Bérengère Pradet-Balade

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.

HSP90 and the R2TP co-chaperone complex: building multi-protein machineries essential for cell growth and gene expression.

HSP90 (Heat Shock Protein 90) is an essential chaperone involved in the last folding steps of client proteins. It has many clients, and these are often recognized through specific adaptors. Recently, the conserved R2TP complex was identified as a key HSP90 co-chaperone. Current evidences indicate that the HSP90/R2TP system assembles multi-molecular protein complexes. Strikingly, these comprise basic machineries of gene expression: (1) nuclear RNA polymerases; (2) the snoRNPs, essential to produce ribosomes; and (3) mTOR Complex 1 and 2, which control translational activity and cell growth. Another important substrate is the telomerase RNP, required for continuous cell proliferation. We discuss here the assembly of RNA polymerases in bacteria and eukaryotes, the role of HSP90/R2TP in this process and in the assembly of snoRNPs and the PIKK family of TORC1 kinase. Finally, we speculate on the roles of R2TP as a master regulator of cell growth under normal or pathological conditions.

CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport.

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm. Instead, CRM1 controls the composition of nucleoplasmic pre‐snoRNP complexes. We observed that Tgs1 long form (Tgs1 LF), the long isoform of the cap hypermethylase, contains a leucine‐rich nuclear export signal, shuttles in a CRM1‐dependent manner, and binds to the nucleolar localization signal (NoLS) of the core snoRNP protein Nop58. In vitro data indicate that CRM1 binds Tgs1 LF and promotes its dissociation from Nop58 NoLS, and immunoprecipitation experiments from cells indicate that the association of Tgs1 LF with snoRNPs increases upon CRM1 inhibition. Thus, CRM1 appears to promote nucleolar transport of snoRNPs by removing Tgs1 LF from the Nop58 NoLS. Microarray/IP data show that this occurs on most snoRNPs, from both C/D and H/ACA families, and on the telomerase RNA. Hence, CRM1 provides a general molecular link between nuclear events and nucleocytoplasmic trafficking.

Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

During small nucleolar ribonucleoprotein complex assembly, a pre-snoRNP complex consisting only of protein components forms first, followed by displacement of the ZNHIT3 subunit when C/D snoRNAs bind and dynamic loading and unloading of RuvBL AAA+ ATPases.

Drosophila spag is the homolog of RNA polymerase II-associated protein 3 (RPAP3) and recruits the heat shock proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries

Background: Mammalian RNA polymerase II-associated protein 3 (RPAP3) recruits heat shock protein 90 (Hsp90) to assemble cellular machineries such as RNA polymerases. Results: Spaghetti encodes the Drosophila homolog of RPAP3. Spaghetti is essential for development. Spag protein binds and stimulates Hsp90 and Hsp70. Conclusion: RPAP3 function is conserved among metazoans. Significance: Our data suggest that Hsp70 assists RPAP3 in complex assembly. The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA+-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.

Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions

Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post‐fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap‐dependent translation repressor 4E‐BP (eukaryotic initiation factor 4E‐binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap‐dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E‐BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin‐sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug did not alter neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element‐binding protein from eIF4E. Mol. Reprod. Dev. 75: 1617–1626, 2008.

SnoRNPs, ZNHIT proteins and the R2TP pathway

HSP90 and its R2TP co-chaperone play central roles in building machineries important for RNA and DNA metabolism (see (1) for a review). These include the nuclear RNA polymerases, complexes containing PIKKs (mTOR, ATM/ATR, DNA-PK, SMG1, and TRRAP), as well as a number of ribonucleoprotein particles, such as the telomerase RNP, the spliceosomal U4 snRNA and the snoRNPs, which are essential to produce ribosomes. Given the known functions of these machineries in gene expression, protein synthesis, and DNA maintenance, it has been hypothesized that the R2TP co-chaperone carries some of the oncogenic functions of HSP90 [1]. In agreement, two R2TP components, the essential and related AAA+ ATPases RUVBL1 and RUVBL2, are overexpressed in hepatocarcinomas and colorectal cancers, and are also necessary for tumorigenesis in mouse cancer models [2]. Yet, RUVBL1 and RUVBL2 are associated to several other cellular complexes and it has not been formally demonstrated that their oncogenic activity is related to their function within the R2TP chaperone. How the R2TP assists HSP90 in the assembly of protein complexes is still poorly understood. We and others took advantage of the box C/D snoRNPs, the R2TP smallest substrate, to decipher the mechanisms involved. To form a functional particle, box C/D snoRNAs have to be assembled with four core proteins: 15.5K, NOP58, NOP56 and Fibrillarin. In eukaryotes, attempts to reconstitute in vitro such a particle from isolated components have been so far unsuccessful. Thus, we studied the C/D snoRNP assembly pathway in vivo, by performing quantitative proteomic experiments using a variety of snoRNP proteins and assembly factors as baits. Importantly, we characterized a protein-only complex that preassembles 15.5K and NOP58 in the absence of snoRNA [3]. This complex contains the assembly factors NUFIP, ZNHIT3 and ZNHIT6 (also called BCD1 - see Figure ​Figure1).1). The key RUVBL1 and RUVBL2 ATPases were present in this complex but, surprisingly, not the other components of the R2TP chaperone: PIH1D1, RPAP3 and their associated prefoldins. Figure 1 Schematic representation of the 6 different ZNHIT proteins found in human, with indication of the HIT (green) and other domains To further decipher the mechanism of box C/D snoRNP assembly, we dissected the interactions between substrates and co-factors by yeast two-hybrid assays and in vitro reconstitution experiments. This revealed that NUFIP forms a stable heterodimer with ZNHIT3 and interacts with the core protein 15.5K [3, 4]. Structural studies further suggested that NUFIP binding to 15.5K prevents premature activation of the catalytic activity of snoRNPs during their biogenesis [3]. One exciting hypothesis that would reconcile currently available data is that the role of the R2TP complex would be to load the essential RUVBL1/2 ATPase on the C/D core proteins NOP58 and 15.5K, thereby holding them together before their incorporation into the nascent snoRNP. In agreement with this hypothesis, RUVBL1/2 make mutually exclusive, ATP-dependent contacts with R2TP components and C/D core proteins: they bind 15.5K when loaded with ATP, and PIH1D1/RPAP3 otherwise [5, 6]. NUFIP/ZNHIT3 and ZNHIT6 could further stabilize the RUVBL1/2:NOP58:15.5K complex. Accordingly, ZNHIT6 also make ATP-dependent contacts with RUVBL1/2. RUVBL1/2 are essential proteins that form heterohexamers or hetero-dodecamers. While these AAA+ ATPases are present in many seemingly unrelated complexes, one unifying possibility would be that some of these complexes are in fact clients of the R2TP chaperone, on which RUVBL1/2 have been loaded in order to stabilize them. In this regard, it is interesting to note that HIT-finger proteins appear to have evolved specific links with RUVBL1/2. There are six such proteins in the human genome (ZNHIT1 to ZNHIT6, Figure ​Figure1).1). ZNHIT3 and ZNHIT6 are associated with RUVBL1/2 during box C/D snoRNP biogenesis. ZNHIT1 and ZNHIT4, as well as RUVBL1/2, are key part of the chromatin remodeling complexes SRCAP and INO80, respectively. Remarkably, recent structural data of the yeast INO80 complex indicates that the ortholog of ZNHIT4, Ies2p, makes direct physical contacts with RuvBL1/2 and plays a central role in connecting them to the rest of the complex [7]. The two remaining HIT-finger proteins, ZNHIT2 and ZNHIT5/DDX59, have not yet been characterized. Yet, ZNHIT2 has already been found to be associated with RUVBL1/2 and RPAP3 proteins in high-throughput proteomic assays. Altogether, these data suggest that HIT-finger proteins are key partners of RUVBL1/2, possibly in relation with R2TP. They may regulate their activity or contribute to their substrate specificity (Figure ​(Figure11).

Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex.

RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90. We also established the 3D structure of an RPAP3:PIH1D1 sub-complex demonstrating the need for a 34-residue insertion, specific of RPAP3 isoform 1, for the tight binding of PIH1D1. We also confirm the existence of a complex lacking PIH1D1 in human cells (R2T), which shows differential binding to certain clients. These results highlight similarities and differences between the yeast and human R2TP complexes, and document the diversification of this family of co-chaperone complexes in human.