Co-translational determination of quaternary structures in chaperone factories

Domain: neuroscience
Published: 2026-01-23
Editorial outcome: minor_revision

Abstract
The HSP90/R2TP quaternary chaperone assembles key cellular machines, including the three nuclear RNA polymerases and many non-coding RNPs. Here, we characterize the RNAs associated with R2TP and find that it binds many proteins co-translationally. Its co-translational interactome reveals potential clients and distinguishes clients bound only co-translationally, only post-translationally, or both. For subunits assembling together in the same complex and bound co-translationally by R2TP, only a marginal proportion of their mRNAs is co-localized and co-translated. Instead, HSP90 and R2TP induce the formation of polysome condensates accumulating these chaperones and specific client mRNAs, thus favoring co-translational interactions between them. During the assembly process, R2TP cycles between co- and post-translational steps and this is regulated by ATP: it binds co-translationally in absence of ATP and becomes released from post-translational assembly intermediates by ATP hydrolysis. This co-translational mechanism, named co-translational chaperone channeling (cha-cha), substitutes for the rarity of co-localized/co-translated mRNAs. In turn, chaperones have the remarkable ability to spatially organize the translational apparatus in the cytoplasm.

Introduction
Non-coding RNPs are complex macromolecular assemblies and studies performed on many systems have established that they require specific factors for their assembly. For instance, a large number of comprehensive studies performed in the last 20 years have revealed that ~200 assembly factors are required to produce functional ribosomes1. Likewise, despite their apparent simplicity, small RNPs like the spliceosomal snRNPs or the nucleolar snoRNPs also require a surprisingly large number of specialized assembly factors2. These factors are evolutionarily conserved, and their number frequently exceeds the set of components of the mature RNP particle, highlighting the generality and importance of this process2,3. Assembly machineries also occur for stable protein complexes devoid of RNAs. For instance, the formation of RNA polymerases requires an assembly machinery that includes more than 14 factors and whose mechanism of action remains poorly understood4. Interestingly, some of these assembly factors, like the HSP90/R2TP chaperone, are linked to the protein folding machinery. Indeed, quaternary protein folding can be seen as an extension of tertiary folding, with the main difference being that it occurs in trans and not in cis. Assembly factors have been shown to chaperone unassembled subunits, improve efficiency and specificity of the assembly process, transport subunits across the cell and perform quality controls1–3.

R2TP is a conserved HSP90 co-chaperone that has a unique and essential role in the cell as a generalist assembly chaperone that helps build multi-subunit complexes5–8. Specifically, R2TP promotes the biogenesis of: (i) small non-coding RNPs such as the box C/D and H/ACA snoRNPs2,9,10; the telomerase RNP11,12; U4 and U5 spliceosomal snRNPs13–15; and miRNPs16; (ii) the three nuclear RNA polymerases4,17; (iii) ciliary dyneins18; (iv) complexes containing PIKKs (mTOR, ATM/ATR, DNA-PK, TRRAP, SMG1)19,209; and (v) the Tuberous Sclerosis Complex (TSC), which controls mTOR activity21. This list of client complexes is likely incomplete, as large-scale interaction assays have revealed additional R2TP partners22–25. In agreement with this diversity of roles, the R2TP subunit RPAP3 is essential in mice, and its targeted deletion in the adult intestine leads to rapid disruption of tissue homeostasis because of a lack of assembly of key complexes such as RNA polymerase II26.

R2TP is highly conserved across evolution, and it can be tracked to the root of the eukaryotic lineage27. It is composed of a heterodimer of RPAP3:PIH1D1 associated with the AAA+ ATPases RUVBL1 and RUVBL224,28,. In humans, R2TP further associates with a set of prefoldins and prefoldin-like subunits to form the PAQosome29. RUVBL1/RUVBL2 form hetero-hexameric and dodecameric rings and are believed to carry a chaperone activity on their own30–32. Compared to other AAA+ ATPases, they have an extra domain called DII, which is located outside the ring and whose conformational changes are believed to play important roles in the assembly function of R2TP7,30,33,34. Indeed, biochemical and structural data have shown that RUVBL1/RUVBL2 make ATP-dependent contacts with clients and cofactors7,35. Because of their hexameric nature, it is thought that this enables them to hold various subunits in place during the assembly process, with nucleotides acting as a switch for client binding or release(36 for a review). Human RPAP3 possesses two TPR domains that bind HSP70 and HSP90 and a C-terminal domain that associates with RUVBL1/RUVBL2 at the opposite side of DII27,33,37,38. RPAP3 forms a heterodimer with PIH1D1, which is important for client recognition and the regulation of the ATPase activities of RUVBL1/RUVBL2 and HSP70/90 during their chaperone cycle37,39,40. The recognition of R2TP clients can occur in several ways. Some clients are recruited with the help of cofactors, such as the TTT complex for PIKKs41–43; ZNHIT2 and ECD for U5 snRNP7,14,15; and NOPCHAP1, NUFIP1, ZNHIT3, and ZNHIT6 for C/D snoRNPs9,25,44. Others are directly recruited by R2TP, and this can occur via the PIH domain of PIH1D1, which contains a basic pocket that binds acidic peptides phosphorylated on the DpSDD/E consensus motif43, or via RPAP3, as in the case of the Dicer co-factor TRBP16 or clients containing Armadillo repeats24.

Recent studies suggested that the assembly of multi-subunit complexes is intimately coupled with translation45–47. For instance, biochemical studies in yeast and mammal have shown that subunits of the same complex can bind each other co-translationally, sometimes reciprocally, and other times in a directional manner45. In mammals, detailed studies of the general transcription factor TFIID and the co-activators SAGA and ATAC have revealed hierarchical co-translational assembly pathways that mix co- and post-translational steps48–50. For TFIID in particular, some subunits join others during their synthesis to create sub-modules, which, after their release from the ribosome, assemble with the largest TAF1 subunit during its translation49. Importantly, the co-translational joining of one subunit with another can occur either by associating and co-translating the two polysomes together (called the co-co pathway) or by transporting one fully translated subunit to its partner polysome (the co-post pathway). Current evidence suggests that the co-co assembly pathway may be common51,52, although single RNA imaging has so far only revealed low frequencies of mRNA co-localization47,52, leaving the exact usage of the co-co pathway an open question. In the case of the co-post pathway, how fully translated subunits are transported to their partner polysomes for assembly is not well understood. In the case of the SMN complex, which assembles the Sm ring on snRNAs, the newly translated Sm proteins remain on the ribosome until they are picked up by the pICln assembly chaperone, which brings them to SMN53. Despite the different mechanisms observed, many pathways converge to the ribosome, indicating that it may function as a hub driving assembly. Here, we analyze co-translational events for chaperone-assisted assembly of macromolecular complexes and show that the HSP90/R2TP assembly chaperone binds many of its clients during their translation. The co-co pathway appears to be marginal, and instead, the chaperone induces the formation of dedicated translation factories that accumulate client mRNAs and thus favor chaperone-client co-translational interactions. R2TP thus channels the formation of macromolecular complexes starting from individual subunit synthesis, likely compensating for the scarcity of subunit mRNA co-localization.

Results
RUVBL1 immunoprecipitates client mRNAs in a translation-dependent manner
The HSP90/R2TP chaperone (Fig. 1A) is specialized in the assembly of macromolecular complexes, and recent evidence has linked this process to translation47 (for a review). We thus tested whether R2TP can bind its client proteins while they are being translated. We performed a RIP-seq experiment using a heterozygous HCT116 cell line having a GFP tag fused at the C-terminus of one allele of RUVBL1. This cell line was characterized by PCR genotyping, Western blot, and immunofluorescence, confirming the proper insertion of the tag and the localization of the RUVBL1-GFP fusion in the cytoplasm and the nucleoplasm as expected (Fig. 1B and Supplementary Fig. 1A, B54). We then immunoprecipitated RUVBL1-GFP using GFP-Trap® beads and sequenced the co-precipitated RNAs. Remarkably, we found 172 RNAs specifically enriched in the GFP immunoprecipitation (IP) over the control (FDR < 0.05), with enrichment values varying from 84 to 2 fold (Fig. 1C; Supplementary Data 1). Some mRNAs coded for known clients of R2TP, such as the PRPF8 and EFTUD2 proteins of U5 snRNP, the large subunits of the three nuclear RNA polymerases (POLR1A, POLR2A, POLR2B, POLR3A), and the PIKK SMG1. Other well-known R2TP clients, such as the snoRNP core proteins DKC1 and NOP58, did not pass the statistical threshold but were nevertheless among the top-enriched mRNAs (ranked 194 and 330, respectively). RUVBL1/RUVBL2 are part of several chromatin complexes (TIP60, SRCAP, INO80), and this is believed to be independent of their participation in the R2TP chaperone. Remarkably, the mRNAs coding for key subunits of these complexes were also highly enriched in the IP (EP400, SRCAP, BRD8, INO80). Most interestingly, many mRNAs encoding proteins not previously known to interact with RUVBL1 were also identified, and several formed clearly identifiable families of related proteins. This was the case for the AGO proteins (AGO1, AGO3, and AGO4, with AGO2 also in the top hits, although below the statistical threshold), the CREB3 family (CREBRF, CREB3, and CREB3L2), and SCAF family members that share a similar domain that interacts with the RNA polymerase II CTD (PCF11, SCAF4, SCAF8, CHERP). It was also noteworthy that RUVBL1-GFP associated with mRNAs that code for subunits assembling together in mature complexes, such as POLR2A and POLR2B in RNA polymerase II, PRPF8 and EFTUD2 in U5 snRNP, and BRD8 and EP400 in the TIP60 chromatin remodeling complex. Other mRNAs coded for seemingly unrelated proteins, although many were long proteins, larger than 1000 amino acids. Finally, we also found an enrichment for several box C/D snoRNAs (SNORD3, SNORD13, SNORD46, and SNORD31), as expected from previous studies54,55. In order to determine if the association of RUVBL1 with mRNAs was translation-dependent, we repeated the RIP-seq experiment after a 30 min treatment with puromycin, which acts as a translation terminator and releases the nascent peptides from the ribosome. We found that most of the mRNAs previously enriched in the RUVBL1-GFP IP were lost or considerably less enriched upon puromycin treatment (Fig. 1D, S1C, E, F), suggesting that RUVBL1-GFP binds to mRNAs via the nascent proteins, while they are translated by ribosomes.

The ATPase activity of RUVBL1/RUVBL2 regulates their binding to polysomes
The AAA+ ATPase activity of RUVBL1/RUVBL2 is central to their chaperone activity and the nucleotide-bound state (ATP, ADP, or none) controls which partners they interact with30,33,35,55. To test how ATP affects their interactions with RNAs, we performed a RIP-seq experiment in the presence of CB-6644, a small molecule that inhibits their ATPase activity and blocks them in the ATP-bound state56,57. Most of the mRNAs previously found in the RUVBL1-GFP IP were lost or less enriched after CB-6644 treatment, including the mRNAs coding for the U5 snRNP proteins, the subunits of the RNA polymerases, and the chromatin remodeling complexes (Fig. 1E, S1D). RUVBL1 may thus bind the corresponding nascent proteins in the ADP or empty state. In contrast, many box C/D snoRNAs were associated more strongly with RUVBL1-GFP upon CB-6644 treatment, suggesting that the drug prevents the release of RUVBL1/RUVBL2 from immature C/D snoRNPs. A few mRNAs also displayed stronger binding with CB-6644. This was the case for the PCF11/SCAF family (with RPRD2 as an additional member), and for WASHC2A and WAHSC2C mRNAs, which encode two almost identical proteins involved in actin dynamics. RUVBL1/RUVBL2 may thus bind more strongly to these nascent proteins when loaded with ATP. Altogether, this indicated that ATP regulates the co-translational binding of RUVBL1 to its clients.

To gain more insight into this process, we performed a quantitative label-free proteomic analysis of the RUVBL1-GFP IPs (AP-MS), with and without CB-6644 treatment. The proteomic analysis revealed the known partners of RUVBL1 as expected, including all subunits of R2TP, the PAQosome and the INO80, SRCAP and TIP60 chromatin remodeling complexes, as well as many subunits of the three nuclear RNA polymerases, the U5 snRNP and the C/D and H/ACA snoRNPs (Fig. 2A; Supplementary Data 2). RUVBL1-GFP was also associated with known R2TP co-factors, such as ZNHIT2, ZNHIT3, NUFIP1, ZNHIT6, NOPCHAP1, and the TTT complex. In the presence of CB-6644, the subunits of the chromatin remodeling complexes became less enriched, while most of the other clients, such as the H/ACA core component DKC1, the RNA polymerase II subunits (POLR2A, POLR2B, POLR2C, POLR2H), U5 snRNP proteins (PRPF8, EFTUD2) and SMG1, as well as cofactors known to be involved in their assembly (POLR2M, RPAP2 for RNA polymerase II; TELO2, TTI1, TTI2 for SMG1; SHQ1 for DKC1), became more enriched (Fig. 2B). As in the RIP-seq, binding to WASHC2A increased dramatically after CB-6644 treatment. This was also the case for the C/D core component SNU13, which directly binds snoRNAs and likely reflects the increased binding of RUVBL1-GFP to C/D snoRNAs observed in the RIP-seq with CB-6644 (Fig. 1E). In contrast, some R2TP cofactors dissociated (NOPCHAP1, DPCD, WDCP). This was consistent with the ATP-dependence observed in vitro for these proteins44,58, suggesting that the differences observed in vitro and in vivo reflect the genuine ATP-dependent affinity of RUVBL1/RUVBL2 for these partners.

Interestingly, when we plotted the fold change induced by CB-6644 in the binding of RUVBL1 to mRNAs versus proteins (Fig. 2C), it became apparent that the inhibition of the RUVBL1/RUVBL2 ATPase activity had a differential effect, with many mRNAs losing binding upon CB-6644 treatment while the corresponding protein showed increased binding (POLR2A, POLR2B, PRPF8, EFTUD2, SMG1, DKC1). This indicated that RUVBL1/RUVBL2 followed an ATPase cycle linking co- and post-translational steps: they bind co-translationally to these client mRNAs in an empty or ADP state and require ATP hydrolysis to be released from later, post-translational, assembly intermediates.

RUVBL1 association to its partners can be strictly co-translational, strictly post-translational or both co- and post-translational
Next, we wanted to determine which partners bind RUVBL1 co- or post-translationally, and we plotted the enrichments measured in the RIP-seq versus the AP-MS proteomic analysis, using the top 300 candidates in each of the two experiments made in untreated conditions (Fig. 2D). This clearly identified three classes of RUVBL1 partners (Fig. 2D, E): (i) only co-translational (41 partners), which spread along the x-axis and were enriched only in the RIP-seq experiments; (ii) only post-translational (84 partners), located along the y-axis and enriched only in the AP-MS proteomic analysis; (iii) co- and post-translational (11 partners), enriched in both experiments (Fig. 2D, E). Interestingly, the latter corresponded to key subunits of the main R2TP client complexes (U5 snRNP, the RNA polymerases), as well as subunits of the chromatin remodeling complexes. In contrast, the post-translational partners corresponded mainly to the other subunits of these chromatin remodeling complexes and to R2TP cofactors, while the strictly co-translational partners corresponded mostly to previously uncharacterized RUVBL1/RUVBL2 partners. This indicated that the co-translational interactome of the R2TP chaperone is quite unique and not easily accessible by traditional proteomic approaches. We verified by IP/Western blot that RUVBL1-GFP interacts with BRD8 (Supplementary Fig. 2A). This is in agreement with the AP-MS and RIP-seq results that categorize BRD8 as a co- and post-translational partner.

RPAP3 binds clients co-translationally and associates with a similar set of polysomes as RUVBL1
RUVBL1/RUVBL2 are not only components of the R2TP complex but also of several other complexes, such as the R2TP-like chaperones27 and the INO80, SRCAP and TIP60 chromatin remodeling complexes29. To determine whether the association of RUVBL1 with polysomes was taking place in the context of R2TP, we performed a RIP-seq experiment with RPAP3, another R2TP subunit. We used CRISPR/Cas9 genome editing to generate a heterozygous HCT116 cell line with a GFP tag fused at the C-terminus of one allele of RPAP3. The cell line was characterized by PCR genotyping and Western blot, which confirmed the proper integration of the tag (Supplementary Fig. 3A, B). RNA sequencing of GFP IPs showed that 153 mRNAs were associated with RPAP3-GFP, as compared to the control IP (FDR < 0.05; Supplementary Data 1; Fig. 3A, C). Comparison of the enrichment values measured in the RPAP3-GFP and RUVBL1-GFP IPs revealed a good correlation between the two RIP-seq experiments (Fig. 3C): mRNAs highly enriched in the RUVBL1-GFP IP were also highly enriched in the RPAP3-GFP IP, with, however, some preferences. For instance, RUVBL1-GFP associated more strongly with C/D snoRNAs, SMG1 and EFTUD2 mRNAs, while RPAP3-GFP bound better the mRNAs coding for SNAPC4, the SCAF4/SCAF8/CHERP family of RNA polymerase II CTD binding proteins, and the CHD family of chromatin remodelers (Fig. 3C). Interestingly, RPAP3 also bound the PCNT and NIN mRNAs, which code for centrosomal proteins and which were recently shown to be locally translated at centrosomes, possibly to enable their co-translational assembly within centrosomes59. Finally, although the presence of RUVBL1 in the INO80, SRCAP and TIP60 chromatin remodeling complexes is thought to be unrelated to R2TP29, RPAP3-GFP was strongly associated with mRNAs coding key subunits of these complexes (EP400, SRCAP, INO80, BRD8; Fig. 3A, C). These mRNAs were the same ones that were bound by RUVBL1-GFP, raising the possibility that the R2TP chaperone could be involved in the biogenesis of these chromatin remodeling complexes, beyond the participation of RUVBL1/RUVBL2 in their mature forms.

Importantly, and as previously observed for RUVBL1, most mRNAs were lost or much less associated with RPAP3-GFP after a 30 min puromycin treatment (Fig. 3B, and Supplementary Fig. 3C; Supplementary Data 1). Only 20 out of the 153 enriched RNAs were associated in puromycin-treated cells as well as in untreated cells, and these generally had low enrichment values over the control IP (<1.5; Supplementary Data 1). One interesting RNA of this class was TERC, the non-coding RNA of telomerase. This RNA carries an H/ACA motif and the R2TP chaperone is involved in telomerase assembly9,11,12,60 suggesting that the translation-independent RNA partners of RPAP3 could also be R2TP clients. In any case, these results demonstrated that the association of RUVBL1 with polysomes reflects an engagement of the full R2TP chaperone, and not a separate function of RUVBL1.

R2TP binds its main mRNA targets via the nascent protein emerging from ribosomes
We then aimed to confirm the association of R2TP with the mRNAs identified above and to unambiguously show that it occurs co-translationally. To this end, we performed RUVBL1-GFP IPs followed by RT-qPCR and compared the association of RUVBL1-GFP with selected mRNAs after translation inhibition with various inhibitors. We observed that puromycin and harringtonine, two translation inhibitors that remove nascent protein chains from the ribosome, abolished the binding of RUVBL1-GFP to POLR2A, POLR2B, EFTUD2, PRPF8, EP400 and BRD8 mRNAs. In contrast, cycloheximide, a translation inhibitor that freezes ribosomes and keeps the nascent polypeptides attached, increases the association of RUVBL1-GFP with these mRNAs (Fig. 3D). This indicates that it is not translation inhibition per se that prevents binding of RUVBL1 to its target mRNAs, but the removal of the nascent protein chain, indicating that this is the binding determinant of RUVBL1.

Next, we performed a polysome fractionation and tested the presence of R2TP by Western blot (Supplementary Fig. 2B). Indeed, a small amount of RUVBL2 could be detected in light polysomal fractions, indicating that it interacts with the translation machinery as expected from its translation-dependent interactions with some mRNAs. Finally, we developed reporters to evaluate the role of translation independently of global translation inhibitors. Using the RUVBL1-GFP cell line, we stably expressed mCherry-POLR2A and mCherry-EFTUD2 fusion proteins, as well as variants in which a STOP codon was inserted immediately after the mCherry. We then IP’ed RUVBL1-GFP and analyzed by RT-qPCR the presence of these mRNAs. We found that mCherry-POLR2A and mCherry-EFTUD2 mRNAs associated with RUVBL1-GFP, while the mRNAs containing the internal STOP codon did not (Fig. 3E, F and Supplementary Fig. 2C, D). Altogether, these results indicated that the association of RUVBL1 with its client mRNAs depends on polysome integrity and, in particular, the translation of the corresponding protein, hence confirming that RUVBL1 is recruited co-translationally by the nascent protein chains of its client subunits.

Artificial R2TP condensates recruit mRNAs coding for client proteins
It has previously been observed that interactions can form artifactually in extracts after cell lysis61. To validate the interaction of R2TP with its target mRNAs, we thus used a co-recruitment assay to evaluate these interactions directly in intact cells14,17. We generated artificial cytoplasmic condensates by using a fragment of DDX6 (amino-acids 289-483, or DDX6fm) that is sufficient to target the protein to P-bodies, and fusing it to DsRed2 on one side and either RUVBL1 or RPAP3 on the other side. By fusing RUVBL1 and RPAP3 to DDX6fm, we artificially created a high accumulation of R2TP in P-bodies and could then check by smFISH whether the candidate mRNAs or proteins were recruited into these artificial condensates (Fig. 4). We tested some of the mRNAs most enriched in the previous RIP-seq experiments, coding for: the RNA polymerase subunits POLR2A, POLR2B, POLR1A, the U5 snRNP component PRPF8 and the subunits EP400 and BRD8 of the TIP60 histone acetylase complex. Remarkably, the endogenous POLR2A and PRPF8 mRNAs were efficiently recruited to the artificial P-bodies formed by DsRed2-DDX6fm-RUVBL1 or DsRed2-DDX6fm-RPAP3, revealing a specific interaction in cells with RUVBL1 and RPAP3, respectively (Fig. 4A, B). In addition, this recruitment was lost after a 30 min puromycin treatment. It also did not occur with DsRed2-DDX6fm-KPNA3, an unrelated protein used as a negative control, or with native P-bodies (Fig. 4C, D and Supplementary Fig. 6C).

To further confirm that the mRNAs recruited to these artificial P-bodies were being translated, we performed a triple color experiment, adding an immunofluorescence labeling of the POLR2A protein (Fig. 4D). Indeed, this protein also accumulated into the DsRed2-DDX6fm-RUVBL1 foci together with the POLR2A mRNA, and the protein labeling mostly disappeared after a 30 min incubation with puromycin. This confirmed that the recruited POLR2A mRNAs were translated. The other tested mRNAs were also recruited to the artificial DsRed2-DDX6fm-RUVBL1 cytoplasmic foci, although with a lower amount of mRNA per foci (Supplementary Fig. 4). Altogether, these results validated the interactions observed in the RIP-seq experiments.

Single-molecule imaging reveals R2TP factories accumulating POLR2A and PRPF8 mRNAs
Next, we wanted to quantify the interactions of R2TP with mRNAs in a more native context, and we thus tagged the endogenous RPAP3 with the SunTag. Indeed, this is a repeated epitope that enables single protein detection in native cells with a recombinant mono-chain antibody fused to GFP62. Genotyping and Western blots confirmed the proper heterozygous integration of the SunTag in HeLa cells and revealed that RPAP3-SunTag was much less expressed than its untagged counterpart (~50 fold less; Supplementary Fig. 5A, B). Interestingly, fluorescence microscopy showed that RPAP3-SunTag could be observed not only as single proteins in the cytoplasm but also as foci containing multiple molecules (Fig. 5A). We then performed smFISH against POLR2A and PRPF8 mRNAs. Surprisingly, both mRNAs were present as single molecules and also as foci, which colocalized with the RPAP3-SunTag foci (Fig. 5A). Counting both the mRNAs present as foci and single molecules, we measured that an average of 30% and 35% of the PRPF8 and POLR2A mRNAs colocalized with RPAP3-SunTag, respectively (Fig. 5B). After treatment with puromycin for 30 minutes, the foci containing RPAP3-SunTag, POLR2A and PRPF8 mRNAs disappeared and the colocalization of these mRNAs with RPAP3-SunTag dropped significantly (Fig. 5B). Importantly, the colocalization of POLR2A and PRPF8 mRNAs with RPAP3-SunTag occurred not only in the foci but also when these mRNAs were present as single molecules (Supplementary Fig. 5C). This colocalization appeared lower than in the foci (20% versus 75%), but it was still significant as it decreased after puromycin treatment (Supplementary Fig. 5C). The relatively high remaining colocalization in puromycin-treated cells (16% for POLR2A mRNA) likely reflected background events due to the density of the RPAP3-SunTag labeling. Indeed, similar values were obtained with α-catenin mRNA, a control unrelated to R2TP (Fig. 5B). The other mRNAs did not form foci (POLR2B, EFTUD2, BRD8, EP400). Nevertheless, we could detect a puromycin-dependent colocalization with RPAP3-SunTag for all of them, which was not the case for the control α-catenin mRNA (Fig. 5B). Their colocalization was lower than for POLR2A and PRPF8 mRNAs (15-25% vs. 30-35%), but the large excess of untagged RPAP3 (50 fold) is likely to underestimate the true colocalization values.

The foci observed with either RPAP3-SunTag or the POLR2A and PRPF8 mRNAs were intriguing as they suggested the existence of structures dedicated to the translation and co-translational binding of POLR2A and PRPF8 proteins to the R2TP chaperone. To explore this possibility, we used native, untagged HeLa Kyoto cells and labeled endogenous RPAP3 by immunofluorescence together with POLR2A mRNA by smFISH. Remarkably, both POLR2A mRNA and RPAP3 protein formed foci and these foci co-localized (Fig. 6A). On average, cells contained four RPAP3 foci and 75% of the POLR2A mRNA foci colocalized with RPAP3 (Supplementary Fig. 5D, E).

To further characterize these naturally occurring POLR2A mRNA foci, we checked whether they contained other components by IF and smFISH. Interestingly, we observed that the ribosomal protein RPS6 and the chaperone HSP90, which have a diffuse localization in the cytoplasm, were both present, although they did not accumulate, in POLR2A mRNA foci (Supplementary Fig. 6A, B). In addition, the POLR2A protein accumulated in these foci, suggesting that the POLR2A mRNAs were indeed translated there (Supplementary Fig. 6C). In agreement, treatment with puromycin or harringtonine led to the near total disappearance of these foci (Supplementary Figs. 5E, 6E, 7A). In contrast, they were preserved upon cycloheximide treatment, a translation inhibitor that does not induce polysome dissociation, indicating that their formation is likely driven by the nascent protein chains. We then tested PRPF8 mRNA, which also formed foci in the RPAP3-SunTag cells (Fig. 5A, B). Indeed, this mRNA formed foci in native, untagged HeLa Kyoto cells, which were also puromycin sensitive and which frequently co-localized with POLR2A mRNA foci (~50%; Fig. 6B and Supplementary Figs. 5F, 7B). Several mRNAs were recently reported to form translation factories, which are small cytoplasmic foci accumulating specific mRNAs undergoing translation and sensitive to puromycin inhibition63. Dual-color smFISH showed that the POLR2A mRNA foci did not colocalize with the translation factories of DYNC1H1, which is not a client of R2TP (Supplementary Fig. 6D). Likewise, the POLR2A mRNA foci were unrelated to P-bodies (Supplementary Fig. 6C). Because the POLR2A mRNA foci accumulate both the nascent POLR2A protein and RPAP3, they appear to be specialized translation sites requiring R2TP activity.

RNAs coding for pairs of subunits that assemble together rarely colocalize
Recent evidence suggests that mRNAs coding for proteins that assemble together can, in some cases, be colocalized and co-translated, a pathway termed co-co assembly45,52. The colocalization of POLR2A and PRPF8 mRNAs in foci thus prompted us to test additional mRNAs, as our R2TP RIP-seq experiments revealed several mRNAs coding for pairs of subunits belonging to the same complex, such as POLR2A/POLR2B for RNA polymerase II, PRPF8/EFTUD2 for U5 snRNP and EP400/BDR8 for the TIP60 chromatin remodeling complex (Fig. 7A). Surprisingly, dual-color smFISH showed only a very limited colocalization between these mRNA pairs (Fig. 7B, C). The highest level was observed for BRD8 and EP400, with an average of 5% of BRD8 mRNA colocalizing with EP400 mRNA (Fig. 7C). This low level nevertheless appeared significant because it dropped to less than 1% after a 30 min puromycin treatment, reaching a level similar to the JUP control mRNA. We also observed a low level of colocalization, but still significant, between POLR2B and POLR2A mRNAs (3.5%; Fig. 7B, C), while no significant colocalization was observed between EFTUD2 and PRPF8 mRNAs. Altogether, these data are not in favor of the co-co assembly model where polysomes of subunit pairs associate together. This was further reinforced by the fact that POLR1A mRNAs colocalized with POLR2A mRNAs to the same extent as POLR2B mRNAs, even though they belong to different protein complexes (Supplementary Fig. 8A, B). This suggested that the low level of mRNA colocalization that we observed is not related to co-co assembly.

The HSP90 and RUVBL1/2 ATPase activity is required for localized mRNA translation
The accumulation of the RPAP3 protein in cytoplasmic foci, together with POLR2A and PRPF8 mRNAs, suggests that these structures function as R2TP factories to enhance interaction of nascent proteins with the chaperone via a proximity effect. The formation of these structures depends on translation (Supplementary Figs. 5E, 6E, 7A and B), and we wondered whether the chaperones, which interact with the nascent proteins, would themselves be involved in their formation. To address this question, we inhibited RUVBL1/RUVBL2 ATPase activity with CB-6644 and analyzed POLR2A mRNA localization (Fig. 8A, B). Interestingly, this led to a diminution of the number of POLR2A mRNA foci after 4 h and 8 h of treatment, and their complete disappearance after 16 h. The same effect was observed for PRPF8 mRNAs and, remarkably, the mRNA foci also disappeared when the ATPase activity of HSP90 was inhibited with Geldanamycin for 1 h (Fig. 8C, D, E). Thus, the HSP90 and RUVBL1/RUVBL2 chaperones play an essential role in the formation of the POLR2A and PRPF8 translation factories, indicating that they not only interact with the client proteins but also organize their localized translation.

Interestingly, the previous RIP-seq experiments raised the possibility that the R2TP chaperone could be involved in another case of local translation, as RPAP3-GFP pulled down several mRNAs localized near the centrosomes: NIN, PCNT and AKAP9 (Fig. 3A). Moreover, other centrosomal mRNAs were among the top hits in the RPAP3 RIP-seq (Supplementary Data 1), although their enrichment was not individually statistically significant: CEP350 (ranked 321 out of ~18000; Supplementary Data 1), CCDC88C (534), BICD2 (1111), NUMA1 (1415) and ASPM (1673). In fact, out of the 8 mRNAs known to localize to human centrosomes in a translation-dependent manner59, all but one were found in the top hits, representing a very significant enrichment for the entire mRNA family (pValue < 0.001). To test whether R2TP plays a role in the local translation of centrosomal mRNAs, we measured by smFISH the centrosomal localization of NIN mRNAs with and without CB-6644 treatment (Fig. 9A, B). We found a nearly 2-fold decrease in the amount of NIN mRNAs at centrosomes upon treatment, indicating a role of the RUVBL1/RUVBL2 ATPases in its localized translation. Altogether, these data demonstrated an important role of the R2TP chaperone in the local translation of its mRNA targets.

Methods
Cell culture and drug treatments
HCT116 cells CCL247 are from ATCC source, HeLa H9 cells were a gift from E. Emiliani and HeLa Kyoto a gift from I. Poser69. HCT116 cells and HeLa cells were grown in McCoy’s 5A medium (Gibco) or Dulbecco’s modified Eagle’s Medium DMEM (Gibco), respectively, containing 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco) at 37°C, 5% CO2. All cells were routinely tested for the absence of mycoplasma. For cell treatments, drugs were used as follows: puromycin at 100 µg/mL for 30 min (Invivogen), CB-6644 at 500 nM for 4 to 16 h (MedChemExpress), geldanamycin at 2 µM for 1 h (Invivogen), cycloheximide at 200 µg/mL for 20 min (Thermo Scientific), and harringtonine at 3 µg/mL for 30 min (MedChemExpress).

Cloning, plasmids and transfection
Plasmids were constructed using the Gateway technology (Invitrogen) with donor vectors from the human ORFeome70. Detailed maps are available upon request. DsRed2-DDX6fm was designed and used as described before14. Stable cell lines expressing DsRed2-DDX6fm-RUVBL1 or DsRed2-DDX6fm-RPAP3 were obtained using the Flp-In system (Thermo-Fischer Scientific) with hygromycin selection (50 mg/mL). Transient transfection was performed for the DsRed2-DDX6fm-KPNA3 construct. All transfections were performed using JetPrime according to the manufacturer's instructions: 200 µL JetPrime Buffer (Polyplus), 2 µg vector DNA, and 4 µL Jetprime reagent (Polyplus) for cells cultured in 2 mL in 6-well plates. Unless indicated otherwise, oligos were provided by Integrated DNA Technologies (IDT).

Generation of stable cell lines using PiggyBac transposon system
POLR2A and EFTUD2 cDNA WT or containing a STOP were fused at the C-terminus of a mCherry tag in the pDest-PiggyBac vector under the control of the UbC promoter. The constructs were transfected in the HCT116-RUVBL1-GFP cell line. 0.3 µg of each plasmid, alongside 1.7 µg of a construct expressing the transposase71 were transfected overnight with JetPrime (Polyplus). The day after, the transfection medium was replaced by fresh medium. Cells positively expressing mCherry were sorted using Melody FACS. mCherry-POLR2A protein expression and localization were further verified by fluorescent microscopy and Western blots.

Insertion of GFP and SunTag cassettes by CRISPR/Cas9 knock-in
HCT116 cells were CRISPR/Cas9 edited to insert an mAID-GFP-IRES-Neomycin cassette in fusion to the C-termini of RPAP3 and RUVBL1. The recombination cassettes contained 500 bases of homology arms flanking a cassette composed of mAID-GFP, a stop codon, an IRES containing a start codon fused to a Neomycin resistance gene, and a stop codon. HeLa Flp-In cells stably expressing the SunTag scFv-sfGFP were CRISPR/Cas9 edited to insert a SunTag*32 cassette at the C-terminus of RPAP3. Cells were selected on 800 µg/mL G418 neomycin for a few weeks. Individual clones were then picked and analyzed for GFP expression by fluorescence microscopy, Western blotting and PCR genotyping. The sequences targeted by the guide RNAs were (PAM sequences are lowercase): TCAGCAGTAAGAGACTCCCCagg for RUVBL1 guide 1, ACTTCATGTACTTATCCTGCtgg for RUVBL1 guide 2; GAGCTGAAAAAGAGGTATGGtgg for the RPAP3 guide. PCR genotyping was conducted using Phusion polymerase (New England Biolabs) and genomic DNA prepared with Wizard® Genomic DNA Purification Kit (Promega). The sequences of the oligonucleotides were as follows (5’ to 3’): Fw1 in the gene before the homology arms: GGTGAAGTCCCCATGTCCTG for RUVBL1 and TGTCGACCGATCAGTGGTAGTAA for RPAP3; Rev1 in the mAID-GFP cassette: CGGTGCTCCGTCCATTGATA; Rev2 in the SunTag*32 cassette: ACTAGTGACCGGTCCTCTAGA; Fw2 in the repair cassettes: GTTCATAAACGCGGGGTTCG; Rev2 in the gene after the homology arm: CGCAGCTCTCATCCTACTGG for RUVBL1 and ACGCTACACTCACATCCAACA for RPAP3.

Western blots
Proteins were resuspended in Laemmli 1X, boiled for 5 min, separated by 10% SDS-PAGE, and transferred to a nitrocellulose membrane (Protran). Membranes were blocked with 5% non-fat milk (weight/volume) in TBST (TBS with 0.5% Tween-20) and incubated in 1% non-fat milk in TBST with the primary antibodies overnight at 4°C followed by three times 5 min washes in TBST at RT, and incubation with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at RT, and finally washed three times 10 min in TBST at RT. Enzymatic activity was detected using the SuperSignal West Pico/Femto Chemiluminescent Substrate (Genetex) or Immobilon (Roche). The primary antibodies used were: anti-RPAP3 (Sigma SAB1411438-100UG, 1/1000 dilution), anti-RUVBL1 (Proteintech 10210-2-AP, 1/500 dilution), anti-GAPDH (Abcam 8245, 1/20000 dilution), anti-GFP (Roche 11814460001, 1/1000 dilution), anti-mCherry (abcam ab213511, 1/1000), anti-BRD8 (Proteintech 10476-1-AP, 1/1000 dilution). Secondary antibodies were anti-rabbit (Abcam ab6721, dilution 1/10000) and anti-mouse (Abcam ab6728, dilution 1/5000) coupled to HRP.

Immunofluorescence
2.105 to 3.105 cells were grown on Ø12 mm glass coverslips, treated as indicated and after 48 h washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at RT then washed with PBS. Permeabilization was done in 0.1% Triton X-100 (Thermo-scientific) in PBS for 10 min at RT. After 3 PBS washes, coverslips were incubated in PBS/BSA 1% or PBS/FBS 1% with primary antibodies for 1 h at RT, washed three times in PBS, incubated with secondary antibody for 1 h at RT, and washed three times again in PBS. Primary antibodies used were anti-POLR2A (PB-7C2, Euromedex, 1/100 dilution), anti-RPAP3 (Sigma SAB1411438-100UG, 1/100 dilution), anti-HUR (Santa Cruz Biotechnology sc-5261, 1/100 dilution) and anti-HSP90α/β (Santa Cruz Biotechnology sc-13119, 1/100 dilution) and anti-p70S6 Kinase (Santa Cruz Biotechnology, sc-8418, 1/100 dilution). Secondary antibodies were: anti-mouse Cy3 diluted 1/1000 (#115-165-003); anti-rabbit Cy3 diluted 1/1000 (#711-166-152); anti-mouse FITC diluted 1/200 (#115-546-006); anti-rabbit FITC diluted 1/200 (#111-096-047) anti-mouse Cy5 diluted 1/200 (#115-176-003) and anti-rabbit Cy5 diluted 1/200 (#111-175-144). Coverslips were mounted in Vectashield medium containing DAPI (Vector Laboratories).

Single molecule fluorescent in situ hybridization (smFISH) with DNA probes
Cells were grown on glass coverslips or glass-bottom 96-well plates (Greiner 655892), fixed with 4% paraformaldehyde in PBS for 20 min at RT and permeabilized with 70% ethanol overnight at 4°C. To detect a specific mRNA, 24–48 unlabeled primary DNA probes complementary to the RNA of interest were used (Supplementary Data 3). In addition to hybridizing to their targets, these probes contained a FLAP sequence that was pre-hybridized to a complementary secondary probe coupled to a fluorophore, as described previously71. Pre-hybridization was done in NEBuffer 3 (NEB, 10 mM NaCl, 5 mM Tris-HCl, 1 mM MgCl2, pH 7.9) according to the ratios indicated in Supplementary Data 3 and using a thermocycler with the following program: 85°C for 3 min, 65°C for 3 min, and 25°C for 5 min. Cells on coverslips were first incubated in 1x SSC, 5 M urea (Sigma-Aldrich) for 15 min at RT, and then incubated with the hybridization mixture containing the probe duplexes (2 µl of pre-hybridization mix per 100 µl of final volume), with 1X SSC, 0.34 mg/mL tRNA, 5 M urea, 2 mM VRC, 0.2 mg/mL RNAse-free BSA, 10% Dextran sulfate, overnight in a wet chamber at 37°C. The day after, the samples were washed three times for 30 min in the wash buffer (1x SSC, 5 M urea) at 37°C and rinsed twice in PBS at RT. The 96-plates were incubated in the dark with 5 µg/mL DAPI for 40 min at RT, then mounted in Vectashield medium without DAPI (Vector Laboratories, Inc.).

SmFISH in 96-well plates with RNA probes
Cells were grown in a 96-well plate with glass bottom (Greiner 655892), fixed with 4% paraformaldehyde in PBS at RT for 20 min and permeabilized with 70% ethanol overnight at 4°C. Probe sets of DNA oligonucleotides (GenScript) targeting the mRNAs of interest were generated based on the Oligostan script72. Each oligo is composed of a specific hybridization sequence flanked by 2 readout sequences and a barcode shared among the gene probeset (Supplementary Data 3). Gene-specific probes were amplified by PCR with the gene-specific barcodes before being transcribed in vitro to obtain transcript-specific primary RNA probe sets, as described previously73. For each mRNA of interest, 50 ng of primary RNA probes and 25 ng of each fluorescent secondary DNA probes (TYE-650-labeled LNA/DNA oligonucleotides, Qiagen)73 targeting the readout sequences were pre-hybridized in 100 µl of a solution containing 7.5 M urea (Sigma-Aldrich), 0.34 mg/ml tRNA (Sigma-Aldrich) and 10% Dextran sulfate. Pre-hybridization was performed in a thermocycler, with the following program: 90°C for 3 min, 53°C for 15 min. Cells in 96-well plates were washed with PBS and incubated for 15 min at RT in hybridization buffer (1X SSC, 7.5 M urea) before adding 100 µL of the hybridization mixture to each well. Hybridization was performed at 48°C overnight. The next day, the plate was washed 8 times for 20 min each in 1X SSC, and 7.5 M urea at 48°C. Finally, cells were washed twice with PBS at RT for 10 min each, stained with 5 µg/mL DAPI, and mounted in 90% glycerol (VWR), 1 mg/mL p-Phenylenediamine (Sigma-Aldrich), in PBS pH 8.

Image acquisition
Coverslip images were acquired using a 63x oil immersion objective (1.4 NA, PL Apo, oil) on a Leica DM6B microscope. DAPI, GFP, CY3, and CY5 channels were imaged using the following filter combinations: 405/60, 455, 470/40; 470/40, 495, 525/50; 545/30, 570, 610/75; 620/60, 660, 700/75. Alternatively, coverslips were imaged using a 63X objective (Plan Apochromat, 1.4 NA, oil) on a Zeiss Axioimager Z2 widefield microscope. DAPI, GFP, CY3, and CY5 channels were then imaged using the following filter combinations: <365, FT 395, 445/50; 470/40, FT 495, 525/50; 545/25, 570, 605/70; 610/30, 660, 640/50. Images were taken as Z-stacks with one plane every 0.24 µm (Zeiss Z2) or every 0.376 µm (Leica Thunder). 96-well plates were imaged on an Opera Phenix High-Content Screening System (PerkinElmer), with a 63× water-immersion objective (NA 1.15). Three-dimensional images were acquired, consisting of 18 slices with a spacing of 0.6 µm. RPS6 and HSP90 staining were acquired using a Zeiss LSM980 Airyscan confocal microscope equipped with a 63X Plan Apo oil 1.4 NA objective.

Image analysis
Image quantifications were based on an image analysis pipeline that included cell segmentation, threshold-based spot detection, dense region deconvolution, and a Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm. Firstly, nuclei and cytoplasm segmentation were inferred in 2D on image stack mean projection using Cellpose neural network74. Secondly, single molecules were detected as 3D spots using the Big-FISH package75, which relies on an intensity Laplacian of Gaussian (LoG) filter followed by a local maxima filter and a user-adjusted threshold. During analysis, we ensured that the same thresholds were applied to images made for the same RNA, for all cell treatments. As local maxima detection can lead to a misestimation of single molecule number in bright regions where individual molecules cannot be distinguished, a dense region deconvolution step was added. It consists of fitting the median detected spot with a 3D-gaussian to construct a reference spot and then reconstructing the bright region signal using multiple reference spots, thus providing the number of single RNAs in bright regions. Thirdly, a DBSCAN was used to assign spots to clusters (foci). At this point, detected spots were divided in clustered spots (spots assigned to a cluster) or free spots. Finally, a centroid calculation was performed to assign coordinates to each cluster. This single-molecule quantification process was repeated on all smFISH or SunTag channels, enabling quantification of RNA colocalization. During analysis, filtering was used to discard aberrations: (i) cells without nucleus or with more than one nuclei; (ii) any cell whose mask laid on the border of the field of view and that was thus considered incomplete; (iii) any spot detected outside of segmented cells; (iv) any regions containing an abnormally high average of 2 spots per pixel in a sphere of 1400 nm. Distances between spots were computed using the exact Euclidian distance. Finally, spots were considered colocalizing when found closer together than a cutoff distance of 310 nm.

A pipeline named SmallFish was also developed to quantify single mRNAs and clusters, and evaluate the percentage of colocalization between clusters. It provides a ready-to-use graphical interface to combine the Python packages used for cell analysis. In the pipeline, cell segmentation (2D) was performed using Cellpose (https://github.com/MouseLand/cellpose), while spot detection was carried out using BigFish (https://github.com/fish-quant/big-fish). Clusters were considered colocalizing when found closer together than a cutoff distance of 310 nm. Boxplots graphs were constructed on Python using Seaborn package76 and Statannotations package77.

RNA immunoprecipitation for RIP-seq
HCT116 cells with GFP-tagged RUVBL1 or RPAP3 alleles were seeded in 15 cm plates (1 plate/condition). At 70% confluency, cells were left untreated or treated as follows: with puromycin for 30 min at 100 µg/mL; with CB-6644 for 16 h at 500 nM; with both CB-6644 for 16 h and puromycin for 30 min. All treatments were done at 37°C. After treatments, cells were put on ice and washed twice with ice-cold PBS and then lysed with 0.5 mL HNTG lysis buffer (20 mM Hepes pH 7.9, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, and 1 mM EGTA) supplemented with a cocktail of protease inhibitors (Roche). Cells were incubated for 30 min on a tube roller at 4°C before being centrifuged for 10 min at 16,000×g at 4°C. The supernatant was subjected to immunoprecipitation for 1.5 h at 4°C using GFP-Trap agarose beads (Chromotek-gta-20). Agarose control beads (Chromotek-bab-20) were used for non-specific control IP. All experiments were done in duplicates. Beads were washed 5 times with ice-cold HNTG. RNeasy minikit (Qiagen-74106) was used to purify RNAs. DNA contamination was eliminated by a TURBO-DNA Free kit, as recommended by the manufacturer (Invitrogen-AM1907).

RNA-sequencing and analysis
SMART-Seq Stranded Kit (Takara Bio) was used to generate cDNA libraries for high-throughput sequencing using NovaSeq 6000 Illumina. RNA and DNA libraries' quality was subjected to quality control checks periodically (or after each step) using a fragment analyzer (Kit high sensitivity NGS) and qPCR (Roche LightCycler 480). Ribosomal cDNAs were cleaved with scZapR enzyme in the presence of scRProbes designed against mammalian ribosomal RNAs. Libraries were then sequenced with NovaSeq Reagent Kits on a flow cell SP (single read, −100 nucleotides) using the 2-channel sequence-by-synthesis method. Before demultiplexing, sequences were mixed with PhiX sequences as an Illumina control (not indexed). Demultiplexing was realized using Illumina bcl2fastq software (v2.20.0.422), and all PhiX sequences were removed.

FastaQC software (v0.11.9) provided a tool to check read quality after high-throughput sequencing, and the MultiQC program (V1.12) served to group data that emerges from all samples. FastQ screen software served to detect contaminating genomes. Sequences were aligned on 15 species genomes, mycoplasma, and ribosomal RNAs using Bowtie2 software. No contamination was detected.

TopHat2 software (v2.1.1) was used to align the obtained reads on the human genome (hg38 version) and to annotate them according to NBCI database. FeatureCounts software was utilized to count reads per gene. All reads that overlap on two genes or show alignment at different positions on the genome were discarded. Counts were normalized in each sample to eliminated differences due to manipulation. This was done by a normalization method known as relative log expression (RLE). Differential expression genes were identified using EdgeR (v3.34.1). Tests are based on a generalized linear model and allow count modeling using a negative binomial distribution. To calculate the reads per kilobase per million mapped reads (RPKM), the formula below was used: (read counts aligned on a specific gene / (total aligned counts x gene length) x 109.

Label-free quantitative proteomics
HCT116 cells with RUVBL1 or RPAP3 endogenously tagged with GFP were seeded in 15 cm plates (1 plate/condition). At 70% confluency, cells were treated or not with CB-6644 for 16 h at 500 nM. Cells were washed with ice-cold PBS, then lysed with 0.5 mL HNTG lysis buffer supplemented with proteinase inhibitor cocktail (Roche; see above for the composition of HNTG). Cells were scraped and incubated for 15 min at 4°C before being centrifuged for 10 min at 16,000×g at 4°C. The supernatant was subjected to immunoprecipitation using GFP-Trap agarose beads (Chromotek-gta-20). Agarose control beads (Chromotek-bab-20) were used for non-specific control IP. All experiments were done in triplicates. Extracts were incubated with beads for 1.5 h at 4°C and washed 5 times with ice-cold HNTG. Proteins were then eluted in Laemmli buffer and heated for 10 min at 95°C.

For sample digestion, proteins were loaded onto SDS-PAGE (10% polyacrylamide, Mini-PROTEAN® TGX™ Precast Gels, Bio-Rad) and stained with Protein Staining Solution (Euromedex). Gel lanes were cut into one single band and destained with 50 mM TriEthylAmmonium BiCarbonate (TEABC, Merck), followed by three washes in 100% acetonitrile. After reduction (with 10 mM dithiothreitol in 50 mM TEABC at 60°C for 30 min) and alkylation (with 55 mM iodoacetamide in 50 mM TEABC at room temperature for 30 min), proteins were digested in-gel using trypsin (1 g/band, Gold, Promega). Digest products were vacuum-dried before LC-MS/MS analysis. For mass spectrometry, a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled with an Ultimate 3000 nano-HPLC (Thermo Fisher Scientific) was used. Peptide samples were solubilized in 0.05% trifluoroacetic acid (TFA) / 2% ACN, and were injected for desalting and pre-concentration on a PepMap® 100 C18 precolumn (0.3 mm x 5 mm; ThermoFisher Scientific). Peptides separation were done on a 50 cm analytical reversed-phase column (75 mm inner diameter, Acclaim Pepmap 100® C18, ThermoFisher Scientific) using a 120 min gradient of 2 to 40% of buffer B (80% ACN, 0.1% formic acid) and a flow rate of 300 nL/min. MS/MS analyses were performed in a data-dependent mode. Full scans (375-1500 m/z) were acquired on the Orbitrap mass analyzer with a 60,000 resolution at 200 m/z. For the full scans, 3e6 ions were accumulated within a maximum injection time of 60 ms and detected in the Orbitrap analyzer. The twelve most intense ions with charge states ≥2 were sequentially isolated to a target value of 1e5 with a maximum injection time of 100 ms and fragmented by HCD (Higher-energy collisional dissociation) in the collision cell (normalized collision energy of 28%) and detected in the Orbitrap analyzer at 30,000 resolution. First mass was set to 100 m/z.

Raw spectra were processed using the MaxQuant environment (v.2.0.3.0), and Andromeda for database search with label-free quantification (LFQ), match between runs, and the iBAQ algorithm (CIT)78. The MS/MS spectra were matched against the UniProt Reference proteome of Homo sapiens (Proteome ID UP000005640; release 2022_03; https://www.uniprot.org/) and a homemade contaminant database. Different release versions were used, depending on the date of analysis. Enzyme specificity was set to trypsin/P, and the search included cysteine carbamidomethylation as a fixed modification and oxidation of methionine, and acetylation (protein N-term) as variable modifications. Up to two missed cleavages were allowed for protease digestion. FDR was set at 0.01 for peptides and proteins and the minimal peptide length at 7. A representative protein ID in each protein group was automatically selected using an in-house developed bioinformatic tool (Leading tool 3.4) as the best annotated protein in UniProtKB (reviewed entries rather than automatic ones79. Analysis of quantification data, statistical analyses and graphical representations were performed using Perseus (v1.6.15). Briefly, label-free quantification (LFQ) intensity-based values were log2 transformed. Imputation of missing values was performed using Perseus with standard parameters (normal distribution, width = 0.3, shift = 1.8). Normalized LFQ intensities were compared between replicates, using a 1% permutation-based false discovery rate (FDR) in a two-tailed Student’s t test (250 randomizations)80.

RT-qPCR
RNA immunoprecipitation was performed as described before except that DNAse Treatment was neglected. The reverse transcription reaction (RT) was carried out by mixing 2 µL of RNA with final 2.5 µM of random primers (Thermofisher Scientific) and 0.5 mM of dNTP mix (Thermofisher Scientific). The RNA-primers mix was incubated for 5 min at 65°C then for 1 min on ice. 100 U of SuperScript IV enzyme was added to the mix in addition to a final 1X SuperScript Buffer, 5 mM DTT (Thermofisher Scientific) and 20 U of the RNase Out recombinant RNase inhibitor (Invitrogen). The final mix (Vf = 20 µL) was incubated for 10 min at 23°C, 10 min at 50°C followed by 10 min at 80°C to inactivate the enzyme. The RT reaction was then diluted 4x by adding 60 µL of RNAse Free H2O.

The qPCR was performed using the LightCycler 480 instrument II (Roche). 2 µL of the RT reaction were mixed with a final 1X of the LightCycler 480 SYBR Green I Master (Roche) and 1 µM of RNA-specific primers (Integrated DNA Technologies or Eurofins Genomics), Vf = 10 µl. Samples were loaded in a LightCycler 480 white multiwell plate 384 (Roche) and sealed well with the LightCycler 480 Sealing foil (Roche). PCR amplicon sizes range between 100 and 120 bp. The sequences of designed primers are listed in the Table 1.

Sucrose gradient and Western Blot
All buffer solutions were treated with diethylpyrocarbonate. HEK293 Trex cells were washed in chilled HKM200 buffer (10 mM Hepes pH 7.6, 5 mM MgCl2, 200 mM KCl) and lysed in the same buffer supplemented with 1% NP-40, 1 µl/mL protease inhibitor cocktail (Calbiochem #535140), 40 units/ml rRNAsin (Recombinant RNasin Ribonuclease Inhibitor, Promega) and 0.5 µM DTT. Cell lysates were clarified by centrifugation 10 min at 4°C at 16,000×g, and loaded on 11–54% sucrose gradients in 20 mM Hepes pH 7.6, 100 mM KCl, 5 mM MgCl2. Ultracentrifugations were performed at 38,000 RPM during 2 h at 4°C in a Beckmann SW41 rotor. One ml fractions were collected manually and proteins were precipitated by TCA and resuspended in SDS loading dye. For Western blots, samples were separated by electrophoresis in 4–12% Tris-glycine SDS/PAGE (Life Technologies) and were transferred onto 0.2 µ nitrocellulose membranes (Protan-BA83; GE Healthcare) using Thermofisher Transblot systems. Membranes were blocked in PBS with 10% (w/v) milk and 0.05% Tween-20 (Euromedex) before incubation with primary antibodies diluted 1:1000 in PBS 0.05% Tween overnight at 4°C. After washing with 1X PBS/0.05% Tween-20, membranes were incubated with primary antibodies anti-RUVBL2 (Proteintech 10195-1-AP, 1/1000 dilution) or anti-RPS3 (Proteintech 66046-1-Ig, 1/1000 dilution) and secondary antibodies coupled to HRP (1/5000 dilution) and visualized using SuperSignal West Femto or Pico (Thermo Scientific) with LAS4000 mini (GE Healthcare).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors explore an important area in protein translation and folding: how do protein complexes assemble? There is a lot of conjecture within the field regarding whether such assembly can occur during translation, and if so, by what mechanism. In this work, the authors have studied R2TP, a HSP90 co-chaperone using a range of cell-based assays (including proteomics, smFISH, and single-molecule imaging).\nThrough these carefully designed and executed experiments, the authors demonstrate a number of key findings. These include a rather complex mechanism for co-translational assembly formation for this ATP-dependent chaperone system, including identifying mRNA clients involved in co- and post-translational interactions, and the formation of “translation factories”. It is on this latter point which is what is remarkable about this study: the authors present a rather unexpected mechanism of “co-chaperone” channelling, which brings translating mRNA into close proximity into biomolecular condensates, and thus offers a very plausible alternative mechanistic understanding for how protein complexes can be formed. This is significant because the current hypothesis is that complexes form because translating polypeptides are brought into close proximity via polysomes. The specifics of this mechanism are (in my opinion) a little more tenuous and could benefit from more clarification (see below), however overall, there is a very plausible (and exciting) hypothesis that is being put forward.\nIn summary, this manuscript offers a set of important findings that will attract broad interest across several fields (including protein translation, chaperone interactions, protein folding, and biomolecular condensate formation), and will no doubt inspire related works.\nA few considerations that may help to improve the clarity of the manuscript for a broader audience:\n1. Puromycin treatment offers a reliable means to detect active translation, however, do the authors detect and ribosomal proteins or rRNA within their proteomic analysis?\n2. On a related point, do the authors have evidence for polysome formation or of ribosomal complexes? Polysome formation is of course the default mode by which translation would occur within cells, however some biochemical evidence may provide some unequivocal proof.\n3. Within the “translational factories” (cha-cha) mechanism it was not clear to me whether the biomolecular condensates (P-bodies) contained actively translating ribosomes or whether they were simply sequestering mRNA? Often P-bodies are associated with compromises within proteostasis (e.g. regulation), however here the authors suggest a more functional role for localised translation. The authors show that condensates can form from mRNA and local translation is likely to occur. What is missing (or at least not clear to me) is whether there is protein-based evidence that the condensate formation results in assembly formation. This may need to clarify the biomolecular condensate description further to give unequivocal credence to the cha-cha and “translational factories” concepts. I anticipate that the authors are correct, but viewing the mechanism from the lens of mRNA localisation alone might considered limiting.\n4. For the non-specialist reader, a description of the significance of the recruitment of POLR2A and PRPF8 mRNAs would be helpful.", "reviewer_2": "The manuscript “Co-translational determination of quaternary structures in chaperone factories” by Manon Philippe#, Soha Salloum# et al., investigates the function of HSP90/R2TP chaperones in co-translational protein complex assembly. Manon Philippe#, Soha Salloum# et al. find evidence for HSP90/R2TP co-translational interactions with many canonical as well as new partners. Furthermore, they find that the chaperone induces the formation of dedicated translation factories, accumulating mRNAs encoding for complex partner subunits and thus favoring chaperone-client co-translational interactions.\nThis manuscript offers a significant contribution to the field of protein synthesis and maturation pathways by proposing a potential new mechanism, termed cha-cha (co-translational chaperone channeling), for macromolecular complex assembly. However, additional validation is necessary to fully support its central claims.\nFirst, the RIP-seq experiments were not performed in the presence of Cycloheximide treatment to prevent ribosomal runoff. Cycloheximide, a small molecule, inhibiting translation elongation by ribosomal E-site binding (\"Structural basis for the inhibition of the eukaryotic ribosome\" de Loubresse,et al., Nature, 2014) is commonly used during selective ribosome profiling experiments capturing co-translational interactions, in vivo (For example: Ingolia N.T.et al.; Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009; Selective ribosome profiling to study interactions of translating ribosomes in yeast, Galmozzi C.G at al., 2019; A rapid protocol for ribosome profiling of low input samples, 2023, Meindl Aet al., NAR). As ribosomes dissociates highly rapidly in response to various stresses and nutrients` deprivation occurring during sample preparation, it is imperative to clarify how the RIP-Seq experiments done under these conditions maintain co-translational interactions. Alternatively cross-linking can preserve native co-translational interactions, but there is no mention of this either. It is important to clarify this issue, as Puromycin treatment in living cells can cause a stress response which may indirectly result in decreased chaperone binding. For example, stress granule formation, limiting mRNA accessibility. These results can suggest, that the RIP-Seq results capture an ensemble of chaperone-mRNA interactions, both translation-dependent and independent ones, under conditions where there is no translation stress, and mRNAs are not sequestered.\nTo further support this possibility, Puromycin treatments seem to have a low impact also in the PARP-3-SUN-TAG experiments (Figure 5), again suggesting translation independent functions. This point needs to be discussed and addressed.\n*In the following figures, an enlargement of the foci images is required (as presented in figure 4A for example):\nFigure 5A, 6B, all of figure 7, figure S4F, figure S5A. These enlargements are important to evaluate co-localization analysis.\n*It is not clear from the text why RPAP3 needs to be in its nascent form to promote condensates of the U5 snRNP component PRPF8 (figure 5). Is RPAP3 co-translationally folding, assembling and functional? or does an exposure of RNA binding domain sufficient?\n*There is a mistake in references numbering, from reference 45 on (Pal, M. et al. Structural Basis for Phosphorylation-Dependent Recruitment of Tel2 to Hsp90 by Pih1.Structure 22, 805–818 (2014)). References are shifted, 45 should be 46, 46 should be 47 and so on.", "reviewer_3": "This study provides evidence for the co-translational role of the HSP90/R2TP chaperone complex in assembling multi-subunit macromolecular complexes in eukaryotic cells. It proposes an interesting mechanism termed “cha-cha” (co-translational chaperone channeling), whereby R2TP facilitates protein complex assembly not via mRNA co-localization, but through localized condensates — termed R2TP factories — that act as hubs for translation and assembly. These factories accumulate client mRNAs and facilitate efficient subunit interaction via chaperone mediation.\nI have some doubts about the validity of the conclusions:\nStrictly speaking, RIP-seq and FISH just shows that the tagged protein binds RNA species in a more or less specific manner.\n- The conclusion that this is co-translational is not fully substantiated. Given that puromycin only reduces (some of) the mRNAs association, that would require additional controls, like showing ribosomal proteins and RNA are co-precipitated. Ribosome profiling or ribosome-nascent chain complex IP would lend further credibility.\n- Also, Puromycin acts fast and globally. Using translation initiation inhibitors (like harringtonine) would confirm that effects are not due to general ribosome collapse.\n- The claim that mRNAs were found that code for proteins that assemble into complexes needs statistical testing to see if this is above what you would randomly expect to find from pulling down that many mRNAs, in particular for related proteins it could just be based on sequence or property similarity.\n- The CB-6644 drugs specificity is unclear to me. Complementing knockout/knockdown with ATPase-dead mutants would solidify ATP-dependence interpretations.\n- For the RNAs that are seen to remain associated with R2TP in the absence of translation (by addition of puromycin), the authors could stain for ribosomal proteins, to see if entire ribosome nascent chain complexes are attached to R2TP.\n- The proteomics data looks at the peptide level, Authors should verify if for co-translationally attached proteins, mainly the N-terminal part is present in the peptides, while for post-translational attached proteins the entire protein should be present in the different detected peptides. This is important to distinguish co-translationally interacting proteins or post-translationally (Figure 2D-E).\nAdditional controls:\n- The control condition of the RIP-seq is poorly chosen, the GFP-trap beads have single-chain antibody against GFP, the authors use as a control just naked agarose beads. At the very least this should be an off-target nanobody and preferably, the experiment should also have been done with the GFP-trap beads in cells where the target is not tagged.\n- In the context of co-translational events, the ribosome is a key player. It’s noteworthy that the authors discuss co-translational processes and interactions without discussing/addressing ribosomal RNA or ribosomal proteins throughout the paper. I suggest using an antibody against a ribosomal protein to stain HeLa cells and look for signals in the foci discussed. Additionally, if applicable, mention that in the label-free proteomics, ribosomal proteins were found in the IP which disappeared when treated with puromycin. This confirms that when the NCs are released, the rRNA and ribosomal proteins should also be reduced significantly. Furthermore, additional western blots should be conducted to verify and confirm the presence of ribosomes in the IP pulldown step with anti-GFP beads.\n- In general, there are very few or no western blots performed for proteins in the lysate versus IP.\nMinor comments:\n- The current title is quite general and broad. I think a more specific title would be suitable, including the HSP90/R2TP chaperone. Possible suggestion: \"HSP90/R2TP-Mediated Co-Translational Chaperone Channeling: Insights into the RNA Interactome”\n- In the abstract: why use the word dubbed?\n- Check citations: Wrong citations?: “For instance, biochemical studies in yeast and mammals have shown that subunits of the same complex can bind each other co-translationally, sometimes reciprocally and other times in a directional manner 45” Wrong citation 45: should be maybe 46 citation of Shiber? 46. Shiber, A. et al. Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling. Nature 561, 268–272 (2018).\n-. p8: I think they switched in vitro and in vivo\n- Figure 2 legend: spelling mistake in RIP-Seq\n- Figure 3 legend: “An” RNA instead of a RNA\n- Figure 4 legend: they could have added also a ribosomal antibody?\n- Page 11/ Figure S4, does it show this?: revealed that the RPAP3-SunTag was much less expressed than its untagged counterpart, or are the labels wrong in Figure S4B? (~50 fold less, Figure S4). What is the difference between column 1 and 2.\n- Legend figure 6, an ‘s’ too much\n- In methods: both SmFish with DNA probes and SmiFISH with RNA probes. Used smFish to determine whether the mRNAs of interest were recruited to these foci. Where did they use SmiFISH?\n- p12: keep consistent, “dual-color smFISH”", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the Reviewer for his/her interest in our discoveries and agree that the manuscript could benefit from some clarifications. We believe that the new results listed below and the clarification of some parts of the manuscript should answer the Reviewer’s concerns. Overall, we believe that the revised manuscript is significantly improved.\nA few considerations that may help to improve the clarity of the manuscript for a broader audience:\n1. Puromycin treatment offers a reliable means to detect active translation, however, do the authors detect any ribosomal proteins or rRNA within their proteomic analysis?\nThe Reviewer raises an interesting point.\nWe always detect ribosomal proteins in our R2TP AP-MS experiments. However, they are never above the background noise and therefore can not be considered as specific interactants. Our hypothesis is that there are too few ribosomes bound to R2TP in an ocean of cellular ribosomes, and therefore the R2TP-bound ribosomes do not emerge from the background. We tried to estimate this effect more quantitatively. The estimated number of ribosomes is around 3 000 000 per cell (https://bionumbers.hms.harvard.edu/bionumber.aspx?s=n&v=1&id=107552), while R2TP is moderately expressed. Assuming that the abundance of R2TP is reflected by its mRNA levels, this yields 30 000 copies per cell as the mRNA ratio of RPAP3 vs ribosomal proteins is 0.01. Assuming that 1% of R2TP is bound co-translationally to polysomes, itself having on average ~10 ribosomes, this implies that only 0.1% of ribosomes would interact with R2TP. Even if 10% of R2TP is bound co-translationally to polysomes, it still means that only 1% of ribosomes would interact with R2TP. These numbers are likely beyond the detection threshold. Of note, the search for rRNA in our RUVBL1-GFP and RPAP3-GFP RIP-seq experiments was not possible as rRNAs are systematically depleted during library preparation.\nWe therefore did two new experiments to detect the interaction of R2TP with ribosomes. First, we generated a RPS6-GFP knock-in cell line and performed smFISH to test whether ribosomes are present in the POLR2A mRNA foci where R2TP also accumulates (Fig. R1 below). Using high resolution confocal imaging, we observed neither an accumulation nor an exclusion of RPS6-GFP from the R2TP-containing POLR2A mRNA foci. This result suggests that ribosomes are present at low levels in the mRNA foci, in agreement with the model that we describe in the manuscript. These new data have been added in the revised manuscript in Fig. S6A.\nAs a second approach to address the association of R2TP with ribosomes, we performed polysome profiling. We fractionated ribosomes and polysomes on a sucrose gradient and analyzed the presence of RUVBL2 in the different fractions by Western blot (Fig. R2 below). RUVBL2 was detected in fractions 5 to 8, indicating that R2TP is associated with small polysomes in cells. These new data have been added in the revised manuscript (Fig. S2B).\n2. On a related point, do the authors have evidence for polysome formation or of ribosomal complexes? Polysome formation is of course the default mode by which translation would occur within cells, however some biochemical evidence may provide some unequivocal proof.\nThe polysome fractionation experiment of Fig. R2 above provides direct biochemical evidence for an association between R2TP and polysomes. We detect association with light polysomes, but because R2TP associates with only few mRNAs that are moderately expressed, its association with larger polysomes may be beyond our detection limit. Alternatively, the mRNAs associated with R2TP may have only a few ribosomes.\n3. Within the “translational factories” (cha-cha) mechanism it was not clear to me whether the biomolecular condensates (P-bodies) contained actively translating ribosomes or whether they were simply sequestering mRNA? Often P-bodies are associated with compromises within proteostasis (e.g. regulation), however here the authors suggest a more functional role for localised translation. The authors show that condensates can form from mRNA and local translation is likely to occur. What is missing (or at least not clear to me) is whether there is protein-based evidence that the condensate formation results in assembly formation. This may need to clarify the biomolecular condensate description further to give unequivocal credence to the cha-cha and “translational factories” concepts. I anticipate that the authors are correct, but viewing the mechanism from the lens of mRNA localisation alone might be considered limiting.\nWe have now clarified the point concerning the molecular condensates and in particular the issue of P-bodies vs. translation factories. Indeed, we performed experiments with two different types of condensates:\n• a first type that is artificially created using a P-body protein fused to a R2TP component.\n• a second type that naturally occurs in cells: the POLR2A translation factories.\nThe artificial condensate forms in the cytoplasm when we use a fragment of a P-body protein: amino-acids 289-483 of DDX6 (referred to as DDX6fm). Indeed, this fragment can be fused to a protein of interest and the resulting fusion protein will artificially accumulate in P-bodies. In the past, we have used this system to look at the co-recruitment of a second protein to these artificial P-bodies, which then provides evidence for an in vivo interaction between the two proteins (see Boulon et al, 2010; Maurizy et al, 2018). In the present manuscript, we adapted the system to look at the recruitment of target mRNAs to the artificial P-bodies. We fused DDX6fm to DsRed2 and either RUVBL1 or RPAP3. RUVBL1 and RPAP3 are not normally present in P-bodies. However, by fusing them to DDX6fm, we artificially concentrate these proteins in P-bodies where they can interact and recruit candidate mRNAs or proteins (Fig.4 and S4 of the revised manuscript). We observed that the artificial concentration of RUVBL1 or RPAP3 in P-bodies leads to the co-recruitment of POLR2A and PRPF8 mRNAs to these artificial structures, indicating an interaction between the two. Importantly, POLR2A and PRPF8 mRNAs do not localize to P-bodies in absence of either the DsRed2-DDX6fm-RUVBL1 or the DsRed2-DDX6fm-RPAP3 fusion proteins (see Fig. S6C of the revised manuscript and also below in Fig. R3). Likewise, they are not recruited to the artificial P-bodies formed by DsRed2-DDX6fm-KPNA3, a negative control unrelated to R2TP (Fig. 4 of the revised manuscript).\nIn the revised manuscript, we clarified the text by adding the sentence: “By fusing them to DDX6fm, we artificially created a high local accumulation of R2TP in P-bodies and could then check by smFISH whether the candidate mRNAs or proteins are recruited into these artificial condensates.”\nIn contrast to these artificial R2TP condensates, the translation factories are natural endogenous condensates that exist in cells in absence of any treatment, and which concentrate POLR2A mRNA and protein, PRPF8 mRNA and R2TP (Fig. 6 and S6E of the revised manuscript). To avoid any confusion, we performed immunofluorescence experiments to verify that the endogenous POLR2A translation factories are not P-bodies (see Fig. R3 below). As expected, the POLR2A mRNA and protein condensates are distinct from P-bodies. These new data have been added to the revised manuscript (Fig. S6C).\nOf note, we believe that actively translating ribosomes are present in both types of condensates, the artificial condensates and the natural POLR2A translation factories. Indeed, for the artificial P-body condensates accumulating R2TP, the POLR2A protein can be detected there together with the POLR2A mRNAs (Fig. 4D of the revised manuscript), and the POLR2A mRNAs dissociate from these artificial R2TP condensates upon translation inhibition (Fig.4A, B and D of the revised manuscript). Likewise, the POLR2A protein is present in the POLR2A translation factories (Figure R3 above and Fig. S6C of the revised manuscript), and these factories are dissolved when translation is inhibited (Fig.6 and S6E of the revised manuscript). Interestingly, this latter effect is observed with puromycin or harringtonine, which dissociate nascent proteins from their mRNAs, but not with cycloheximide, which also inhibits translation but maintains the polysomes intact with the nascent protein chains still attached to the mRNA. This indicates that the nascent POLR2A protein likely plays a key role in driving the assembly of the translation factories (Fig. R4 below, now included in Fig. S7A of the revised manuscript). This is in line with the requirement for HSP90 and R2TP in the formation of the factories (Fig. 8 of the revised manuscript), highlighting a model in which these two chaperones bind their client proteins co-translationally and drive assembly of the factories, possibly because of the multivalency of R2TP (RUVBL1/2 form hexamers and dodecamers) and polysomes (which carry multiple nascent chains).\nTo further confirm this model, we performed new microscopy experiments showing that the RPS6 ribosomal protein and HSP90 are present in these foci, even though they do not accumulate there (Fig. R1 above and R5 below, corresponding to Fig. S6A and S6B of the revised manuscript).\nTo better link the POLR2A translation factories with the assembly process, we performed additional experiments using siRNAs against various subunits of the RNA polymerase II. The rationale was to test how a block in assembly, induced by the lack of a subunit, would affect the POLR2A translation factories. The results, shown in Fig. R6 below, indicate that the knock-down of POLR2H, which is nearly always associated with POLR2A during the assembly process (Boulon et al, 2010), leads to the disappearance of the POLR2A mRNA foci. Likewise, the knock-downs of POLR2B or POLR2C, which form another building block of the enzyme (Boulon et al, 2010) also dissolve the POLR2A translation factories. While the mechanisms involved are unclear at this point, they nevertheless reveal a link between the POLR2A translation factories and the presence of its partner subunits, thus revealing a link with the assembly of RNA polymerase II. These new data have not been included in the manuscript because we think that they will be better suited for a future manuscript dedicated to the detailed assembly mechanisms of RNA polymerase II. However, we hope that, together with the dual role of HSP90 and R2TP in the assembly of the enzyme and the formation of the translation factories, these new data will convince the Reviewer that condensate formation is linked to the assembly process.\n4. For the non-specialist reader, a description of the significance of the recruitment of POLR2A and PRPF8 mRNAs would be helpful.\nWe thank the Reviewer for this suggestion. We have added a small paragraph in the discussion of the revised manuscript to describe the significance of the recruitment of POLR2A and PRPF8 mRNAs to translation factories, also referred to as “chaperone factories” in the main text:\n‘POLR2A and PRPF8 are both large proteins (>210 kDa) that must be assembled into large macromolecular complexes, RNA polymerase II and U5 snRNP, respectively. Even though these two proteins are not part of the same complex, they need to be taken in charge and stabilized by the same chaperone, HSP90/R2TP, as soon as they come out of the ribosome. Indeed, we have previously shown that the cytoplasmic unassembled POLR2A becomes unstable upon HSP90 or R2TP inhibition (Boulon et al, 2010). Likewise, PRPF8 is rapidly degraded in absence of its co-chaperones (Erkelenz et al, 2021). The risk for these large proteins, if they are not properly taken in charge by their chaperones, is that they get misfolded, interact with the wrong partners, aggregate or become unstable. Chaperone factories have a high local concentration of R2TP and may thus favor their co-translational interaction with the chaperone, likely preventing them from these unwanted events. It is unclear why the other mRNAs that also associate with R2TP are absent from the R2TP factories. It may be that POLR2A and PRPF8 have particularities favoring polysome condensation, such as a stronger or higher valency of binding to HSP90 and R2TP, or a controlled ribosomal pausing providing more time for binding the chaperones and condensing the ribosomes.’\nReviewer #2 (Remarks to the Author)\nThe manuscript “Co-translational determination of quaternary structures in chaperone factories” by Manon Philippe#, Soha Salloum# et al., investigates the function of HSP90/R2TP chaperones in co-translational protein complex assembly. Manon Philippe#, Soha Salloum# et al. find evidence for HSP90/R2TP co- translational interactions with many canonical as well as new substrates. They find that HSP90/R2TP function is involved in the formation of dedicated translation factories, accumulating mRNAs encoding for complex partner subunits and thus favoring chaperone-client co-translational interactions.This manuscript offers a significant contribution to the field of protein synthesis and maturation pathways by proposing a potential new mechanism, termed cha-cha (co-translational chaperone channeling), for macromolecular complex assembly.\n1. However, additional validation is necessary to fully support its central claims. First, the RIP-seq experiments were not performed in the presence of Cycloheximide treatment to prevent ribosomal runoff. Cycloheximide, a small molecule, inhibiting translation elongation by ribosomal E-site binding (\"Structural basis for the inhibition of the eukaryotic ribosome\" de Loubresse,et al., Nature, 2014) is commonly used during selective ribosome profiling experiments capturing co-translational interactions, in vivo (For example: Ingolia N.T.et al.; Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009; Selective ribosome profiling to study interactions of translating ribosomes in yeast, Galmozzi C.G at al., 2019; A rapid protocol for ribosome profiling of low input samples, 2023, Meindl Aet al., NAR). As ribosomes dissociate highly rapidly in response to various stresses and nutrients` deprivation occurring during sample preparation, it is imperative to clarify how the RIP-Seq experiments done under these conditions maintain co-translational interactions.\nWe have done the experiment suggested and tested several translation inhibitors that act by different mechanisms: cycloheximide, which freezes and stabilizes elongating ribosomes, harringtonine, which prevents initiation, and puromycin, which acts as a chain terminator. We performed IPs of RUVBL1-GFP in our knock-in cell line, followed by RT-qPCR to compare the specific association of a set of mRNAs after these various treatments. The results have been integrated in Fig. 3D of the revised manuscript and are presented in Fig. R7 below. In brief, puromycin and harringtonine, the two translation inhibitors that dissociate nascent chains from ribosomes, eliminate the association of RUVBL1-GFP with the tested mRNAs (POLR2A, POLR2B, EFTUD2, PRPF8, EP400 and BRD8), whereas cycloheximide, which stabilizes polysomes, slightly increases it. This shows that the mRNA-RUVBL1 interactions that we observe are well preserved during the preparation of our sample even in the absence of cycloheximide.\nIn addition, we would like to point out that several published studies have characterized co-translational interaction without using cycloheximide, including using Ribo-Seq. Please find three examples below, and note that ref 3 also describes artifacts that can be induced with cycloheximide:\n1-Toullec D, Elías-Villalobos A, Faux C, Noly A, Lledo G, Séveno M, Helmlinger D. The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKKs prior to complex assembly. Cell Rep. 2021, 37(3):109867. doi: 10.1016/j.celrep.2021.109867. PMID: 34686329.\n2-Hisler V, Bardot P, Detilleux D, Bernardini A, Stierle M, Sanchez EG, Richard C, Arab LH, Ehrhard C, Morlet B, Hadzhiev Y, Jung M, Le Gras S, Négroni L, Müller F, Tora L, Vincent SD. RNA polymerase II transcription initiation in holo-TFIID-depleted mouse embryonic stem cells. Cell Rep. 2024, 43(10):114791. doi: 10.1016/j.celrep.2024.114791. PMID: 39352809.\n3-Daniel A Santos, Lei Shi, Benjamin P Tu, Jonathan S Weissman. Cycloheximide can distort measurements of mRNA levels and translation efficiency, Nucleic Acids Research, Volume 47, Issue 10, 2019, Pages 4974–4985, https://doi.org/10.1093/nar/gkz205\n2-Alternatively cross-linking can preserve native co-translational interactions, but there is no mention of this either. It is important to clarify this issue, as Puromycin treatment in living cells can cause a stress response which may indirectly result in decreased chaperone binding. For example, stress granule formation, limiting mRNA accessibility. These results can suggest that the RIP-Seq results capture an ensemble of chaperone-mRNA interactions, both translation-dependent and independent ones, under conditions where there is no translation stress, and mRNAs are not sequestered.\nWe haven’t crosslinked our samples. However, as mentioned above and shown by the cycloheximide experiment, we believe that the mRNA-nascent protein-RUVBL1 interactions that we observe are well preserved during our sample preparation. Please note that in our RIP-seq and RIP-qPCR experiments, and unlike in Ribo-Seq/IP experiments, the samples are always kept at 4°C and that the total duration of the experiment is only ~3h. We believe that this explains the preservation of the interactions in absence of crosslinking. The other points of the Reviewer are addressed below.\nFirst, we aimed to rule out the possibility that puromycin treatment leads to the formation of stress granules, which may indirectly decrease chaperone binding. To this end, we used a previously developed cell line (Pichon et al., 2016), in which the POLR2A gene is endogenously tagged with a SunTag array at its N-terminus (STx32-POLR2A). In these cells, the colocalization between POLR2A mRNA and protein allows to directly monitor the translation of POLR2A mRNAs in native cells, and relate it to the formation of stress granules.\nUsing HuR as a stress granule marker, we observed that short puromycin treatment as the ones used throughout the manuscript (<1h), did not induce the appearance of stress granules (Fig. R8 below). Please note that this was also previously reported by others (Kedersha and Anderson, 2007; Bounedjah et al, 2014). However, puromycin removed the SunTag signals associated with the POLR2A mRNAs, indicating that it inhibited translation and dissociated polysomes as expected.\nIn contrast, arsenite treatment did induce stress granules. In this case, the POLR2A mRNAs relocalized and accumulated in stress granules. Moreover, as in puromycin-treated cells, the colocalization between the SunTag signal of the nascent POLR2A protein and the POLR2A mRNA was lost , as expected because arsenite also inhibits translation (Fig. R8).\nAltogether, these new results show that, at least in our conditions, puromycin does not induce stress granules.\nSecond, we wanted to confirm that the dissociation of the nascent protein chains from the ribosome is sufficient to prevent the binding of RUVBL1-GFP from its target mRNAs and that this was not due to some side effect of puromycin. As described above in Fig. R7, we performed IPs of RUVBL1-GFP followed by RT-qPCR in untreated cells versus puromycin-, cycloheximide- or harringtonine-treated cells. This showed that harringtonine and puromycin eliminated the interactions of RUVBL1-GFP with the tested target mRNAs (POLR2A, POLR2B, EFTUD2, PRPF8, EP400 and BRD8), while cycloheximide slightly increased it. This shows that interaction of RUVBL1 with mRNAs is not altered by any type of translation inhibition, but solely by translation inhibitors that induce the dissociation of the nascent protein chains from the ribosome and the mRNAs.\nFinally, we developed reporters that enabled us to evaluate the role of the nascent protein chains in the absence of any inhibitors or particular cell treatments. We created vectors expressing mCherry-POLR2A or mCherry-EFTUD2 fusions, as well as variants in which a stop codon was inserted immediately after the coding sequences of mCherry. These reporters are thus identical except for the presence of stop codons that prevent the translation of POLR2A or EFTUD2. We stably expressed these reporters in the RUVBL1-GFP knock-in cell line and then analyzed by IP/RT-qPCR whether these mRNAs associate with RUVBL1-GFP. We found that the mCherry-POLR2A and mCherry-EFTUD2 mRNAs associated with RUVBL1-GFP, while the internal stop codon abolished the interaction (Fig. R9 below). This supports the conclusion that the interaction between POLR2A or EFTUD2 mRNAs and RUVBL1-GFP is strictly dependent on the nascent polypeptide. These results have been added to the revised manuscript in Fig. 3E, F.\nAltogether, these new results indicate that puromycin is a reliable tool to assess the translation-dependency of interactions and that, for the tested mRNAs, there are no detectable translation-independent interactions between the chaperone and the mRNAs.\n3-To further support this possibility, Puromycin treatments seem to have a low impact also in the RPAP3-SUN-TAG experiments (Figure 5), again suggesting translation independent functions. This point needs to be discussed and addressed.\nIn Fig. 5A of the original manuscript, we use a microscopy assay to show that the proportion of mRNA colocalizing with RPAP3-SunTag significantly decreases upon puromycin treatment, for EP400, EFTUD2, PRPF8 and POLR2A mRNAs. Indeed, as pointed out by the Reviewer, the proportion of co-localizing mRNAs does not go down to 0% in puromycin-treated cells. We believe that this is due to the nature of the assay. Let’s imagine that RPAP3-SunTag occupies 50% of the pixels in a cell, then any spot randomly localized would have a basal colocalization rate of 50% with RPAP3-SunTag. Indeed, we measured that ~15% of 𝛂-catenin mRNA colocalizes with RPAP3-SunTag in both untreated and puromycin-treated cells, although this mRNA is a negative control unrelated to RPAP3, which does not appear in our RIP-Seq experiments. Thus, colocalization values of ~15% represent the baseline.\nWe believe that the significance of the colocalization is demonstrated by the puromycin treatment and by the comparison with the 𝛂-catenin negative control, rather than by its absolute value. Also, it is important to note that the RPAP3-SunTag protein is expressed at about a 50 fold lower level than the endogenous protein. The true association rate of these mRNAs with RPAP3 is thus likely to be much higher than measured with the RPAP3-SunTag system.\n*In the following figures, an enlargement of the foci images is required (as presented in figure 4A for example): Figure 5A, 6B, all of figure 7, figure S4F, figure S5A. These enlargements are important to evaluate co-localization analysis.\nWe thank the Reviewer for pointing this out and have now added enlargements of the foci images in Fig. 5A, 6B and 7. The Figures S4F and S5A are now numbered S6D and S8A in the revised manuscript and have been modified too.\n*It is not clear from the text why RPAP3 needs to be in its nascent form to promote condensates of the U5 snRNP component PRPF8 (figure 5). Is RPAP3 co- translationally folding, assembling and functional? or does an exposure of RNA binding domain sufficient?\nWe believe that there is some misunderstanding here and would like to clarify this point. The SunTag system is not limited to the detection of nascent proteins. The SunTag is a repeating peptide array inserted at the N-terminus or C-terminus of the protein of interest, which can recruit multiple copies of a protein single chain variable fragment (scFv) fused to a fluorescent protein (FP), therefore enabling imaging of single protein molecules, whether they are nascent or mature. In our RPAP3 study, the SunTag is inserted at the C-terminus of the protein. Therefore, the RPAP3-SunTag spots are, at least for the vast majority, not polysomes but single molecules of the mature RPAP3 protein, or clusters of them. In Fig. 5, the colocalization between RPAP3-SunTag and the mRNAs only indicates that RPAP3 is present in PRPF8 and POLR2A mRNA condensates.\n*There is a mistake in references numbering, from reference 45 on (Pal, M. et al. Structural Basis for Phosphorylation-Dependent Recruitment of Tel2 to Hsp90 by Pih1.Structure 22, 805–818 (2014)). References are shifted, 45 should be 46, 46 should be 47 and so on.\nWe thank the Reviewer for his/her vigilance. We have corrected the reference numbering.\nReviewer #3 (Remarks to the Author)\nThis study provides evidence for the co-translational role of the HSP90/R2TP chaperone complex in assembling multi-subunit macromolecular complexes in eukaryotic cells. It proposes an interesting mechanism termed “cha-cha” (co- translational chaperone channeling), whereby R2TP facilitates protein complex assembly not via mRNA co-localization, but through localized condensates — termed R2TP factories — that act as hubs for translation and assembly. These factories accumulate client mRNAs and facilitate efficient subunit interaction via chaperone mediation.\nI have some doubts about the validity of the conclusions:\nStrictly speaking, RIP-seq and FISH just shows that the tagged protein binds RNA species in a more or less specific manner.\n1-The conclusion that this is co-translational is not fully substantiated. Given that puromycin only reduces (some of) the mRNAs association, that would require additional controls, like showing ribosomal proteins and RNA are co-precipitated. Ribosome profiling or ribosome-nascent chain complex IP would lend further credibility.\nThis is an interesting point that is also raised by Reviewer 1. As mentioned in the response to Reviewer 1 (see his/her point 1), we have tried to detect an interaction between R2TP and ribosomal proteins by different means (AP-MS and IF). In summary, there are only a few ribosomes bound to R2TP, which are diluted in an ocean of other cellular ribosomes, and therefore the background will be very high in any kind of interaction assay using R2TP as bait, and will preclude detection. We thus performed a series of alternative experiments to rigorously demonstrate the co-translational nature of the interactions detected by RIP-seq.\n-As mentioned above in our response to Reviewer 1, we have now performed polysome profiling experiments and analyzed the presence of RUVBL2 in the different polysomal fractions. We found that RUVBL2 is present in small amounts in light polysome fractions (Fig. R2 above).\n-As suggested by the Reviewer, we have also performed an experiment inspired from the RiboSeq-nascent chain IP method. In this experiment, we immunoprecipitated RUVBL1-GFP from cell extracts and treated the beads with a saturating amount of RNAse I to degrade all the free pieces of RNA and keep only the ribosome protected fragments associated with RUVBL1-GFP. Completion of RNA degradation was ensured by spiking an in vitro translated RNA directly on the IP’ed beads and verifying by gel that it was degraded to pieces of <10 nucleotides. The protected RNA fragments were then sequenced. Below in Fig. R10, we provide browser tracks for POLR2A and PRPF8 mRNAs, the main R2TP mRNA targets, along with two abundant control mRNAs that do not associate with R2TP in the RIP-Seq experiments, EEF1D and EEF1G. We observed that many reads are distributed along the coding sequence of the POLR2A and PRPF8 mRNA, while very few are present on the 5’ and 3’ UTR regions of these mRNAs, or on the control mRNA that does not bind R2TP (Fig. R10A). Moreover, the reads sizes show two peaks of 32 and 38 nucleotides, suggesting that we predominantly detect single ribosome footprints. Interestingly, the reads start near the N-terminus of PRPF8, while they pile-up in the long last exon of POLR2A, suggesting that the timing of R2TP co-translational recruitment is different for these two mRNAs: early during translation for PRPF8, and late for POLR2A.\nBecause the manuscript is already heavy in data and because we would also like to optimize this experiment to obtain more read counts and better define the timing of R2TP co-translational recruitment, these new data have not been incorporated in the revised manuscript. Nevertheless, the results confirm the co-translational nature of the recruitment of RUVBL1 on POLR2A and PRPF8 mRNAs.\n-We developed reporters enabling a direct assessment of the co-translational nature of the interactions made by RUVBL1. As shown in the response to Reviewer 2 and in Fig. R9 above, we created reporter cell lines stably expressing mCherry-POLR2A and mCherry-EFTUD2 fusions, with or without a stop codon immediately after the mCherry coding sequences. We found that mCherry-POLR2A and mCherry-EFTUD2 mRNAs nicely associated with RUVBL1-GFP, while the mCherry-STOP-POLR2A and mCherry-STOP-EFTUD2 mRNAs did not (Fig. R9 above). These mRNAs are identical except for the stop codon, and these results thus provide strong support to our conclusion that the interaction between POLR2A or EFTUD2 mRNAs and RUVBL1-GFP is co-translational and depends on the nascent polypeptide. These results have been added to the manuscript in Fig. 3E and F.\n-Regarding the point raised by Reviewer 3 that puromycin only reduces RUVBL1 association with some of the mRNAs, we show below in Fig. R11 the RPKM counts of the most interesting mRNAs in the GFP and the control IP. Of note, puromycin reduces binding of RUVBL1-GFP to these mRNAs to a level similar to the control IP, indicating that there is little or no interaction in absence of translation. We do agree that it is possible that some mRNAs species may associate with RUVBL1-GFP independently of translation, but this is clearly not the case for the main hits found in the RIP-Seq experiments. Also, please note that such putative translation-independent mRNA targets are beyond the scope of the present study.\n2-Also, Puromycin acts fast and globally. Using translation initiation inhibitors (like harringtonine) would confirm that effects are not due to general ribosome collapse.\nWe thank the Reviewer for this advice. We have now performed RUVBL1-GFP IPs followed by RTqPCR to compare the effects of puromycin, cycloheximide and harringtonine. The results are presented in Fig. R7 above. In brief, puromycin and harringtonine, two translation inhibitors that dissociate nascent chains from polysomes, abolish the association of RUVBL1-GFP with POLR2A, POLR2B, EFTUD2, PRPF8, EP400 and BRD8 mRNAs, whereas cycloheximide, a translation inhibitor that freezes ribosomes and stabilizes polysomes, slightly increases it. These results indicate that the nascent protein chain must remain associated with the mRNA to enable RUVBL1 binding. These results have been added to Fig. 3D of the manuscript.\n3-The claim that mRNAs were found that code for proteins that assemble into complexes needs statistical testing to see if this is above what you would randomly expect to find from pulling down that many mRNAs, in particular for related proteins it could just be based on sequence or property similarity.\nWe do not claim that the RIP-Seq experiments show by themselves a role of R2TP in client assembly. We only observed that most mRNAs found in the RIP-Seq experiments code for proteins that are known to be assembled together by R2TP. In particular, this was previously shown for RNA polymerase II (Boulon et al, 2010; Forget et al, 2013; Lynham and Houry, 2022) and U5 snRNP (Malinova et al, 2017; Cloutier et al, 2017). The text was clarified.\n4-The CB-6644 drug specificity is unclear to me. Complementing knockout/ knockdown with ATPase-dead mutants would solidify ATP-dependence interpretations.\nRUVBL1 and RUVBL2 are essential genes. Therefore, knockout cannot be used to answer this question and developing degrons has proved to be unsuccessful so far as well. RUVBL1 and RUVBL2 ATPase-dead mutants show a significant decrease in the ATPase activity of the RUVBL1/2 heterohexamer in vitro (up to 80% activity inhibition with only one mutant in the complex and 90% when both mutants are combined; Assimon et al, 2019). However, previous studies in yeast have shown that these mutants are very toxic and thus would be very difficult to use in the mammalian system (King et al., 2001). Importantly, the drug CB-6644 shows an inhibition of ~80% of RUVBL1/2 ATPase activity in vitro, and displays a similar effect as the ATPase-dead mutants (Garcia-Martin et al, 2024). In addition, CB-6644 has been considered selective for RUVBL1/2, since it does not have any activity on related AAA ATPases (Assimon et al, 2019), and furthermore, while CB-6644 inhibits cell growth, these effects can be reverted by a single point mutant of the RUVBLs that prevents CB-6644 binding (Assimon et al, 2019).\nThe mechanism of action of the drug has now been well described, thanks to recent cryo-EM studies (Garcia-Martin et al, 2024). It was shown that CB-6644 recognizes the ATP-bound conformation of the RUVBL1/2 heterohexamer by interacting at the interface between two RUVBL1 and RUVBL2 subunits, thereby trapping the ATP-bound conformation of the complex and preventing ATP hydrolysis (Garcia-Martin et al, 2024). Importantly, the drug CB-6644 inhibits the activity of the RUVBL1/2 complex but not the activity of each individual protein. Therefore we are confident that the drug CB-6644 is a powerful tool to specifically and rapidly inhibit RUVBL1/2 activity in cells. Given the toxicity of the ATPase dead mutants of the RUVBLs, it would be very difficult to devise a suitable expression system to perform RIP-seq experiments with them, and results may also be prone to artefacts.\n5-For the RNAs that are seen to remain associated with R2TP in the absence of translation (by addition of puromycin), the authors could stain for ribosomal proteins, to see if entire ribosome nascent chain complexes are attached to R2TP.\nThis is an interesting point. Indeed, translation-independent mRNAs may in fact still be interacting via the nascent chain if the ribosomes are stuck in long pauses. We feel that this could be the topic of future studies since the present manuscript is focused on the co-translational recruitment of the HSP90/R2TP chaperone by certain clients.\nWe have performed microscopy experiments to test the colocalization of RPS6-GFP ribosomal protein with POLR2A mRNA. Even using a high resolution Airyscan confocal microscope, we detect high levels of RPS6-GFP throughout the cytoplasm and it is thus difficult to assess the significance of the RPS6-GFP signals that overlap single molecules of POLR2A mRNAs (Fig. R1).\n6-The proteomics data looks at the peptide level, Authors should verify if for co-translationally attached proteins, mainly the N-terminal part is present in the peptides, while for post-translational attached proteins the entire protein should be present in the different detected peptides. This is important to distinguish co-translationally interacting proteins or post-translationally (Figure 2D-E).\nThis is an interesting point. However, strictly co-translationally interacting proteins are, according to our definition, not found in our AP-MS data and therefore, we cannot check whether the peptides identified for these proteins are mainly located in the N-terminus of the corresponding proteins. As for proteins interacting both co- and post-translationally with the R2TP chaperone, we observed that for POLR2A and PRPF8, the peptides identified by AP-MS experiments are located along the entire length of these proteins (see Fig. R12 below). Likewise, peptide intensity is relatively homogeneous along the POLR2A and PRPF8 proteins and we do not see an enrichment at their N-termini (Fig. R12C).\nAdditional controls:\n7-The control condition of the RIP-seq is poorly chosen, the GFP-trap beads have single-chain antibody against GFP, the authors use as a control just naked agarose beads. At the very least this should be an off-target nanobody and preferably, the experiment should also have been done with the GFP-trap beads in cells where the target is not tagged.\nWe thank the Reviewer for pointing this out. Each type of IP internal control has its advantages and disadvantages and it is indeed better to perform both. The use of naked agarose beads with RUVBL1-GFP extracts allowed us to bypass the proteome variations that could be due to clonal variation between cell lines. Conversely, using GFP-TRAP beads with the parental cells would allow us to bypass the risk that naked agarose beads do not bind exactly the same contaminants as the GFP-TRAP beads.\nWe have thus performed new RUVBL1-GFP IP/RT-qPCR experiments to test different types of internal controls, i.e., parental cells with GFP-TRAP beads (IP-GFP HCT116-WT), RUVBL1-GFP cells with FLAG beads (IP-FLAG HCT116-RUVBL1-GFP) and RUVBL1-GFP cells with naked agarose beads (IP-CTL HCT116-RUVBL1-GFP). The results are presented in Fig. R13 below and show that all three controls are similar. In addition, we can confirm interactions of RUVBL1-GFP with the mRNAs coding for POLR2A, POLRB, EFTUD2, PRPF8, EP400 and BRD8, while POLR2C and POLR2H are not enriched in the GFP IPs as compared to the controls.\n8- In the context of co-translational events, the ribosome is a key player. It’s noteworthy that the authors discuss co-translational processes and interactions without discussing/addressing ribosomal RNA or ribosomal proteins throughout the paper. I suggest using an antibody against a ribosomal protein to stain HeLa cells and look for signals in the foci discussed. Additionally, if applicable, mention that in the label-free proteomics, ribosomal proteins were found in the IP which disappeared when treated with puromycin. This confirms that when the NCs are released, the rRNA and ribosomal proteins should also be reduced significantly. Furthermore, additional western blots should be conducted to verify and confirm the presence of ribosomes in the IP pulldown step with anti-GFP beads.\nThis is similar to the points 1 and 5 of the Reviewer 1.\nAs mentioned above (see point 1 of Reviewer 1), there are only a few ribosomes bound to R2TP, which are diluted in a vast amount of non-interacting ribosomes. These non-interacting ribosomes generate background and detecting an enrichment of ribosomes in a R2TP IP is thus extremely difficult if not impossible.\nLikewise, ribosomes are present throughout the entire cytoplasm with a concentration of ~1 𝜇M (https://tinyurl.com/vkav59yb), it is therefore very difficult to detect any enrichment of ribosomes on single mRNAs (see example images in Fig. R1).\nHowever, as discussed above, we have now performed a series of experiments rigorously demonstrating the co-translational nature of the interaction of R2TP with its target mRNAs:\n• Polysome profiling experiments show the presence of RUVBL2 in light polysome fractions (Fig. R2 above).\n• Experiments inspired from the IP/RiboSeq method showed that we can detect ribosome protected-fragments in RUVBL1-GFP IPs, which are located along the POLR2A and PRPF8 mRNAs but not control mRNAs (Fig. R10 above).\n• The use of multiple translation inhibitors (puromycin, harringtonine and cycloheximide), demonstrate that the nascent polypeptide is essential to promote the association of RUVBL1 with its target mRNAs.\n• The use of mCherry fusion reporters bearing or not a stop codon in between the mCherry and POLR2A (or EFTUD2), demonstrates that the POLR2A and EFTUD2 RNA sequences must be translated to promote association with RUVBL1 (Fig. R9 above).\nAltogether, we believe that these new results demonstrate beyond doubt that RUVBL1 interacts with its main mRNA targets via the nascent protein chains.\n9- In general, there are very few or no western blots performed for proteins in the lysate versus IP.\nWe would like to point out that most proteins identified in our AP-MS experiments were previously shown to interact with RUVBL1/2 (Maurizy et al. 2018, Cloutier et al. 2017, Rizzolo et al. 2017, Cho el al. 2021). Therefore, we believe that it was not necessary to confirm those interactions by WB.\nWe performed several Western blots to check the new partners found in our RIP-seq experiments. We tested BRD8, which is found in both the RIP-seq and the AP-MS, and indeed we could detect an interaction between RUVBL1-GFP and the BRD8 protein by Western blot (Fig. R14A and Fig. S2A of the revised manuscript). In contrast, SCAF4 was only found in the RIP-seq and not the IP-MS, and we could not detect any interaction with RUVBL1-GFP by Western blot (Fig. R14B, not shown in the manuscript).\nMinor comments:\n- The current title is quite general and broad. I think a more specific title would be suitable, including the HSP90/R2TP chaperone. Possible suggestion: \"HSP90/R2TP-Mediated Co-Translational Chaperone Channeling: Insights into the RNA Interactome”\nWe thank the Reviewer for his/her advice. Indeed, the title is quite broad but we would prefer to keep it this way because it conveys the main message of the study. We believe that the abstract provides the requested specifics in particular with regards to HSP90, R2TP, their clients and the cellular model.\n- In the abstract: why use the word dubbed?\nWe changed the word in the abstract and used “named” instead.\n- Check citations: Wrong citations?: “For instance, biochemical studies in yeast and mammals have shown that subunits of the same complex can bind each other co- translationally, sometimes reciprocally and other times in a directional manner 45” Wrong citation 45: should be maybe 46 citation of Shiber? 46. Shiber, A. et al. Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling. Nature 561, 268–272 (2018).\nWe thank the Reviewer for his/her vigilance and modified the reference numbering accordingly.\n-. p8: I think they switched in vitro and in vivo: done (we clarified the whole sentence in the revised manuscript).\n- Figure 2 legend: spelling mistake in RIP-Seq: done\n- Figure 3 legend: “An” RNA instead of a RNA: done\n- Figure 4 legend: they could have added also a ribosomal antibody? done in Fig. S6A\n- Page 11/ Figure S4, does it show this?: revealed that the RPAP3-SunTag was much less expressed than its untagged counterpart, or are the labels wrong in Figure S4B? (~50 fold less, Figure S4). What is the difference between column 1 and 2. done in Fig. S5B of the revised manuscript. There are 3 lanes: lane 1 is a high exposure version of lane 2 (HeLa stably expressing RPAP3-SunTag*32), displaying two bands revealed by anti-RPAP3 antibody, with the upper band being RPAP3-SunTag*32 and the lower band being the untagged RPAP3 (the cell line is heterozygous). Lane 3 is a control (parental HeLa cells stably expressing scFv-GFP), which only displays the lower band, corresponding to endogenous RPAP3. Both the legend and the figure itself have been clarified.\n- Legend figure 6, an ‘s’ too much: done\n- In methods: both SmFish with DNA probes and SmiFISH with RNA probes. Used smFish to determine whether the mRNAs of interest were recruited to these foci. Where did they use SmiFISH? done. We removed the “SmiFISH” term.\n- p12: keep consistent, “dual-color smFISH” : done"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you to the authors for the revised manuscript. You have addressed the comments that I had in a really careful and considered way. The additional experimental data you generated is particularly impressive too.\nI do not have any further comments.", "reviewer_2": "The revised manuscript “Co-translational determination of quaternary structures in chaperone factories” by Manon Philippe#, Soha Salloum# et al., investigates the function of HSP90/R2TP chaperones in co-translational protein complex assembly, presenting evidence for HSP90/R2TP co-translational interactions with many canonical as well as new substrates. They find that HSP90/R2TP function is involved in the formation of dedicated translation factories, accumulating mRNAs encoding for complex partner subunits and thus favoring chaperone-client co-translational interactions.\nThe revised manuscript has been substantially improved. The authors have performed extensive new experiments that significantly validate the main conclusions. Overall, the extensive experimental additions and textual revisions have enhanced the scientific rigor and impact of the work. This manuscript offers a significant contribution to the field of protein synthesis and maturation pathways by proposing a co-translational chaperone channeling pathway for macromolecular complex assembly.", "reviewer_3": "The authors have substantially strengthened the manuscript by performing a series of additional, highly relevant experiments, particularly to clarify the presence of ribosomal proteins—an essential aspect when discussing co-translational interactions. The integration of polysome profiling, improved IP/RiboSeq approaches, the use of different translation inhibitors, and innovative fusion reporters significantly enhances the overall quality of the study. The polysome profiling convincingly demonstrates an association of R2TP with ribosomes; however, the data suggest that the protein is primarily linked to monosomes rather than larger polysomes. The authors provide plausible explanations for this observation, citing potential detection limits, the scarcity of large ribosomal assemblies, or a specific association of R2TP with mRNAs containing only a few ribosomes. Importantly, they addressed prior concerns by incorporating additional internal controls to rule out off-target or non-specific interactions in IP/RT-qPCR experiments and validated newly identified partners from RIP-seq analyses through western blotting. All remaining minor comments were carefully considered. Overall, these clarifications and methodological improvements result in a stronger and more convincing manuscript.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter