15 min (n=186), 30 min (n=130), 40 min (n=210), 50 min (n=352)), (Δcho2: 0 min (n=195), 15 min (n=191), 30 min (n=126), 40 min (n=98) , 50 min (n=150)), (Δopi3: 0 min (n=148), 15 min (n=200), 30 min (n=224), 40 min (n=221), 50 min (n=216).

D. WT, Δcho2 and Δopi3 cells were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were harvested after 3 h of starvation, fixed, embedded and observed by EM. V-vacuole, N-nucleus. Scale bar 1 µm.

E. PC levels were quantified by shotgun lipidomics in WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 during log phase in SD-URA. Statistical analysis was done by Anova multiple comparisons test- Dunnett's, compared to WT (**, p≤0.005, ****, p≤0.0001), error bars represent SEM of 3 independent experiments.
Phospholipid imbalance impairs autophagosome completion (2022)
Figure 3

Figure 3: Addition of choline rescues autophagy in Δopi3 cells

A, B. WT and Δopi3 cells expressing GFP-Atg8 were grown to log phase in SD-URA, and shifted to SD-N for 4 h, choline (1 mM) was not supplemented (-choline) or supplemented to SD and SD-N (+choline SD, SD-N) or only to SD-N (+choline, SD-N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel- Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns-not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD-N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared to WT (ns-not significant, *, p≤0.05, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.

C. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT and Δopi3 cells expressing GFP-Atg8 were grown in SD-URA and shifted to SD-N. Choline (1mM) was either excluded (-choline), added to growth and starvation medium (+choline SD, SD-N) or added only to starvation medium (+choline, SD-N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 µm. Right panel- Quantification of cells with GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; -Choline: WT (n=487), Δopi3 (n=316), Choline SD, SD-N: WT (n=251), Δopi3 (n=361), Choline SD-N: WT (n=320), Δopi3 (n=267).

D. PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP-Atg8, with or without choline (1 mM) supplementation to SD-N. Samples were taken after 4 h in SD-N medium. Statistical analysis was done by Anova multiple comparisons test- Sidak's (****, p≤0.0001, ns- not significant), error bars represent SEM of at least 3 independent experiments.
Phospholipid imbalance impairs autophagosome completion (2022)
Figure 6

Figure 6: Phospholipid imbalance leads to accumulation of elongated unsealed phagophores

A. Representative Images of Atg5-mNG with sfTq2-Atg8 during SD-N. Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium, shifted to SD-N for 4 h, and observed by Widefield microscopy (left panel), scale bar 1 µm. Right panel- quantification of percentage of cells with Atg5 puncta during different time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's ns- not significant), error bars represent SEM of 2 independent experiments. Number of cells analyzed for each strain and time point (0 hour: Δatg3 (n=296), Δatg3Δopi3 (n=111), 1 hour: Δatg3 (n=325), Δatg3Δopi3 (n=110), 2 hours: Δatg3 (n=338), Δatg3Δopi3 (n=135), 4 hours: Δatg3 (n=309), Δatg3Δopi3 (n=167).

B. Representative images of WT and Δopi3 cells expressing TagBFP-Ape1 under the native APE1 promoter and Ape1 under the CUP1 promoter. Cells were grown to log phase in SD-LEU medium in the presence of 500 µM copper sulfate, and observed by Widefield microscopy, scale bar 1 µm.

C. Representative images of WT and Δopi3 cells expressing GFP-Atg8. Cells were grown to log phase in SD-URA medium, shifted to SD-N for 3 h and observed by Airyscan microscopy (left panel). White dashes indicate cell boundaries, yellow dashes indicate phagophores. For each cell - Magnification of yellow dashed area (top right), schematic representation (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 structures imaged by Airyscan microscopy. Statistical analysis was done by student's t-test (unpaired) (** p≤0.05), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=45), Δopi3 (n=110).

D. Representative CLEM images of WT and Δopi3 cells expressing GFP-Atg8, grown to log phase in SD-URA medium and shifted to SD-N for 3h. Cells were harvested, deep frozen and processed for CLEM (left panel), V-vacuole. For Δopi3 cell - Magnification of yellow dashed area of GFP-Atg8 positive phagophore (top right), TEM image only (middle right), schematic representation of a phagophore (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 positive cup shaped membrane structures imaged by CLEM. Statistical analysis was done by student's t-test (unpaired) (*** p≤0.005), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=20), Δopi3 (n=21).

E. Tomogram slice of CLEM Δopi3 image (D), focused on the phagophore, thickness of 120 nm, scale bar 500 nm.
PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. (2010)
Figure 1.

Mitochondrial localization of Parkin is etiologically important. (A) HeLa cells expressing HA-Parkin were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies. (B) HeLa cells stably expressing HA-Parkin or intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments. I, C, and M indicate input, cytosol-rich supernatant, and mitochondria-rich membrane pellet, respectively. (C) Schematic diagram of disease-relevant mutants of Parkin used in this study. IBR, in between RING; Ubl, ubiquitin like. (D) Polarized mitochondria stained with MitoTracker red (red arrowheads) were not labeled by Parkin. In contrast, damaged mitochondria marked by Parkin (green arrowheads) were not stained with MitoTracker red. (E) HeLa cells expressing HA-Parkin with various pathogenic mutations were treated with CCCP, followed by immunocytochemistry. (A, D, and E) Higher magnification views of the boxed areas are shown in the insets. (F) Parkin colocalization with mitochondria was analyzed in >100 cells per mutation. Example figures indicative of robust colocalization (counted as 1), weak colocalization (counted as 0.5), and no colocalization (counted as 0) are shown. Error bars represent the mean ± SD values of at least three experiments. Bars: (A, E, and F) 10 µm; (D) 30 µm.
PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. (2010)
Figure 2.

Parkin exerts E3 activity only when the mitochondrial membrane potential is decreased. (A) HeLa cells expressing wild-type Parkin or E3-inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies. When E3-inactivating mutations were introduced into Parkin, the mitochondrial ubiquitylation signal disappeared. (B and C) HeLa cells expressing HA-Parkin (B) or expressing both Mt-GFP and HA-Parkin (C) were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies. (D) Localization of GFP-Parkin to the mitochondria after CCCP treatment. (A–D) Higher magnification views of the boxed areas are shown in the insets. (E) HeLa cell lysates expressing GFP-Parkin were immunoprecipitated by anti-GFP antibody, followed by immunoblotting with the indicated antibodies. (F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane. (G) GFP-Parkin–expressing HeLa cells with various pathogenic mutations (Fig. 1 C) were treated with CCCP and subjected to immunoblotting. Asterisks show ubiquitylation of GFP-Parkin. Vertical black lines indicate that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation. Bars, 10 µm.
PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. (2010)
Figure 3.

PINK1 is constitutively degraded in a mitochondrial membrane potential–dependent manner and localizes to depolarized mitochondria. (A) The number of HeLa cells with N-terminal– or C-terminal–tagged PINK1 localized to the mitochondria was counted in >100 cells. (B and C) Exogenous nontagged PINK1 (B) or endogenous PINK1 (C) in HeLa cells was immunostained with the indicated antibodies. (D) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A. (A and D) Error bars represent the mean ± SD values of least three experiments. (E) Endogenous PINK1 gradually accumulated after CCCP treatment. The first through the fifth lanes show endogenous PINK1, and the sixth lane shows overexpressed untagged PINK1. Note that the asterisk indicates a cross-reacting band because it was not affected by overproduction of untagged PINK1. (F) Subcellular fractionation of endogenous PINK1. Intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments (same sample as Fig. 1 B). I, C, and M indicate input, cytosol-rich supernatant, and the mitochondria-rich membrane pellet, respectively. (G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band. (H) N-terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP. The top panel shows control HeLa cells expressing only GFP. (B, C, and H) Higher magnification views of the boxed areas are shown in the insets. Bars, 10 µm.
PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. (2010)
Figure 4.

PINK1 recruits cytoplasmic Parkin to damaged mitochondria. (A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A. (C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C) or for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation. (E) The number of MEFs without anti-Tom20 antibody–detectable mitochondria was counted as in Fig. 3 A. In the example figure (left), blue arrowheads indicate cells without anti-Tom20 antibody–detectable mitochondria, and red arrowheads indicate cells harboring anti-Tom20 antibody–detectable mitochondria. (B and E) Error bars represent the mean ± SD values of least three experiments. Bars: (A) 10 µm; (D) 50 µm; (E) 150 µm.
PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. (2010)
Figure 5.

Kinase activity and mitochondrial targeting of PINK1 is imperative for mitochondrial localization of Parkin. (A) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study. MTS, mitochondria-targeting sequence; TMD, transmembrane domain. (B) Subcellular localization of Parkin in PINK1−/− cells complemented by various pathogenic and deletion mutants of PINK1-Flag. Cells were treated with CCCP. Higher magnification views of the boxed areas are shown in the insets. (C) The number of cells with Parkin-positive mitochondria was counted as in Fig. 3 A. Error bars represent the mean ± SD values of least three experiments. (D) PINK1−/− MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti-Parkin or anti-Flag (tag of PINK1) antibodies. IB, immunoblot; Ub, ubiquitylation. Bars, 10 µm.
Requirements for multivalent Yb body assembly in transposon silencing in Drosophila (2019)
Figure 1

Figure 1. Hierarchy of protein components in Yb body assembly

A. The Armi complex immunoisolated from OSCs using anti-Armi antibodies contains Yb, SoYb, and Vret.

B. The Vret complex immunoisolated from OSCs using anti-Vret antibodies contains Yb, Armi, and SoYb.

C. Silver staining of protein components in the Vret complex in (B). The lane α-Vret (left) contained only the anti-Vret antibodies used in these experiments.

D. Immunoprecipitation and western blotting were performed in OSCs upon RNAi treatment (kd). Even without the association of Yb, Armi, SoYb, and Vret associated. Yb and Armi remained bound to each other upon SoYb or Vret depletion. The level of SoYb (input) decreased upon Vret depletion. Likewise, the level of Vret (input) decreased upon SoYb depletion.

E. Upon Armi depletion, SoYb and Vret were still bound to each other but this heterodimer no longer associated with Yb.

F. Immunofluorescence analyses in OSCs. Yb bodies disappeared upon Yb depletion, which led Armi, SoYb and Vret to be localized in the cytosol. Yb bodies were detected in OSCs upon Armi depletion, but SoYb and Vret localized in the cytosol. Armi was detected at Yb bodies upon SoYb or Vret depletion. Yb bodies are indicated by white arrows. Nuclei are shown in blue. Scale bar: 2 μm.

G. The hierarchy of Yb body assembly proposed in this study. Domain structures of Yb, Armi, SoYb, and Vret are shown on the right. Hel-C, Helicase C-terminal; eTud, extended Tudor; ZnF, zinc finger; RRM, RNA-recognition motif; MYND, MYND-type zinc finger.
Requirements for multivalent Yb body assembly in transposon silencing in Drosophila (2019)
Figure 2

Figure 2. Behaviors of Yb mutants ∆Hel-C and ∆eTud in OSCs

A. Domain structures of WT Yb and deletion mutants ∆Hel-C and ∆eTud.

B. In Yb-lacking OSCs (Yb kd), WT Yb and ∆eTud bound to Armi but ∆eTud did not. Note that WT Yb and both mutants were engineered to be RNAi-resistant by mutations. β-gal was used as a negative control.

C. In normal OSCs, WT Yb and ∆eTud bound with Yb but ∆Hel-C did not.

D. Flag-Hel-C copurified with Myc-Hel-C but not with BSD (negative control).

E. Subcellular localization of WT Yb and deletion mutants ∆Hel-C and ∆eTud. Myc-Yb is shown in green. Endogenous Armi is shown in red. DAPI shows nuclei (blue). Scale bar: 5 μm. Western blotting (right) showing WT Yb and deletion mutants ∆Hel-C and ∆eTud expressed in Yb-lacking OSCs.

F. CLIP shows that WT Yb and ∆Hel-C bind RNA in vivo but ∆eTud does not.
Requirements for multivalent Yb body assembly in transposon silencing in Drosophila (2019)
Figure 3

Figure 3. Functional analyses of ∆Hel-C and ∆eTud

A. Rescue experiments showing that ∆Hel-C but not ∆eTud restored piRNA biogenesis attenuated by endogenous Yb depletion. Flag-Piwi was expressed upon endogenous Yb depletion.

B. The levels of transposon mdg1 in (A). Bars and error bars represent means ± SEM values of three independent experiments. p values were calculated by t-test. *: p < 0.05.

C. Mapping of flam-piRNAs produced in the presence and absence of the Hel-C domain in OSCs.

D. Mapping of tj-piRNAs produced in the presence and absence of the Hel-C domain in OSCs.

E. Scatterplot showing changes in the abundances of transposon-repressing piRNAs and non-transposon-repressing piRNAs in the presence and absence of the Hel-C domain. Orange and blue dots indicate the number of normalized piRNA reads mapped uniquely to transposons in the reverse orientation and coding genes in the sense orientation, respectively.

F. The relative enrichments of RNAs coimmunopurified with WT Yb and ∆Hel-C. Bars and error bars represent means ± SEM values of three independent experiments. In addition to tj, three genic piRNA sources that were characterized in previous studies (dm/Myc [59], c-Fos/kay [60], and CG9257 [34]) are also shown. rp49 was used as a negative control. p value was calculated by t-test. ***: p < 0.001.

G. Summary of requirements of Hel-C and eTud domains of Yb in Yb body formation and transposon-repressing and non-transposon-repressing piRNA production.
Requirements for multivalent Yb body assembly in transposon silencing in Drosophila (2019)
Figure 4

Figure 4. Yb bodies are phase-separated condensates

A. Yb bodies (Yb in green) disappeared after 1,6-hexanediol treatment of OSCs, as did P bodies (Dcp1 in red). Nuclei are shown in blue. Scale bar: 5 μm.

B. Snapshots of live imaging of GFP-Yb expressed in OSCs (Movie EV1). Fusion- and fission-like dynamics of Yb bodies were observed. Saturated pixels are shown in red. Scale bar: 0.5 μm.

C. Yb Y23A and F129A mutants (green) impaired Yb body formation. Nuclei are shown in blue. Scale bar: 5 μm.

D. Yb Y23A and F129A mutants failed to produce flam-piRNAs. miR-310 was used as a loading control.

E. Mapping of flam- and tj-piRNAs produced in the flamKG mutant and heterozygote ovaries.

F. Summary of the functionality of each domain of Yb. Production of non-transposon-repressing piRNAs requires RNA helicase and eTud domains of Yb. Yb body formation via LLPS requires all three domains of Yb, but Armi, SoYb, and Vret are dispensable. Production of transposon-repressing piRNAs (flam piRNAs) requires all three domains of Yb and also the Yb body components, Armi, SoYb, and Vret. The Hel-C domain is responsible for Yb−Yb interaction (self-assembly of Yb). The RNA helicase and eTud domains are required for Yb−RNA binding. The eTud domain is also required for Armi binding. Yb binds the SoYb−Vret heterodimer Armi-dependently.

Expanded View Figure legends