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• W2015808797 abstract "The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2–7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis. The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2–7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis. Eukaryotic genes are transcribed as long precursors that need to undergo post-transcriptional processing to produce functional, protein-coding mRNAs. The spliceosome, a large dynamic macromolecular assembly of hundreds of proteins and a few RNAs, is responsible for the proper excision of introns and ligation of exons during pre-mRNA splicing (1Jurica M.S. Moore M.J. Mol. Cell. 2003; 12: 5-14Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Spliceosomal small nuclear ribonucleoproteins (snRNPs) 2The abbreviations used are: snRNP, small nuclear ribonucleoprotein; SMN, survival motor neuron; SMA, spinal muscular atrophy; RNAi, RNA interference; siRNA, small interfering RNA; PBS, phosphate buffered saline; GST, glutathione S-transferase. are a class of abundant cellular particles and the essential components of the spliceosome. Major snRNPs are composed of an snRNA molecule (U1, U2, U4/U6, and U5) and a set of common (Sm proteins) and snRNP-specific proteins (2Will C.L. Luhrmann R. Curr. Opin. Cell Biol. 2001; 13: 290-301Crossref PubMed Scopus (552) Google Scholar). The hallmark of snRNPs is the presence of a heptameric ring of Sm proteins, known as the Sm core, around a conserved sequence called the Sm site (3Kambach C. Walke S. Young R. Avis J.M. de la Fortelle E. Raker V.A. Luhrmann R. Li J. Nagai K. Cell. 1999; 96: 375-387Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Although snRNPs function in the nucleus, the biogenesis of spliceosomal snRNPs in higher eukaryotes follows a complex pathway that requires the functions of many cellular proteins and includes nuclear as well as cytoplasmic phases (2Will C.L. Luhrmann R. Curr. Opin. Cell Biol. 2001; 13: 290-301Crossref PubMed Scopus (552) Google Scholar). The survival motor neuron (SMN) protein has emerged in recent years as a key player in the biogenesis of snRNPs (4Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 5Yong J. Wan L. Dreyfuss G. Trends Cell Biol. 2004; 14: 226-232Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Understanding the molecular function(s) of SMN also bears direct relevance to human disease because reduced levels of SMN expression, due to homozygous mutations or deletions of the SMN1 gene, cause the fatal neurodegenerative disease spinal muscular atrophy (SMA) (6Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. Le Paslier D. Frezal J. Cohen D. Weissenbach J. Munnich A. Melki J. Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (3049) Google Scholar). SMN localizes in the cytoplasm, in the nucleoplasm, and is highly concentrated in Gems, nuclear structures that are often associated with Cajal bodies (7Liu Q. Dreyfuss G. EMBO J. 1996; 15: 3555-3565Crossref PubMed Scopus (647) Google Scholar). At least six additional proteins (Gemin2–7) are stably associated with SMN in large macromolecular complexes and display a similar subcellular distribution, including co-localization with SMN in Gems (8Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, 9Charroux B. Pellizzoni L. Perkinson R.A. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 1999; 147: 1181-1194Crossref PubMed Scopus (226) Google Scholar, 10Charroux B. Pellizzoni L. Perkinson R.A. Yong J. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 2000; 148: 1177-1186Crossref PubMed Scopus (204) Google Scholar, 11Gubitz A.K. Mourelatos Z. Abel L. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 5631-5636Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 12Pellizzoni L. Baccon J. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 7540-7545Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Baccon J. Pellizzoni L. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 31957-31962Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Therefore, Gemin2–7 proteins are considered as integral components of the SMN complex. The SMN complex associates transiently with several additional proteins and RNAs and is thought to function in multiple cellular pathways, which are often related to RNA metabolism (4Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 14Paushkin S. Gubitz A.K. Massenet S. Dreyfuss G. Curr. Opin. Cell Biol. 2002; 14: 305-312Crossref PubMed Scopus (287) Google Scholar). The best characterized function of the SMN complex is the assembly of the heptameric core of Sm proteins on spliceosomal snRNAs (4Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 5Yong J. Wan L. Dreyfuss G. Trends Cell Biol. 2004; 14: 226-232Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Newly translated Sm proteins bind first to pICln and the PRMT5 complex, which carries out the symmetrical dimethylation of specific arginine residues of a subset of Sm proteins, and then to the SMN complex (15Friesen W.J. Paushkin S. Wyce A. Massenet S. Pesiridis G.S. Van Duyne G. Rappsilber J. Mann M. Dreyfuss G. Mol. Cell Biol. 2001; 21: 8289-8300Crossref PubMed Scopus (319) Google Scholar, 16Friesen W.J. Wyce A. Paushkin S. Abel L. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 8243-8247Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 17Meister G. Eggert C. Buhler D. Brahms H. Kambach C. Fischer U. Curr. Biol. 2001; 11: 1990-1994Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). The SMN complex bound to unrip and the seven Sm proteins is the macromolecular machine poised for snRNP assembly in the cytoplasm (18Carissimi 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, 19Grimmler M. Otter S. Peter C. Muller F. Chari A. Fischer U. Hum. Mol. Genet. 2005; 14: 3099-3111Crossref PubMed Scopus (66) Google Scholar, 20Meister G. Buhler D. Pillai R. Lottspeich F. Fischer U. Nat. Cell Biol. 2001; 3: 945-949Crossref PubMed Scopus (248) Google Scholar, 21Pellizzoni L. Yong J. Dreyfuss G. Science. 2002; 298: 1775-1779Crossref PubMed Scopus (435) Google Scholar, 22Meister G. Fischer U. EMBO J. 2002; 21: 5853-5863Crossref PubMed Scopus (163) Google Scholar). Through an ordered series of events, this SMN complex interacts directly with newly exported snRNAs, which are transcribed as precursors with short extensions at the 3′-end by RNA polymerase II in the nucleus, and mediates the ATP-dependent assembly of the Sm core (21Pellizzoni L. Yong J. Dreyfuss G. Science. 2002; 298: 1775-1779Crossref PubMed Scopus (435) Google Scholar, 22Meister G. Fischer U. EMBO J. 2002; 21: 5853-5863Crossref PubMed Scopus (163) Google Scholar). Additional cytoplasmic steps of snRNP biogenesis are the hypermethylation of the m7G-cap structure at the 5′-end of snRNAs, trimming of the 3′-end extensions, and binding of nuclear import factors (2Will C.L. Luhrmann R. Curr. Opin. Cell Biol. 2001; 13: 290-301Crossref PubMed Scopus (552) Google Scholar). The SMN complex remains associated with snRNPs until their import back into the nucleus and likely plays additional roles in snRNP biogenesis beyond formation of the Sm core (23Massenet S. Pellizzoni L. Paushkin S. Mattaj I.W. Dreyfuss G. Mol. Cell. Biol. 2002; 22: 6533-6541Crossref PubMed Scopus (102) Google Scholar, 24Narayanan U. Ospina J.K. Frey M.R. Hebert M.D. Matera A.G. Hum. Mol. Genet. 2002; 11: 1785-1795Crossref PubMed Google Scholar). Indeed, the association of the SMN complex with the Sm core is important for nuclear import of snRNPs (25Narayanan U. Achsel T. Luhrmann R. Matera A.G. Mol. Cell. 2004; 16: 223-234Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Once in the nucleus and before functioning in pre-mRNA splicing, snRNPs undergo further steps of maturation, including the association with snRNP-specific proteins as well as methylation and pseudouridylation of specific nucleotides (26Kiss T. J. Cell Sci. 2004; 117: 5949-5951Crossref PubMed Scopus (168) Google Scholar). Several individual components of the SMN complex interact with specific subsets of Sm proteins and collectively contribute to the association of Sm proteins with the SMN complex (4Meister G. Eggert C. Fischer U. Trends Cell Biol. 2002; 12: 472-478Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 14Paushkin S. Gubitz A.K. Massenet S. Dreyfuss G. Curr. Opin. Cell Biol. 2002; 14: 305-312Crossref PubMed Scopus (287) Google Scholar). The SMN complex also interacts directly with specific domains of snRNAs, and this interaction is important for the specificity of snRNP assembly (5Yong J. Wan L. Dreyfuss G. Trends Cell Biol. 2004; 14: 226-232Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 21Pellizzoni L. Yong J. Dreyfuss G. Science. 2002; 298: 1775-1779Crossref PubMed Scopus (435) Google Scholar). This mode of action of the SMN complex applies to the formation of Sm cores on snRNAs of the major (U1, U2, U4, and U5) and of the minor (U11 and U12) spliceosomes as well as on viral small RNAs encoded by the lymphotropic herpesvirus saimiri (21Pellizzoni L. Yong J. Dreyfuss G. Science. 2002; 298: 1775-1779Crossref PubMed Scopus (435) Google Scholar, 27Yong J. Pellizzoni L. Dreyfuss G. EMBO J. 2002; 21: 1188-1196Crossref PubMed Scopus (71) Google Scholar, 28Yong J. Golembe T.J. Battle D.J. Pellizzoni L. Dreyfuss G. Mol. Cell. Biol. 2004; 24: 2747-2756Crossref PubMed Scopus (72) Google Scholar, 29Golembe T.J. Yong J. Battle D.J. Feng W. Wan L. Dreyfuss G. Mol. Cell. Biol. 2005; 25: 602-611Crossref PubMed Scopus (29) Google Scholar). The SMN complex also assembles U7 snRNP, which is required for 3′-end processing of histone pre-mRNAs and whose Sm core contains a unique combination Sm and Lsm proteins (30Pillai 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). Hence, the SMN complex is the general macromolecular assembly machine employed by cells to assemble the Sm core. As part of our efforts to characterize the protein composition of SMN complexes, we have identified by peptide microsequencing by mass spectrometry a novel protein that we term Gemin8. Here we report the characterization of Gemin8 as a novel integral component of the SMN complex that localizes to nuclear Gems, interacts with the Gemin6-Gemin7 heterodimer, and functions in snRNP biogenesis. DNA Constructs—The open reading frame of Gemin8 was generated by PCR amplification from the Ultimate open reading frame clone IOH3877 (Invitrogen) with appropriate primers and was cloned into the EcoRI and XhoI sites of pet28, pGEX-5X, and pcDNA3 vectors downstream of the sequences encoding for the GST, His6, and FLAG epitopes, respectively. For in vitro translation experiments, the Gemin8 cDNA was also cloned without any tags into a pcDNA3 vector. A 6xHis-Gemin8 construct was generated by cloning into a pDEST 17 vector using Gateway technology (Invitrogen). All constructs were analyzed by automated DNA sequencing. Plasmids encoding for epitope-tagged Gemin2, Gemin6, Gemin7, and unrip were described previously (12Pellizzoni L. Baccon J. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 7540-7545Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Baccon J. Pellizzoni L. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 31957-31962Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18Carissimi 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). Antibodies—For production of monoclonal antibodies, BALB/c females were primed with ImmunEasy adjuvant (Qiagen) and 25 μg of either GST-unrip or 6xHis-Gemin8 purified recombinant proteins. Following two boosts at 2-week intervals, SP2 myeloma cells were fused with mouse splenocytes, and hybridoma supernatants were analyzed onto antigen-coated aminosilane-modified slides using an LS400 scanner (Tecan) and the GenePix Pro 4.1 software as described previously (31De Masi F. Chiarella P. Wilhelm H. Massimi M. Bullard B. Ansorge W. Sawyer A. Proteomics. 2005; 5: 4070-4081Crossref PubMed Scopus (69) Google Scholar). The antibodies used are as follows: anti-Gemin8 1F8 (this work); anti-unrip 3G6 (this work); anti-SMN clone 8 (BD Transduction Laboratories); anti-SMN 7F3 (18Carissimi 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); anti-Gemin2 2E17 (8Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar); anti-Gemin3 12H12 (9Charroux B. Pellizzoni L. Perkinson R.A. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 1999; 147: 1181-1194Crossref PubMed Scopus (226) Google Scholar); anti-Gemin4 17D10 (10Charroux B. Pellizzoni L. Perkinson R.A. Yong J. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 2000; 148: 1177-1186Crossref PubMed Scopus (204) Google Scholar); anti-Gemin5 10G11 (11Gubitz A.K. Mourelatos Z. Abel L. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 5631-5636Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar); anti-Gemin6 rG6 (27Yong J. Pellizzoni L. Dreyfuss G. EMBO J. 2002; 21: 1188-1196Crossref PubMed Scopus (71) Google Scholar); anti-hnRNP A1 4B10 (32Pinol-Roma S. Choi Y.D. Matunis M.J. Dreyfuss G. Genes Dev. 1988; 2: 215-227Crossref PubMed Scopus (324) Google Scholar); anti-Sm Y12 (Lab Vision Corp.); anti-pICln clone 32 (BD Transduction Laboratories); anti-coilin clone 56 (BD Transduction Laboratories); anti-H1 and core histones (Chemicon); anti-tubulin (Sigma); anti-FLAG M2 (Sigma); anti-T7 tag (Novagen); and purified mouse IgG immunoglobulins (Sigma). Protein Sequencing by Mass Spectrometry—Bands were excised from silver-stained polyacrylamide gels and in-gel digested with trypsin. Tryptic peptides were analyzed by nano-electrospray tandem mass spectrometry as described previously (33Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar). Cell Culture—HeLa and 293T cells were cultured in Dulbecco's modified Eagle's medium with high glucose (BioWhittaker) containing 10% fetal bovine serum and penicillin/streptomycin. 293T cells were transfected using the calcium phosphate method and manufacturer's instructions (Clontech). Following overnight incubation with DNA, medium was replaced, and cells were harvested after 1 additional day in culture. Immunofluorescence Analysis—HeLa PV cells grown on 35-mm tissue culture plates were washed briefly with phosphate-buffered saline, fixed in 50% methanol, 50% acetone for 5 min at -20 °C, and air-dried for 30 min. Cells were then blocked with 3% bovine serum albumin in phosphate-buffered saline for 1 h at room temperature. Single- or double-label immunofluorescence experiments were carried out by separate sequential incubations of each primary antibody followed by the specific fluorescently labeled secondary antibody as described previously (12Pellizzoni L. Baccon J. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 7540-7545Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Indirect epifluorescence microscopy was performed with an Olympus AX70 microscope equipped with a SPOT digital camera (Diagnostic Instruments). Protein Production and in Vitro Binding Experiments—In vitro translated proteins were produced in the presence of [35S]methionine (Amersham Biosciences) using a coupled transcription-translation system according to the manufacturer's protocol (Promega). All His6- or GST-tagged recombinant proteins were expressed in Escherichia coli BL21(DE)pLysE cells (Invitrogen) and purified by affinity chromatography on nickel-chelated agarose (Pierce) or glutathione-Sepharose (Amersham Biosciences), respectively. GST-TEV-Gemin6 and 6xHis-Gemin7 (or GST-TEV-Gemin7 and 6xHis-Gemin6) proteins were co-expressed and purified as described previously (13Baccon J. Pellizzoni L. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 31957-31962Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 34Ma Y. Dostie J. Dreyfuss G. Van Duyne G.D. Structure (Camb.). 2005; 13: 883-892Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Gemin6-Gemin7 heterodimers were purified by a tandem affinity purification strategy. Briefly, following cleavage of GST-TEV-Gemin6/6xHis-Gemin7 complexes bound to glutathione-Sepharose beads with recombinant AcTEV protease (Invitrogen) according to the manufacturer's instructions, Gemin6/6xHis-Gemin7 heterodimers were further purified by affinity chromatography on nickel-chelated agarose (Pierce). For in vitro binding experiments, 2 μg of GST or GST-tagged proteins bound to glutathione-Sepharose beads were incubated either with in vitro translated [35S]methionine-labeled proteins or with 1 μg of purified recombinant proteins in binding buffer (200 mm NaCl, 50 mm Tris-HCl, pH 7.4, 2 mm EDTA) containing 0.1% Nonidet P-40 and EDTA-free protease inhibitor mixture (Roche Applied Science) for 2 h at 4°C. Following extensive washing with the same buffer, bound proteins were eluted by boiling in SDS-PAGE sample buffer and were analyzed by SDS-PAGE on 12% polyacrylamide gels. Extract Preparation, Sucrose Gradient Centrifugation, and Immunoprecipitation Experiments—Mouse tissues from C57BL/6 embryos at stage 18 were collected by manual dissection, quickly frozen in liquid nitrogen, and stored at -80 °C until further processing. Whole tissue extracts for Western blot analysis were prepared by homogenization of tissues in SDS-PAGE sample buffer, followed by sonication, boiling, and centrifugation for 15 min at 10,000 rpm at room temperature. For total cell extract preparation, HeLa or 293T cells were resuspended in RSB-100 buffer (100 mm NaCl, 10 mm Tris-HCl, pH 7.4, 2.5 mm MgCl2) containing 0.1% Nonidet P-40, EDTA-free protease inhibitor mixture (Roche Applied Science), and phosphatase inhibitors (20 mm NaF, 0.2 mm Na3VO4), briefly sonicated three times on ice, and centrifuged for 15 min at 10,000 rpm at 4 °C. For sucrose gradient centrifugation experiments, HeLa total cell extracts were fractionated on 10 ml of 10–30% sucrose gradients in RSB-100 buffer by centrifugation for 4 h at 38,000 rpm in an SW 41 rotor at 4 °C. Immunoprecipitation experiments were carried out from HeLa or 293T total cell extracts using either antibodies bound to protein G-Sepharose (Sigma) or FLAG M2-agarose (Sigma) in RSB-100 buffer containing 0.1% Nonidet P-40 for 2 h at 4°C. Following five washes with the same buffer, bound proteins were eluted by boiling in SDS-PAGE sample buffer or analyzed by snRNP assembly. Immunoprecipitations of snRNP assembly reactions with anti-Sm (Y12) antibodies bound to protein G-Sepharose were carried out for 2 h at 4°Cin RSB-500 buffer (500 mm NaCl, 10 mm Tris-HCl, pH 7.4, 2.5 mm MgCl2) containing 0.1% Nonidet P-40 and protease inhibitors. Following five washes with the same buffer, bound RNAs were recovered from immunoprecipitates by proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation. RNAs were then analyzed by electrophoresis on denaturing polyacrylamide gels and autoradiography. Purification of SMN Complexes—Parental HeLa Tet-On cells (Clontech) constitutively expressing a tetracycline-regulated transcriptional transactivator and the HeLa Tet-On stable cell line containing FLAG-Gemin2 under the control of a tetracycline-inducible promoter were grown in the presence of doxycycline (5 μg/ml) to induce FLAG-Gemin2 expression (12Pellizzoni L. Baccon J. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 7540-7545Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Native SMN complexes were affinity-purified using the high salt-wash procedure established previously and described below (12Pellizzoni L. Baccon J. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 7540-7545Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Cells were resuspended in ice-cold RSB-100 buffer containing 0.1% Nonidet P-40 and protease and phosphatase inhibitors, passed five times through a 27-gauge needle, and sonicated briefly. After centrifugation for 15 min at 10,000 rpm at 4 °C, extracts were passed through a 0.2-μm filter and incubated with anti-FLAG M2-agarose beads (Sigma) for 2 h at 4°C. Beads were first extensively washed with RSB-100 containing 0.1% Nonidet P-40 and then three times with 10 bead volumes of RSB-500 containing 0.02% Nonidet P-40 for 15 min at 4 °C. Native SMN complexes were eluted under mild conditions by competitive displacement of FLAG-Gemin2 with 0.5 mg/ml of 3X-FLAG peptide (Sigma) in RSB-100 buffer containing 0.02% Nonidet P-40 for 1 h at 4 °C. RNA Interference—For silencing experiments, HeLa cells were transfected with 21-nucleotide-long small interfering RNA duplexes (siRNAs) using Lipofectamine 2000™ (Invitrogen) according to the manufacturer's instructions and harvested 72 h after transfection. Preliminary time course analysis showed that the greatest reduction in the levels of Gemin8 is observed at 72 h after transfection, and this time point was consequently used in the experiments (data not shown). Furthermore, indirect immunofluorescence experiments indicated that over 90% of cells were transfected with siRNAs and displayed reduced expression of the target proteins (data not shown). The following siRNAs (sense strand), selected using the BLOCK-iT™ RNAi Designer, were synthesized, purified, and annealed with the corresponding antisense RNA oligonucleotides by Invitrogen: Gemin8a (5′-GCAAGAUACUGGCAACAUUdTdT-3′) and Gemin8b (5′-GCAAUGGCUUGGAUGCAAAdTdT-3′) against Gemin8; (5′-GAAGAAUACUGCAGCUUCCdTdT-3′) against SMN (35Feng W. Gubitz A.K. Wan L. Battle D.J. Dostie J. Golembe T.J. Dreyfuss G. Hum. Mol. Genet. 2005; 14: 1605-1611Crossref PubMed Scopus (84) Google Scholar); and (5′-GCUGGAGAGCAACUGCAUAdTdT-3′) against firefly luciferase as a control. In Vitro snRNP Assembly—U1 and U1ΔSm snRNAs were transcribed in vitro from linearized template DNAs in the presence of [α-32P]UTP (3000 Ci/mmol) and purified from denaturing polyacrylamide gels according to standard procedures. Extracts for snRNP assembly were prepared as described previously with minor modifications (21Pellizzoni L. Yong J. Dreyfuss G. Science. 2002; 298: 1775-1779Crossref PubMed Scopus (435) Google Scholar). HeLa cells were resuspended in ice-cold reconstitution buffer (20 mm Hepes-KOH, pH 7.9, 50 mm KCl, 5 mm MgCl2, 0.2 mm EDTA, 5% glycerol) containing 0.01% Nonidet P-40, passed five times through a 25-gauge needle, and then centrifuged at 10,000 rpm for 15 min at 4 °C. Supernatants were collected and either used directly or stored in frozen aliquots at -80 °C. For snRNP assembly experiments, beads-bound protein complexes were immunopurified from 293T total cell extracts using FLAG M2-agarose (Sigma) as described above and washed for five additional times with ice-cold reconstitution buffer containing 0.01% Nonidet P-40. snRNP assembly reactions with HeLa cell extracts or beads-bound protein complexes were carried out for 1 h at 30°C in a volume of 20 μl of reconstitution buffer containing 0.01% Nonidet P-40, 10,000 cpm of in vitro transcribed [α-32P]UTP-labeled U1 snRNA, 2.5 mm ATP, and 10 μm E. coli tRNA. Reactions were then analyzed by immunoprecipitation experiments or by electrophoresis on 6% polyacrylamide native gels at 4 °C and autoradiography as described previously (18Carissimi 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). Identification of Gemin8 by Mass Spectrometry—We purified native SMN complexes from a stable HeLa cell line that expresses FLAG-tagged Gemin2 using affinity chromatography on anti-FLAG-agarose beads and the high salt-wash procedure established previously (12Pellizzoni L. Baccon J. Rappsilber J. Mann M. Dreyfuss G. J. Biol. Chem. 2002; 277: 7540-7545Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). A parallel purification was carried out from the parental HeLa cell line as a control. Bound proteins were eluted from beads by competition with a molar excess of FLAG peptides and analyzed by SDS-PAGE and silver staining (Fig. 1). In addition to the known integral components of the SMN complex, peptide microsequencing by mass spectrometry revealed three distinct peptides whose amino acid sequences perfectly match several expressed sequences in the data base and belong to a novel 32-kDa protein, which migrates close to Gemin2 and which we term Gemin8 (Fig. 1 and supplemental Fig. S1). Gemin8 cDNA contains both start and stop codons as well as an in-frame stop codon upstream of the initial methionine, indicating that the open reading frame is complete (supplemental Fig. S1). Gemin8 cDNA encodes for a protein of 242 amino acids with a predicted molecular mass of 28.6 kDa and an isoelectric point of 6.8. Protein analyses in silico using various web-based search engines indicated that Gemin8 neither contains identifiable protein motifs nor shares significant homology with other proteins that may hint to a possible function. We then performed BLAST search of the data base and identified several putative orthologues of human Gemin8 in diverse organisms. Fig. 2 shows an alignment of the amino acid sequences of Gemin8 from Homo sapiens, Canis familiaris (73.2% identity, 79.7% similarity), Rattus norvegicus (66.5% identity, 74.2% similarity), Mus musculus (66% identity, 72.9% similarity), Bos taurus (58.9% identity, 62.1% similarity), Xenopus laevis (50% identity, 54.5% similarity), Xenopus tropicalis (47.6% identity, 51.7% similarity), Danio rerio (45.5% identity, 54.1% similarity), and Caenorhabditis elegans (26.6% identity, 30% similarity). The high degree of evolutionary conservation suggests that Gemin8 may perform important cellular functions. However, we did not identify Gemin8 orthologues in organisms such as Drosophila melanogaster or Schizosaccharomyces pombe that contain SMN and whose genomes have been entirely sequenced. This situation is also observed for several other Gemin proteins and may indicate either lack of Gemin8 in these organisms or, as is the case for Gemin2 (36Hannus S. Buhler D. Romano M" @default.
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• W2015808797 title "Gemin8 Is a Novel Component of the Survival Motor Neuron Complex and Functions in Small Nuclear Ribonucleoprotein Assembly" @default.
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