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

Boulon S, Marmier-Gourrier N, Pradet-Balade B, Wurth L, Verheggen C, Jády BE, Rothé B, Pescia C, Robert MC, Kiss T, Bardoni B, Krol A, Branlant C, Allmang C, Bertrand E, Charpentier B - J. Cell Biol. (2008)

Bottom Line: 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.This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics of Montpellier, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Montpellier Cedex 5, France.

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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Related in: MedlinePlus

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.
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fig3: 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.

Mentions: Rsa1 was initially found in a Y3H screen with an RNA bait that bound Snu13, suggesting that it interacted with an Snu13–RNA complex. To verify this, we reconstituted the complex in vitro using gel-shift assays (Fig. 3 A, left). Snu13 alone could bind U14 snoRNA, whereas two His-tagged fragments of Rsa1 (N3C1 and yPEP) did not. However, when Snu13 was added, complexes of higher molecular weight were obtained with both Rsa1 fragments. These complexes were supershifted by anti-His antibodies (Fig. 3 A), demonstrating that they contained N3C1 and yPEP. To check whether Nufip could also associate with Snu13–RNA complexes, we used Y3H assays (Fig. 3 A, right). As expected, we found that Nufip could specifically interact with the B/C motif in a PEP-dependent manner. These results demonstrate that Nufip and Rsa1 can form ternary complexes with Snu13 bound to RNA.


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

Boulon S, Marmier-Gourrier N, Pradet-Balade B, Wurth L, Verheggen C, Jády BE, Rothé B, Pescia C, Robert MC, Kiss T, Bardoni B, Krol A, Branlant C, Allmang C, Bertrand E, Charpentier B - J. Cell Biol. (2008)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: Rsa1 was initially found in a Y3H screen with an RNA bait that bound Snu13, suggesting that it interacted with an Snu13–RNA complex. To verify this, we reconstituted the complex in vitro using gel-shift assays (Fig. 3 A, left). Snu13 alone could bind U14 snoRNA, whereas two His-tagged fragments of Rsa1 (N3C1 and yPEP) did not. However, when Snu13 was added, complexes of higher molecular weight were obtained with both Rsa1 fragments. These complexes were supershifted by anti-His antibodies (Fig. 3 A), demonstrating that they contained N3C1 and yPEP. To check whether Nufip could also associate with Snu13–RNA complexes, we used Y3H assays (Fig. 3 A, right). As expected, we found that Nufip could specifically interact with the B/C motif in a PEP-dependent manner. These results demonstrate that Nufip and Rsa1 can form ternary complexes with Snu13 bound to RNA.

Bottom Line: 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.This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics of Montpellier, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Montpellier Cedex 5, France.

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.

Show MeSH
Related in: MedlinePlus