2006/12/19 by Simon Otter, Matthias Grimmler, Nils Neuenkirchen +3 · 26 citations
Medicine · Biochemistry, Genetics and Molecular Biology · #Neurogenetic and Muscular Disorders Research #RNA modifications and cancer #RNA Research and Splicing
paper · pdf · doi:10.1074/jbc.m608528200
Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs. Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs. Several nuclear RNA-protein complexes (RNPs) 3The abbreviations used are: RNP, ribonucleoprotein; U snRNP, uridine-rich small ribonucleoprotein; snRNA, small nuclear ribonucleic acid; SMN, survival of motor neurons; Gemins 2-8, components of gems number 2-8, respectively; GST, glutathione S-transferase; HA, hemagglutinin; siRNA, short interfering RNA; RNAi, RNA interference; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. involved in the processing of mRNAs, such as the snRNPs of the major (U1, U2, U4/6, and U5) and minor (U11, U12, U5, and U4/6atac) spliceosome and the histone-mRNA processing U7 snRNP contain a set of evolutionary conserved proteins of the Sm/LSm class (1Seraphin B. EMBO J. 1995; 14: 2089-2098Crossref PubMed Scopus (248) Google Scholar, 2Hermann H. Fabrizio P. Raker V.A. Foulaki K. Hornig H. Brahms H. Luhrmann R. EMBO J. 1995; 14: 2076-2088Crossref PubMed Scopus (215) Google Scholar). This group of proteins has the propensity to form heptameric rings in the presence of their respective target snRNA. Sm and LSm/Sm rings (also called “cores”) can form spontaneously in vitro on their target RNAs (3Raker V.A. Plessel G. Luhrmann R. EMBO J. 1996; 15: 2256-2269Crossref PubMed Scopus (195) Google Scholar, 4Raker V.A. Hartmuth K. Kastner B. Luhrmann R. Mol. Cell. Biol. 1999; 19: 6554-6565Crossref PubMed Scopus (112) Google Scholar, 5Achsel T. Stark H. Luhrmann R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3685-3689Crossref PubMed Scopus (107) Google Scholar). However, assembly in vivo occurs in a highly regulated manner and is assisted by trans-acting factors. One well characterized entity in this pathway is the SMN complex, whose name-giving component SMN is the product of the spinal muscular atrophy (SMA) disease gene (6Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (3049) Google Scholar, 7Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (875) Google Scholar). This entity recruits all Sm proteins and promotes their transfer onto the U snRNAs (8Meister G. Buhler D. Pillai R. Lottspeich F. Fischer U. Nat. Cell Biol. 2001; 3: 945-949Crossref PubMed Scopus (248) Google Scholar, 9Meister G. Fischer U. EMBO J. 2002; 21: 5853-5863Crossref PubMed Scopus (163) Google Scholar, 10Pellizzoni L. Yong J. Dreyfuss G. Science. 2002; 298: 1775-1779Crossref PubMed Scopus (435) Google Scholar). Likewise, assembly of the U7 snRNP is facilitated by a specialized SMN complex that is charged with the unique set of Sm and LSm proteins of this particle (11Pillai R.S. Grimmler M. Meister G. Will C.L. Luhrmann R. Fischer U. Schumperli D. Genes Dev. 2003; 17: 2321-2333Crossref PubMed Scopus (172) Google Scholar). With a sedimentation coefficient of 25-40 S and an estimated molecular mass exceeding 1 megadalton, the SMN complex represents a macromolecular machine of great complexity. So far, nine major proteins termed SMN, Gemins 2-8, and unrip as well as nine Sm/LSm protein “substrates” (i.e. B/B′, D1, D2, D3, E, F, G, LSm10, and LSm11) have been identified as components of this assembly machinery (12Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Gubitz A.K. Feng W. Dreyfuss G. Exp. Cell Res. 2004; 296: 51-56Crossref PubMed Scopus (214) Google Scholar). Our knowledge about the architecture of the core SMN complex (i.e. the SMN complex without substrate proteins) is still limited and relies mostly on in vitro interaction assays using recombinant proteins. These studies have placed the SMN protein in the center with interactions to Gemins 2, 3, 5, and 7, as well as substrate Sm proteins. In contrast, other factors such as Gemins 2, 4-6, and 8 and unrip appeared to be peripheral and interact only with few other components of the complex (see Refs. 12Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar and 13Gubitz A.K. Feng W. Dreyfuss G. Exp. Cell Res. 2004; 296: 51-56Crossref PubMed Scopus (214) Google Scholar for reviews; Refs. 14Carissimi C. Baccon J. Straccia M. Chiarella P. Maiolica A. Sawyer A. Rappsilber J. Pellizzoni L. FEBS Lett. 2005; 579: 2348-2354Crossref PubMed Scopus (64) Google Scholar, 15Grimmler M. Otter S. Peter C. Muller F. Chari A. Fischer U. Hum. Mol. Genet. 2005; 14: 3099-3111Crossref PubMed Scopus (66) Google Scholar, 16Carissimi C. Saieva L. Baccon J. Chiarella P. Maiolica A. Sawyer A. Rappsilber J. Pellizzoni L. J. Biol. Chem. 2006; 281: 8126-8134Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The relevance of these interactions in the context of the native SMN complex remained unclear from these studies. To address this issue, we applied a combination of in vitro and in vivo approaches (in vitro binding assays, co-immunoprecipitations and a yeast two-hybrid interaction system) to establish a comprehensive interaction map of the SMN complex. Furthermore, by use of a novel assay we were able to reconstitute the SMN complex in reticulocyte lysate. Interestingly, complex formation is severely compromised by an SMA-causing mutation in the SMN protein. Finally, we have assessed the contribution of the peripheral component Gemin5 to the activity of the SMN complex. DNA Constructs—Plasmids encoding full-length cDNAs corresponding to the open reading frames of SMN, unrip, and Gemins 2-7 have been described previously (15Grimmler M. Otter S. Peter C. Muller F. Chari A. Fischer U. Hum. Mol. Genet. 2005; 14: 3099-3111Crossref PubMed Scopus (66) Google Scholar). The full-length open reading frame of Gemin8 was purchased from RZPD (Clone IRAUp969F1069D6) and subcloned into the vectors pGEX6P-1 (GE Healthcare), pET28a (Novagen), and pHA (an N-terminal HA-tag containing derivative of pcDNA3.1; Invitrogen). Recombinant Proteins and in Vitro Protein Binding Assays—Expression and purification of single proteins or protein complexes were performed as described (15Grimmler M. Otter S. Peter C. Muller F. Chari A. Fischer U. Hum. Mol. Genet. 2005; 14: 3099-3111Crossref PubMed Scopus (66) Google Scholar). [35S]Methionine-labeled proteins were produced using the TNT-T7 quick coupled transcription/translation system (Promega). In vitro co-translations of the entire SMN complex were carried out in one single TNT reaction with [35S]methionine, using a mixture of vectors encoding for the different SMN complex components. In the case of GST binding assays, in vitro translated proteins were incubated with ∼2 μg of purified GST fusion proteins, immobilized on glutathione-Sepharose (GE Healthcare), and allowed to bind in lysis buffer (50 mm Tris/HCl, pH 7.5, 200 mm NaCl, 0.01% Igepal, 1 mm dithiothreitol, 5 mm EDTA, 5 mm EGTA, 1 μg/ml bovine serum albumin) at 4 °C for 1 h. After washing the resin extensively, bound proteins were eluted by boiling in 2× SDS sample buffer, resolved by SDS-PAGE, and analyzed by Coomassie staining and autoradiography of the dried gel. Preparation of HeLa Cell Extract, Antibodies, and was as described M. L. M. R. Meister G. Fischer U. EMBO 2005; PubMed Scopus Google Scholar). Gemins were by of full-length proteins into were purified on with the respective and were purchased from of the SMN complex were carried out in HeLa using a SMN (8Meister G. Buhler D. Pillai R. Lottspeich F. Fischer U. Nat. Cell Biol. 2001; 3: 945-949Crossref PubMed Scopus (248) Google Scholar). SMN complex was with buffer (50 mm Tris/HCl, pH 7.5, 1 mm EDTA, 0.01% of 1 SMN complexes were eluted by 2× SDS buffer and resolved on of SMN complexes with an immobilized complex was with buffer and incubated with recombinant purified as described C. S. R. Raker V.A. Luhrmann R. J. K. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). After complexes were eluted by 2× SDS buffer and resolved in RNA were by of HeLa with and were purchased from and with the of the of was assessed by of using a from these were performed as described using for SMN by with of Gemin5 and in Vitro Assembly of U Gemin5 protein was using the system Nat. 2004; PubMed Scopus Google Scholar). were 5 by and with mm 200 mm NaCl, pH The was to and eluted with mm were and 0.01% Igepal, and 1 mm To the of Gemin5 in U snRNP an SMN complex in the presence and of recombinant was incubated with μg of recombinant Gemin5 in an assembly assay using snRNA (15Grimmler M. Otter S. Peter C. Muller F. Chari A. Fischer U. Hum. Mol. Genet. 2005; 14: 3099-3111Crossref PubMed Scopus (66) Google Scholar). Sm core formation was assessed by native of buffer containing 4 and as described (8Meister G. Buhler D. Pillai R. Lottspeich F. Fischer U. Nat. Cell Biol. 2001; 3: 945-949Crossref PubMed Scopus (248) Google Scholar). of the in Vitro SMN vitro SMN complexes were with to a of on a and for in an were in from to yeast and the yeast two-hybrid interaction assay were carried out as described J. H. R. Cell. Full Text PDF PubMed Scopus Google Scholar, J. R. in 1999; 4 Scholar). In to all interactions the SMN complex, the complex components (i.e. SMN, Gemins 2-8, and were subcloned into of fusion proteins and into the to proteins were in the were by staining of on containing were on as a of Recombinant SMN in a of we used recombinant proteins to the interactions with in vitro translated components of the SMN complex, produced in reticulocyte In only SMN and Gemins and be produced in as full-length proteins, Gemins and unrip were binding of SMN, Gemins 2, 3, and 8 to immobilized was of translated protein was to GST be a complex of and was used as that to bound SMN only other protein of the SMN complex in this assay we analyzed and 7, have been to form a A.K. Feng W. Dreyfuss G. Exp. Cell Res. 2004; 296: 51-56Crossref PubMed Scopus (214) Google Scholar). In with we binding of translated Gemin7 to and and to bind to other binding of Gemin7 to Gemins 2, and 8 and unrip, and binding to SMN be In the assay, bound to SMN as well as In this that Gemin8 the proteins Gemins and and unrip with the of the SMN complex (see 1 for binding assays that this The data from this set of are in of Gemins 3, and proteins of the SMN complex, Gemins 3, and 5 be produced in as full-length proteins in a manner and in to the described the interactions of these proteins were analyzed by of and components. In a set of the interactions of were in was with single proteins of the SMN complex, with an Gemins 2, and and or a The co-translations were via the respective and analyzed by autoradiography (see for this assay, we of with SMN, Gemins 2, and 5 other component of the SMN complex was in this and 2, 3, and interactions. binding of with proteins of the SMN complex was by the (see for and to a Gemin5 be with and with and Finally, Gemin5 was analyzed binding was to and and the other components to interact in the assay and To the interaction and of were by in HeLa in of different to the a protein SMN and Gemin7 were Gemin5 was 1 with and from these were with an and components by Gemins 2, 5, and be with SMN from from the of SMN with and Gemin5 was in the at Gemin7 was 1 with and These data that Gemin5 is to the SMN complex via an interaction with in This was by and studies with of Gemin5 These the and the of Gemin5 as the binding for of Protein the SMN by a described interactions of individual SMN complex components to other in set to interactions the SMN complex in vivo using a yeast two-hybrid assay J. H. R. Cell. Full Text PDF PubMed Scopus Google Scholar, J. R. in 1999; 4 Scholar). this a of were encoding for of the of the and SMN, Gemins 2-8, or unrip the without a fusion These were with encoding for of the and the proteins as in the vectors the Binding of to fusion proteins was assessed in a assay upon of fusion of SMN from the are in In with data and in 1 and 2, SMN with and Gemin8 and these interactions can be SMN is as protein (see and The activity in yeast with SMN and most likely a as the fusion protein is and data In the we binding of with and Gemin7 with Gemins and Furthermore, we novel protein of Gemins and as well as Gemin5 that interactions such as SMN and Gemins and and SMN can be to this most data in vitro in However, binding of unrip that be in vitro were identified in the yeast system to in this the of the SMN studies described on interactions of components. an to address the interactions in the context of the SMN complex and to its To this all proteins of the complex, including SMN were translated and in reticulocyte lysate. by the proteins were to and this to an of Gemins and proteins were from a mixture SMN 8 and the are in 1 and Gemin5 and unrip are into the complex and a of the SMN complex all core components are This is by studies in SMN complex components to the complex and data the formation of a In contrast, SMN is from the reaction the sedimentation of all Gemins is in a molecular 5, and that or single translated proteins can be in 5, and were performed in the of single components. with the in vitro binding assays all other complex components still be with was from the is a peripheral protein that is to the complex via In contrast, in the of to the formation of a complex that of on the of the other proteins into the complex These data are with the that and 4 interact and with the SMN complex via G. Buhler D. B. M. Lottspeich F. Fischer U. Hum. Mol. Genet. PubMed Scopus Google Scholar, B. Pellizzoni L. Yong J. A. M. Dreyfuss G. J. Cell Biol. PubMed Scopus Google Scholar). however, a binding of to the SMN complex in the of and data This that with protein, Gemin8 as has been in the in vitro binding and in the yeast interaction assay and in the of Gemin7 binding of and unrip and of Gemin8 to the of Gemins and and unrip to the complex These data in with in the that Gemin8 the the and the of the SMN complex. These are in of the in vitro translated SMN complexes by SMN complex components were and as fusion proteins with or without SMN in the TNT reticulocyte The were on were from to in sample of was analyzed by and autoradiography of the dried is The is by of or of SMN protein. was to in SMN with its into the complex. To address we the well characterized mutation that has previously been to and Sm protein binding S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (875) Google Scholar, C.L. J. B. T. Nat. Genet. 19: PubMed Scopus Google Scholar, L. B. Dreyfuss G. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). the was with all SMN complex components only be binding to all other components was severely impaired or formation of the SMN complex be a in carrying this and other for Gemin5 in the Assembly assembly map Gemin5 in the of the complex with an interaction to Gemin5 to be for the of the SMN complex, we that have a in the U snRNP assembly To this we a to SMN complexes This was by of immobilized purified SMN complex with of most components of the complex to D3, and Gemin5 at mm and NaCl, (see for a of the purified complex, for of the with and SMN complexes were for their to formation of the Sm core on snRNA. formation was assessed by native The native SMN complex the formation of the Sm core as by the of the snRNA in the native and upon of the and Interestingly, the complex B/B′, and the formation of an that be with and This most likely to the particle that has been previously to form in vitro Sm proteins D1, D2, E, F, and and are incubated with snRNA (3Raker V.A. Plessel G. Luhrmann R. EMBO J. 1996; 15: 2256-2269Crossref PubMed Scopus (195) Google Scholar). we Sm core formation be upon of the Sm proteins and a recombinant was bound by the complex by Sm core formation be that the of snRNA in the native be upon of and was by Sm proteins and by an an SMN complex containing the set of Sm proteins Gemin5 can formation Sm core To address the the of this SMN complex be upon of this protein was in and purified to of recombinant Gemin5 to the assembly mixture the assembly as by the formation of a complex that be with and In contrast, upon of a of the of the Gemin5 protein, the SMN complex was and E, RNA In these data are with the that Gemin5 an essential in the assembly of U most likely in the transfer of Sm proteins onto the U snRNA. The of the SMN complex relies on the of the nine components SMN, Gemins 2-8, and studies have a great number of interactions these proteins using in vitro and in vivo assays (12Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Gubitz A.K. Feng W. Dreyfuss G. Exp. Cell Res. 2004; 296: 51-56Crossref PubMed Scopus (214) Google Scholar, 14Carissimi C. Baccon J. Straccia M. Chiarella P. Maiolica A. Sawyer A. Rappsilber J. Pellizzoni L. FEBS Lett. 2005; 579: 2348-2354Crossref PubMed Scopus (64) Google Scholar, 15Grimmler M. Otter S. Peter C. Muller F. Chari A. Fischer U. Hum. Mol. Genet. 2005; 14: 3099-3111Crossref PubMed Scopus (66) Google Scholar, 16Carissimi C. Saieva L. Baccon J. Chiarella P. Maiolica A. Sawyer A. Rappsilber J. Pellizzoni L. J. Biol. Chem. 2006; 281: 8126-8134Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). However, as most interaction assays are is that data the interactions the core SMN complex have been To this we have an interaction map that relies on different and interaction protein that be in at were in the of an interaction Our studies have a of the SMN complex with SMN, and Gemin8 as its These three proteins provide a binding for the other components of the complex via multiple SMN to Q. Fischer U. F. Dreyfuss G. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google B. Pellizzoni L. A. M. Dreyfuss G. J. Cell Biol. 1999; PubMed Scopus Google Scholar, L. P. Hum. Mol. Genet. PubMed Scopus Google and Gemin8, in with Gemins 4 and Finally, Gemin7 recruits unrip and via interactions C. Baccon J. Straccia M. Chiarella P. Maiolica A. Sawyer A. Rappsilber J. Pellizzoni L. FEBS Lett. 2005; 579: 2348-2354Crossref PubMed Scopus (64) Google Scholar, 15Grimmler M. Otter S. Peter C. Muller F. Chari A. Fischer U. Hum. Mol. Genet. 2005; 14: 3099-3111Crossref PubMed Scopus (66) Google Scholar, J. Pellizzoni L. Rappsilber J. M. Dreyfuss G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). a interaction map from data in Refs. 12Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar and 13Gubitz A.K. Feng W. Dreyfuss G. Exp. Cell Res. 2004; 296: 51-56Crossref PubMed Scopus (214) Google and from in this most components of the SMN complex form a the peripheral protein Gemin5 can be upon with This its into the complex via interactions by and on data presented here and in we are that we the architecture of the SMN complex. we the that have or were based on the described In with the of the detailed we are in a to map the binding of other peripheral components of the SMN complex. These substrate Sm/LSm proteins (12Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Gubitz A.K. Feng W. Dreyfuss G. Exp. Cell Res. 2004; 296: 51-56Crossref PubMed Scopus (214) Google the nuclear factors and U. Hum. Mol. Genet. 2002; PubMed Google Scholar, U. T. Luhrmann R. Mol. Cell. 2004; 16: Full Text Full Text PDF PubMed Scopus Google and a binding of SMN in nuclear Genes Dev. 2001; 15: PubMed Scopus Google Scholar). These studies provide mechanistic into the U snRNP of are by the of SMN protein. However, of disease are of the protein. The of and in the for SMN interaction with Sm proteins S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (875) Google Scholar, C.L. J. B. T. Nat. Genet. 19: PubMed Scopus Google Scholar, L. B. Dreyfuss G. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). Here, we provide that the SMN mutation is into the SMN complex. This be to the of SMN to form the to bind other proteins such as Gemin8, of SMN, the mutation is (see and studies based on the novel assay here be to the other SMN in the formation of SMN complexes and this is the for the of the disease in data the of Gemin5 have been Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, W. A.K. L. J. Dreyfuss G. Hum. Mol. Genet. 2005; 14: PubMed Scopus Google Scholar, L. H. F. Dreyfuss G. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In RNA was used to show that Gemin5 is for the assembly reaction Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, W. A.K. L. J. Dreyfuss G. Hum. Mol. Genet. 2005; 14: PubMed Scopus Google Scholar). In contrast, for an essential of Gemin5 in the formation of U snRNPs has been in L. H. F. Dreyfuss G. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In this Gemin5 was to bind snRNAs in a its in the of these to the SMN complex. Our that an SMN complex Gemin5 to U snRNP assembly is with this However, we out of Gemin5 in the assembly reaction such as transfer of Sm proteins onto the U snRNA. The of Gemin5 in to address these are to T. J. L. G. and F. for and to A. and for and with