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. 2008 Feb 11;180(3):579-95.
doi: 10.1083/jcb.200708110.

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

Séverine Boulon  1 , Nathalie Marmier-Gourrier, Bérengère Pradet-Balade, Laurence Wurth, Céline Verheggen, Beáta E Jády, Benjamin Rothé, Christina Pescia, Marie-Cécile Robert, Tamás Kiss, Barbara Bardoni, Alain Krol, Christiane Branlant, Christine Allmang, Edouard Bertrand, Bruno Charpentier
Affiliations

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

Séverine Boulon et al. J Cell Biol. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Composition of L7Ae RNPs. The various RNPs containing the L7Ae family members (yellow) studied here are depicted. The yeast names are indicated below the human names.
Figure 2.
Figure 2.
Nufip and Rsa1 are related proteins that bind L7Ae family members through a short peptide motif. (A) Alignment of Rsa1 with Nufip. The conserved motif, named PEP, is represented. (B) Y2H tests between the indicated proteins. Rsa1, Nufip, and their various domains are represented by lines. Growth on −Leu −His −Trp (−L −H −T) indicates an interaction. (C) GST pull-down experiments.15.5K and SBP2 were translated in vitro in bacterial S30 lysate and assayed for binding to the indicated recombinant GST fusions. GFP-hNhp2 was translated in rabbit reticulocyte lysate, but similar results were obtained with bacterial lysate (not depicted). Snu13 was purified from E. coli and stained with Coomassie blue. (D) In vivo co-IP assays. GFP-15.5K and GFP-hNHP2 were expressed in HeLa cells and purified with anti-GFP antibodies. Endogenous SBP2 was immunopurified from nuclear HeLa cell extracts using antipeptide antibodies. Bound proteins were analyzed by Western blots with anti-Nufip antibodies. I, input (10% of total); Pt, pellet; NI, preimmune control serum; Ct, control beads without antibodies. The sizes of the various products are indicated on the side of the gels.
Figure 3.
Figure 3.
Nufip binds box C/D and H/ACA snoRNAs, a B/C-containing RNA, U4 snRNA, and mRNAs coding for selenoproteins. (A) In vitro interactions of Rsa1 and Nufip with Snu13–RNA complexes. (left) Gel-shift assays show that the Rsa1 N3C1 and PEP domains interact with Snu13 bound to RNA. Radiolabeled yeast U14 snoRNA was incubated with the indicated recombinant proteins and anti-His antibodies when indicated. (right) Y3H assays show that Nufip interacts with yU3-B/C RNA in a PEP-dependent manner. Plate −Leu −Ura (−L −U) shows the growth of the test strain. Growth on −Leu −Ura −His (−L −U −H) indicates a positive interaction. (B) In vivo association of Rsa1 with U3 precursors in yeast. Extracts from TAP-Rsa1 or wild-type (−) isogenic strains were purified on IgG beads and analyzed by RT-PCR with primers specific for U3 precursors. (C) In vivo interactions of Nufip with rat U3B.7 and other box C/D snoRNAs. HeLa cells were transfected with the indicated snoRNA gene either alone (top) or with an Nufip-GFP vector (bottom). Extracts were purified with anti-Nufip (top) or anti-GFP (bottom) antibodies or beads as a control, and bound RNAs were analyzed by RNase protection. U3ΔC′ and U3ΔCΔC′ are mutated in the C′ and in the C and C′ boxes. dBB is an artificial intronic C/D snoRNA (see Results). I, input (10% of total); M*, mature species. (D) In vivo association of Nufip with endogenous U4 snRNA (right) and a transfected, tagged U4 snRNA (left and middle). Legend as in C. (E) In vivo binding of Nufip with H/ACA snoRNAs. HeLa nuclear extracts were immunoprecipitated with anti-Nufip antibodies and analyzed by RNase protection with the indicated probes. Legend as in C. (F) Nufip associates with mRNAs coding for selenoproteins. Anti-GFP IP of extracts of 293FT cells transfected with SBP2 alone (lanes Ct) or together with Nufip-GFP (lane Pt). U3 and β-actin are positive and negative controls, and type 2 deiodinase and glutathione peroxidase 4 are two selenoproteins. Input, 10% of total.
Figure 4.
Figure 4.
Nufip and Rsa1 are required for the production of snoRNAs. (A) Nufip is required for the production of U3. HeLa cells were treated with siRNA duplexes against Nufip. Nufip depletion was verified by Western blotting (top), and the levels of the indicated RNAs were monitored by Northern blotting (bottom) or by qPCR (graph). The qPCR values represent ratios of the indicated RNA to GAPDH mRNA and are expressed as fractions of the levels obtained with the control siRNAs (Ct). (B) Primer extension was used to measure the levels of small RNAs in yeast cells lacking Rsa1 (ΔRsa1). Numbers represent the amount of RNA relative to wild type (W303). (C) Processing of a modified U3del gene was analyzed in strains deleted for Rsa1, Pih1, and Tah1 (BY4741 background). This U3 gene accumulates precursor species (I and I′), and this defect is more pronounced in mutant strains. (left) Northern blot. (right) Quantification of each species after normalization to levels in wild-type cells. Error bars represent SD.
Figure 5.
Figure 5.
Nufip tethers 15.5K to hPrp31, U3-55K, and fibrillarin. (A) Nufip interacts with hPp31, U3-55K, and fibrillarin in GST pull-down experiments. The indicated proteins were translated in vitro in bacterial S30 lysate and assayed for binding to recombinant GST-Nufip. I, input (10% of total). Arrows indicate the full-length protein. The other bands represent incomplete and read-through translation products that occur in the bacterial lysate. (B) Summary of the interactions obtained between Nufip and U4 or box C/D core proteins. Y2H interactions are indicated by arrows, and physical interactions observed in GST pull-downs are indicated by straight lines. (C) Nufip links 15.5K to fibrillarin, hPrp31, and U3-55K in a bridged Y2H assay. Y2H strains containing the indicated pAS and pACT test plasmids were transformed with an ADE2 plasmid expressing Nufip, a mutant lacking the PEP domain, or no protein. Transformants were plated on triple or quadruple dropout media. Growth on −Leu −Trp −His −Ade (−L −T −H −A) indicates the formation of a complex between the indicated proteins. (D) Nufip tethers hPRP31 and the C-terminal domain of fibrillarin to yU3B/C RNA. Y3H strains deleted for Rsa1 were transformed with the indicated plasmid and an ADE2 vector expressing Nufip, a mutant lacking the PEP domain, or no protein. Growth on quadruple dropout media indicates the formation of a complex between the indicated proteins and yU3B/C RNA.
Figure 6.
Figure 6.
Hsp90 and human homologues of R2TP proteins bind U3, U4, and SBP2. (A) U3. Vectors expressing rU3B.7 were transfected in HeLa cells, extracts were immunoprecipitated with the indicated antibody, and RNAs bound to antibody-coated beads or beads alone as a control were analyzed by RNase protection. Precursors and mature forms of U3 are indicated by a bar and an asterisk, respectively. I, input (3% of total). (B) U4. Legend as in A except that binding was tested against the endogenous U4 RNA. I, input (10% of total). (C) SBP2. 293FT cells were transfected with vectors expressing SBP2 alone or with GFP-hPih1, hRvb1-GFP, or hRvb2-GFP, and extracts were immunoprecipitated with anti-GFP (left) or with anti-Hsp90 antibodies (right). Precipitates were analyzed with anti-SBP2 antibodies. I, input (5% of total); Pt, pellets. (D) hRvb1 is present in immunopurified SBP2 complexes. HeLa nuclear extracts were purified with anti-SBP2 antibodies (Pt) or nonimmune serum (NI) and analyzed by Western blotting with anti-hRvb1 antibodies. I, input (0.5% of total).
Figure 7.
Figure 7.
Inhibition of Hsp90 prevents the production of L7Ae RNPs and destabilizes hNop58, 15.5K, hNHP2, and SBP2. (A) Geldanamycin prevents the accumulation of U4, U3, and telomerase RNAs. HeLa cells were cotransfected with a GFP expression vector and either a tagged U4 gene (U4 tag), a rat U3 gene, or a human telomerase gene and were incubated with 2 μM geldanamycin for 16 h. The RNAs produced were then analyzed by RNase protection (left) or by qPCR (right). The different U3 precursors (U3-0 to U3-III) and the mature U3 (U3-m) are indicated. (bottom) Western blots against GFP as controls. The graph represents the levels of hTR RNA normalized to levels of GAPDH mRNA and expressed as the fraction of untreated cells. Values are the averages of three experiments. The error bar represents SD. (B) Geldanamycin inhibits the accumulation of GFP-15.5K, GFP-hNHP2, GFP-hNop58, and SBP2. 293 cells were cotransfected with vectors expressing the indicated proteins and GFP as a control and were incubated with 2 μM geldanamycin for 16 h. Cell extracts were analyzed by Western blotting with anti-GFP antibodies (left) or with the indicated combination of antibodies. NT, nontransfected cells.
Figure 8.
Figure 8.
hSpagh, hPih1, and Nufip mediate the interaction of Hsp90 with hNop58, 15.5K, hNHP2, and SBP2. (A) Interactions between Rsa1, Nufip, and R2TP proteins. (top) Summary of interactions between yeast (left) and human (right) proteins. Y2H interactions are indicated by green arrows, and physical interactions observed in GST pull-downs are indicated by blue lines. (bottom) GST pull-down experiments. Legend as in Fig. 4. For the interaction between hSpagh and hRvb2, a FLAG-hRvb2 fusion was used. (B) SBP2, hNop56, and hNop58 interact with hPih1 in GST pull-down experiments. Legend as in A except that hNop56 and hNop58 were translated in rabbit reticulocyte lysates (RR). The schematic summarizes the interactions of SBP2 with Nufip and R2TP proteins (legend as in A).
Figure 9.
Figure 9.
Nufip and R2TP proteins are present within large cytoplasmic complexes. (A) Sedimentation profile of Nufip, R2TP proteins, and core snoRNP proteins. S100 cytoplasmic extracts were fractionated on 10–30% glycerol gradients, and 23 fractions were collected. Fractions 7 (top)–16 (bottom) are shown. The boxed fractions contain all of the analyzed proteins. (B) Fibrillarin associates with hRvb1 within large cytoplasmic complexes. Fractions 12–14 were pooled, immunoprecipitated with fibrillarin antibodies, and analyzed by Western blots with anti-hRvb1 antibodies.
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