A Cell System with Targeted Disruption of the SMNGene
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m009162200
ISSN1083-351X
Autores Tópico(s)Congenital Anomalies and Fetal Surgery
ResumoThe motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenousSMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.AF322650 The motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenousSMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.AF322650 spinal muscular atrophy survival motor neuron small nuclear ribonucleoprotein particle tetracycline SDS-polyacrylamide gel electrophoresis chicken SMN human SMN wild-type propidium iodide Spinal muscular atrophy (SMA),1 an autosomal recessive disease with characteristics of motor neuron degeneration and muscle atrophy, is a common childhood genetic disorder and the most frequent genetic cause of infant mortality (1Roberts D.F. Chavez J. Court S.D. Arch. Dis. Child. 1970; 45: 33-38Crossref PubMed Scopus (137) Google Scholar, 2Pearn J. Lancet. 1980; 1: 919-922Abstract PubMed Scopus (359) Google Scholar, 3Czeizel A. Hamula J. J. Med. Genet. 1989; 26: 761-763Crossref PubMed Scopus (72) Google Scholar). Based on the age of onset and the severity of the disease, SMA is clinically classified as the severe type I (Werdnig-Hoffman disease), the moderate type II, and the mild type III (Kugelberg-Welander disease). The survival motor neuron (SMN) gene has been established as the disease gene of SMA. The human genome contains two copies of the SMN gene because of an inverted duplication at 5q13. This phenomenon appears to be human-specific, because all other organisms examined to date have a single copy of SMN. Deletions or mutations of the telomericSMN1 gene, which result in reduced SMN protein level, have been found in the vast majority of SMA patients (4Cobben J.M. van der Steege G. Grootscholten P. de Visser M. Scheffer H. Buys C.H. Am. J. Hum. Genet. 1995; 57: 805-808PubMed Google Scholar, 5Bussaglia E. Clermont O. Tizzano E. Lefebvre S. Burglen L. Cruaud C. Urtizberea J.A. Colomer J. Munnich A. Baiget M. et al.Nat. Genet. 1995; 11: 335-337Crossref PubMed Scopus (206) Google Scholar, 6Hahnen E. Forkert R. Marke C. Rudnik-Schoneborn S. Schonling J. Zerres K. Wirth B. Hum. Mol. Genet. 1995; 4: 1927-1933Crossref PubMed Scopus (271) Google Scholar, 7Rodrigues N.R. Owen N. Talbot K. Ignatius J. Dubowitz V. Davies K.E. Hum. Mol. Genet. 1995; 4: 631-634Crossref PubMed Scopus (221) Google Scholar, 8Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. et al.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2932) Google Scholar, 9Chang J.G. Jong Y.J. Huang J.M. Wang W.S. Yang T.Y. Chang C.P. Chen Y.J. Lin S.P. Am. J. Hum. Genet. 1995; 57: 1503-1505PubMed Google Scholar, 10Hahnen E. Schonling J. Rudnik-Schoneborn S. Zerres K. Wirth B. Am. J. Hum. Genet. 1996; 59: 1057-1065PubMed Google Scholar, 11Velasco E. Valero C. Valero A. Moreno F. Hernandez-Chico C. Hum. Mol. Genet. 1996; 5: 257-263Crossref PubMed Scopus (180) Google Scholar).Although motor neurons seem to be the only known cell type that is affected in SMA patients, SMN protein is expressed ubiquitously in all tissues and cell types examined (8Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. et al.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2932) Google Scholar, 12Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (854) Google Scholar, 13Coovert D.D. Le T.T. McAndrew P.E. Strasswimmer J. Crawford T.O. Mendell J.R. Coulson S.E. Androphy E.J. Prior T.W. Burghes A.H. Hum. Mol. Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar). The amino acid sequence of SMN does not share significant homology with any protein with a known function; nor does it contain any domains of known function. Although several lines of evidence have suggested that SMN participates in several divergent cellular processes, the question of how reduction of the SMN level leads to motor neuron degeneration remains open. In addition to its cytoplasmic localization, SMN is found in a novel subnuclear structure, named gems, which are found in the vicinity of, and often overlap with, coiled bodies (14Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). The function of coiled bodies is unknown, but they contain spliceosomal snRNPs (small nuclear ribonucleoprotein particles), which function in pre-mRNA splicing, and components of small nucleolar ribonucleoprotein particles, which are involved in pre-rRNA processing. This has led to the speculation that coiled bodies may play some roles in snRNP and small nucleolar ribonucleoprotein particle metabolism (15Gall J.G. Tsvetkov A. Wu Z. Murphy C. Dev. Genet. 1995; 16: 25-35Crossref PubMed Scopus (156) Google Scholar). The fact that gems and coiled bodies are often associated and contain similar sets of proteins and RNAs suggests that they have related functions. In line with this idea, SMN has been shown to interact with a group of Sm proteins, the core proteins of snRNPs, and a novel protein, Gemin2 (formerly known as SIP1), both in vitro and in vivo (14Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Injection of antibodies against either SMN or Gemin2 into Xenopusoocytes inhibits assembly and import of snRNPs, suggesting that the SMN-Gemin2 complex performs an important function in snRNP metabolism (16Fischer U. Liu Q. Dreyfuss G. Cell. 1997; 90: 1023-1029Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 17Buhler D. Raker V. Luhrmann R. Fischer U. Hum. Mol. Genet. 1999; 8: 2351-2357Crossref PubMed Scopus (218) Google Scholar). A dominant negative mutant of SMN, SMNΔN27, also inhibits snRNP assembly in the cytoplasm (18Pellizzoni L. Kataoka N. Charroux B. Dreyfuss G. Cell. 1998; 95: 615-624Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Moreover, the nuclear pool of SMN protein was found to be required for pre-mRNA splicing, probably by facilitating regeneration or recycling of snRNPs in the nucleus (18Pellizzoni L. Kataoka N. Charroux B. Dreyfuss G. Cell. 1998; 95: 615-624Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Recently, two additional proteins, Gemin3 and Gemin4, that are associated with SMN have been described (19Charroux B. Pellizzoni L. Perkinson R.A. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 1999; 147: 1181-1194Crossref PubMed Scopus (222) Google Scholar, 20Charroux B. Pellizzoni L. Perkinson R.A. Yong J. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 2000; 148: 1177-1186Crossref PubMed Scopus (201) Google Scholar). SMN may also be involved in regulation of gene expression by interacting with transcriptional activators (21Strasswimmer J. Lorson C.L. Breiding D.E. Chen J.J. Le T. Burghes A.H. Androphy E.J. Hum. Mol. Genet. 1999; 8: 1219-1226Crossref PubMed Scopus (94) Google Scholar, 22Campbell L. Hunter K.M. Mohaghegh P. Tinsley J.M. Brasch M.A. Davies K.E. Hum. Mol. Genet. 2000; 9: 1093-1100Crossref PubMed Scopus (84) Google Scholar, 23Williams B.Y. Hamilton S.L. Sarkar H.K. FEBS Lett. 2000; 470: 207-210Crossref PubMed Scopus (54) Google Scholar). The ability of SMN to directly bind RNA, along with its close localization to microtubules in the cytoplasm and neuronal dendrites and axons, raises a possibility that SMN is involved in the transport of RNA (24Lorson C.L. Androphy E.J. Hum. Mol. Genet. 1998; 7: 1269-1275Crossref PubMed Google Scholar, 25Bechade C. Rostaing P. Cisterni C. Kalisch R. La Bella V. Pettmann B. Triller A. Eur. J. Neurosci. 1999; 11: 293-304Crossref PubMed Scopus (78) Google Scholar, 26Bertrandy S. Burlet P. Clermont O. Huber C. Fondrat C. Thierry-Mieg D. Munnich A. Lefebvre S. Hum. Mol. Genet. 1999; 8: 775-782Crossref PubMed Scopus (76) Google Scholar, 27Pagliardini S. Giavazzi A. Setola V. Lizier C. Di Luca M. DeBiasi S. Battaglia G. Hum. Mol. Genet. 2000; 9: 47-56Crossref PubMed Scopus (122) Google Scholar).SMN is evolutionarily conserved throughout eukaryotes, because homologues of SMN have been identified in many organisms. Genetic studies have shown that SMN is an essential gene in mice,Caenorhabditis elegans, and Schizosaccharomyces pombe (28Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (542) Google Scholar, 29Miguel-Aliaga I. Culetto E. Walker D.S. Baylis H.A. Sattelle D.B. Davies K.E. Hum. Mol. Genet. 1999; 8: 2133-2143Crossref PubMed Scopus (103) Google Scholar, 30Owen N. Doe C.L. Mellor J. Davies K.E. Hum. Mol. Genet. 2000; 9: 675-684Crossref PubMed Scopus (54) Google Scholar, 31Hannus S. Buhler D. Romano M. Seraphin B. Fischer U. Hum. Mol. Genet. 2000; 9: 663-674Crossref PubMed Scopus (63) Google Scholar, 32Paushkin S. Charroux B. Abel L. Perkinson R.A. Pellizzoni L. Dreyfuss G. J. Biol. Chem. 2000; 275: 23841-23846Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Mice carrying the human centromericSMN2 transgene under an SMN null background are viable and display phenotypes similar to the symptoms of SMA patients, thereby confirming that SMN is the disease gene of SMA (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar,34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar). However, little progress has been made from these studies to elucidate the SMN function.Homologous recombination occurs at exceptionally high frequencies in the chicken pre-B cell line DT40, which makes DT40 a useful genetic system for targeted gene disruption and for studying gene functions (35Buerstedde J.M. Takeda S. Cell. 1991; 67: 179-188Abstract Full Text PDF PubMed Scopus (480) Google Scholar). Moreover, disruption of an essential gene is possible in DT40 when the gene product is expressed from a conditional promoter (36Wang J. Takagaki Y. Manley J.L. Genes Dev. 1996; 10: 2588-2599Crossref PubMed Scopus (172) Google Scholar). To gain further insight into the function of SMN, we constructed a DT40 cell line in which the endogenous SMN gene is disrupted, and SMN protein is produced from an SMN cDNA under the control of the tetracycline (tet)-repressible promoter. Here we have used this system to show that SMN is essential for cell viability and that human SMN can functionally complement for chicken SMN, even though these two proteins are only ∼60% identical in amino acid sequence. This cell line should provide a powerful system for the characterization of SMN function and for screening for potential therapeutic drugs for SMA.DISCUSSIONBy generating a cell line with a knockout of the SMNgene and conditional expression of the SMN protein, we have demonstrated directly that SMN is required for cell viability. This is consistent with genetic analysis of the SMN gene in organisms. Disruption of SMN expression in mice and C. elegans results in early embryonic lethality, which indicates a requirement of SMN for cell viability in embryos (28Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (542) Google Scholar, 29Miguel-Aliaga I. Culetto E. Walker D.S. Baylis H.A. Sattelle D.B. Davies K.E. Hum. Mol. Genet. 1999; 8: 2133-2143Crossref PubMed Scopus (103) Google Scholar). Loss of the SMN homologue in S. pombe also shows a lethal phenotype (30Owen N. Doe C.L. Mellor J. Davies K.E. Hum. Mol. Genet. 2000; 9: 675-684Crossref PubMed Scopus (54) Google Scholar, 31Hannus S. Buhler D. Romano M. Seraphin B. Fischer U. Hum. Mol. Genet. 2000; 9: 663-674Crossref PubMed Scopus (63) Google Scholar, 32Paushkin S. Charroux B. Abel L. Perkinson R.A. Pellizzoni L. Dreyfuss G. J. Biol. Chem. 2000; 275: 23841-23846Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Therefore, SMN is required for fundamental cellular processes that are conserved from fungi to mammals. Although the mice models for SMA generated recently (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar, 34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar, 40Frugier T. Tiziano F.D. Cifuentes-Diaz C. Miniou P. Roblot N. Dierich A. Le Meur M. Melki J. Hum. Mol. Genet. 2000; 9: 849-858Crossref PubMed Scopus (210) Google Scholar) may be very useful in elucidating the pathology of SMA, the cell-based genetic system we describe here provides a setting in which the function of SMN can be studied more directly, and it offers several unique advantages. First, the SMN level in these cell lines can be modulated precisely and over a broad range,i.e. from none to 3–4-fold overexpression. Second, the homogeneity of a cell line should greatly facilitate characterization of phenotypes at the molecular level. More importantly, expression of huSMN protein in cSMN-depleted cells completely rescued the lethal phenotype, indicating that huSMN performs the same function as cSMN does, at least at the level of supporting cell viability and proliferation. Therefore, the function of cSMN defined by characterization of the phenotypes of cSMN-depleted S5 cells directly reflects the function of huSMN in vivo.The interaction of Gemin2 and SMN has been well characterized (14Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). First, Gemin2 directly interacts with SMN in vitro. Second, Gemin2 can be coimmunoprecipitated with an SMN antibody, and vice versa. Finally, SMN and Gemin2 are colocalized in cells. Here we observed a significant decrease of the Gemin2 level concomitant with cSMN depletion. This confirms and extends to an in vivosetting the biochemically defined interaction between SMN and Gemin2 and further suggests that components of the SMN-Gemin2 complex are stabilized by forming this complex. The effect of the SMN level on the stability of Gemin2 is reminiscent of the rapid degradation of the constituents of TFIID and SAGA complexes upon inactivation of a single TATA box-binding protein-associated factor subunit (41Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 42Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 43Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 44Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 45Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). TFIID and SAGA are well characterized complexes that function in gene transcription. It will be interesting to test whether the level of Gemin2 is also reduced in Type I SMA patients. Additional proteins that are associated with the SMN-Gemin2 complex have been reported (19Charroux B. Pellizzoni L. Perkinson R.A. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 1999; 147: 1181-1194Crossref PubMed Scopus (222) Google Scholar, 20Charroux B. Pellizzoni L. Perkinson R.A. Yong J. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 2000; 148: 1177-1186Crossref PubMed Scopus (201) Google Scholar), and it will also be of interest to study what happens to the amounts of these proteins following SMN depletion.The molecular basis of SMA is a reduction in the level of SMN protein. In the most severe type I SMA patients, significant decrease of SMN protein can be seen in all tissues examined (12Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (854) Google Scholar, 13Coovert D.D. Le T.T. McAndrew P.E. Strasswimmer J. Crawford T.O. Mendell J.R. Coulson S.E. Androphy E.J. Prior T.W. Burghes A.H. Hum. Mol. Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar). However, motor neurons appear to be the only cells that are affected. Here we show that S5 cells that express low levels of cSMN (∼30% of the protein in wt DT40 cells) display slow growth. Considering that DT40 is a pre-B cell line, this result indicates that cells other than motor neurons are also dependent on SMN and are sensitive to SMN levels. Overall growth defects can be seen in SMA mouse models (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar, 34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar). Mice that express low levels of SMN protein and display SMA symptoms are smaller than their normal littermates after birth. Thus, it appears that human cells normally express much more SMN protein than they need, and a fraction of the SMN pool may actually be sufficient to support normal growth of cells except for motor neurons. The S5 cell line, whose growth can be specifically modulated by cSMN levels, provides a powerful system to search for potential SMA therapies. When maintained to express low levels of cSMN, S5 cells should be useful for high throughput screening for molecules that may be able to increase cell growth, presumably by enhancing the activity of cSMN, increasing its production, or slowing its turnover. Furthermore, cSMN-depleted S5 cells should be valuable for high throughput screening for compounds (if such exist) that can completely substitute for cSMN function. Given the high conservation of the SMN function, the chemical compounds that we search for will probably exert the same effect on huSMN and will, therefore, be potential therapeutic drugs for SMA. It should also be possible to carry out such screening on cSMN-depleted S5 cells whose growth is supported only by (low levels of) huSMN. Such compounds should also be useful reagents for further understanding of the normal function of SMN and the pathology of SMA. Spinal muscular atrophy (SMA),1 an autosomal recessive disease with characteristics of motor neuron degeneration and muscle atrophy, is a common childhood genetic disorder and the most frequent genetic cause of infant mortality (1Roberts D.F. Chavez J. Court S.D. Arch. Dis. Child. 1970; 45: 33-38Crossref PubMed Scopus (137) Google Scholar, 2Pearn J. Lancet. 1980; 1: 919-922Abstract PubMed Scopus (359) Google Scholar, 3Czeizel A. Hamula J. J. Med. Genet. 1989; 26: 761-763Crossref PubMed Scopus (72) Google Scholar). Based on the age of onset and the severity of the disease, SMA is clinically classified as the severe type I (Werdnig-Hoffman disease), the moderate type II, and the mild type III (Kugelberg-Welander disease). The survival motor neuron (SMN) gene has been established as the disease gene of SMA. The human genome contains two copies of the SMN gene because of an inverted duplication at 5q13. This phenomenon appears to be human-specific, because all other organisms examined to date have a single copy of SMN. Deletions or mutations of the telomericSMN1 gene, which result in reduced SMN protein level, have been found in the vast majority of SMA patients (4Cobben J.M. van der Steege G. Grootscholten P. de Visser M. Scheffer H. Buys C.H. Am. J. Hum. Genet. 1995; 57: 805-808PubMed Google Scholar, 5Bussaglia E. Clermont O. Tizzano E. Lefebvre S. Burglen L. Cruaud C. Urtizberea J.A. Colomer J. Munnich A. Baiget M. et al.Nat. Genet. 1995; 11: 335-337Crossref PubMed Scopus (206) Google Scholar, 6Hahnen E. Forkert R. Marke C. Rudnik-Schoneborn S. Schonling J. Zerres K. Wirth B. Hum. Mol. Genet. 1995; 4: 1927-1933Crossref PubMed Scopus (271) Google Scholar, 7Rodrigues N.R. Owen N. Talbot K. Ignatius J. Dubowitz V. Davies K.E. Hum. Mol. Genet. 1995; 4: 631-634Crossref PubMed Scopus (221) Google Scholar, 8Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. et al.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2932) Google Scholar, 9Chang J.G. Jong Y.J. Huang J.M. Wang W.S. Yang T.Y. Chang C.P. Chen Y.J. Lin S.P. Am. J. Hum. Genet. 1995; 57: 1503-1505PubMed Google Scholar, 10Hahnen E. Schonling J. Rudnik-Schoneborn S. Zerres K. Wirth B. Am. J. Hum. Genet. 1996; 59: 1057-1065PubMed Google Scholar, 11Velasco E. Valero C. Valero A. Moreno F. Hernandez-Chico C. Hum. Mol. Genet. 1996; 5: 257-263Crossref PubMed Scopus (180) Google Scholar). Although motor neurons seem to be the only known cell type that is affected in SMA patients, SMN protein is expressed ubiquitously in all tissues and cell types examined (8Lefebvre S. Burglen L. Reboullet S. Clermont O. Burlet P. Viollet L. Benichou B. Cruaud C. Millasseau P. Zeviani M. et al.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2932) Google Scholar, 12Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (854) Google Scholar, 13Coovert D.D. Le T.T. McAndrew P.E. Strasswimmer J. Crawford T.O. Mendell J.R. Coulson S.E. Androphy E.J. Prior T.W. Burghes A.H. Hum. Mol. Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar). The amino acid sequence of SMN does not share significant homology with any protein with a known function; nor does it contain any domains of known function. Although several lines of evidence have suggested that SMN participates in several divergent cellular processes, the question of how reduction of the SMN level leads to motor neuron degeneration remains open. In addition to its cytoplasmic localization, SMN is found in a novel subnuclear structure, named gems, which are found in the vicinity of, and often overlap with, coiled bodies (14Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). The function of coiled bodies is unknown, but they contain spliceosomal snRNPs (small nuclear ribonucleoprotein particles), which function in pre-mRNA splicing, and components of small nucleolar ribonucleoprotein particles, which are involved in pre-rRNA processing. This has led to the speculation that coiled bodies may play some roles in snRNP and small nucleolar ribonucleoprotein particle metabolism (15Gall J.G. Tsvetkov A. Wu Z. Murphy C. Dev. Genet. 1995; 16: 25-35Crossref PubMed Scopus (156) Google Scholar). The fact that gems and coiled bodies are often associated and contain similar sets of proteins and RNAs suggests that they have related functions. In line with this idea, SMN has been shown to interact with a group of Sm proteins, the core proteins of snRNPs, and a novel protein, Gemin2 (formerly known as SIP1), both in vitro and in vivo (14Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Injection of antibodies against either SMN or Gemin2 into Xenopusoocytes inhibits assembly and import of snRNPs, suggesting that the SMN-Gemin2 complex performs an important function in snRNP metabolism (16Fischer U. Liu Q. Dreyfuss G. Cell. 1997; 90: 1023-1029Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 17Buhler D. Raker V. Luhrmann R. Fischer U. Hum. Mol. Genet. 1999; 8: 2351-2357Crossref PubMed Scopus (218) Google Scholar). A dominant negative mutant of SMN, SMNΔN27, also inhibits snRNP assembly in the cytoplasm (18Pellizzoni L. Kataoka N. Charroux B. Dreyfuss G. Cell. 1998; 95: 615-624Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Moreover, the nuclear pool of SMN protein was found to be required for pre-mRNA splicing, probably by facilitating regeneration or recycling of snRNPs in the nucleus (18Pellizzoni L. Kataoka N. Charroux B. Dreyfuss G. Cell. 1998; 95: 615-624Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Recently, two additional proteins, Gemin3 and Gemin4, that are associated with SMN have been described (19Charroux B. Pellizzoni L. Perkinson R.A. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 1999; 147: 1181-1194Crossref PubMed Scopus (222) Google Scholar, 20Charroux B. Pellizzoni L. Perkinson R.A. Yong J. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 2000; 148: 1177-1186Crossref PubMed Scopus (201) Google Scholar). SMN may also be involved in regulation of gene expression by interacting with transcriptional activators (21Strasswimmer J. Lorson C.L. Breiding D.E. Chen J.J. Le T. Burghes A.H. Androphy E.J. Hum. Mol. Genet. 1999; 8: 1219-1226Crossref PubMed Scopus (94) Google Scholar, 22Campbell L. Hunter K.M. Mohaghegh P. Tinsley J.M. Brasch M.A. Davies K.E. Hum. Mol. Genet. 2000; 9: 1093-1100Crossref PubMed Scopus (84) Google Scholar, 23Williams B.Y. Hamilton S.L. Sarkar H.K. FEBS Lett. 2000; 470: 207-210Crossref PubMed Scopus (54) Google Scholar). The ability of SMN to directly bind RNA, along with its close localization to microtubules in the cytoplasm and neuronal dendrites and axons, raises a possibility that SMN is involved in the transport of RNA (24Lorson C.L. Androphy E.J. Hum. Mol. Genet. 1998; 7: 1269-1275Crossref PubMed Google Scholar, 25Bechade C. Rostaing P. Cisterni C. Kalisch R. La Bella V. Pettmann B. Triller A. Eur. J. Neurosci. 1999; 11: 293-304Crossref PubMed Scopus (78) Google Scholar, 26Bertrandy S. Burlet P. Clermont O. Huber C. Fondrat C. Thierry-Mieg D. Munnich A. Lefebvre S. Hum. Mol. Genet. 1999; 8: 775-782Crossref PubMed Scopus (76) Google Scholar, 27Pagliardini S. Giavazzi A. Setola V. Lizier C. Di Luca M. DeBiasi S. Battaglia G. Hum. Mol. Genet. 2000; 9: 47-56Crossref PubMed Scopus (122) Google Scholar). SMN is evolutionarily conserved throughout eukaryotes, because homologues of SMN have been identified in many organisms. Genetic studies have shown that SMN is an essential gene in mice,Caenorhabditis elegans, and Schizosaccharomyces pombe (28Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (542) Google Scholar, 29Miguel-Aliaga I. Culetto E. Walker D.S. Baylis H.A. Sattelle D.B. Davies K.E. Hum. Mol. Genet. 1999; 8: 2133-2143Crossref PubMed Scopus (103) Google Scholar, 30Owen N. Doe C.L. Mellor J. Davies K.E. Hum. Mol. Genet. 2000; 9: 675-684Crossref PubMed Scopus (54) Google Scholar, 31Hannus S. Buhler D. Romano M. Seraphin B. Fischer U. Hum. Mol. Genet. 2000; 9: 663-674Crossref PubMed Scopus (63) Google Scholar, 32Paushkin S. Charroux B. Abel L. Perkinson R.A. Pellizzoni L. Dreyfuss G. J. Biol. Chem. 2000; 275: 23841-23846Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Mice carrying the human centromericSMN2 transgene under an SMN null background are viable and display phenotypes similar to the symptoms of SMA patients, thereby confirming that SMN is the disease gene of SMA (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar,34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar). However, little progress has been made from these studies to elucidate the SMN function. Homologous recombination occurs at exceptionally high frequencies in the chicken pre-B cell line DT40, which makes DT40 a useful genetic system for targeted gene disruption and for studying gene functions (35Buerstedde J.M. Takeda S. Cell. 1991; 67: 179-188Abstract Full Text PDF PubMed Scopus (480) Google Scholar). Moreover, disruption of an essential gene is possible in DT40 when the gene product is expressed from a conditional promoter (36Wang J. Takagaki Y. Manley J.L. Genes Dev. 1996; 10: 2588-2599Crossref PubMed Scopus (172) Google Scholar). To gain further insight into the function of SMN, we constructed a DT40 cell line in which the endogenous SMN gene is disrupted, and SMN protein is produced from an SMN cDNA under the control of the tetracycline (tet)-repressible promoter. Here we have used this system to show that SMN is essential for cell viability and that human SMN can functionally complement for chicken SMN, even though these two proteins are only ∼60% identical in amino acid sequence. This cell line should provide a powerful system for the characterization of SMN function and for screening for potential therapeutic drugs for SMA. DISCUSSIONBy generating a cell line with a knockout of the SMNgene and conditional expression of the SMN protein, we have demonstrated directly that SMN is required for cell viability. This is consistent with genetic analysis of the SMN gene in organisms. Disruption of SMN expression in mice and C. elegans results in early embryonic lethality, which indicates a requirement of SMN for cell viability in embryos (28Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (542) Google Scholar, 29Miguel-Aliaga I. Culetto E. Walker D.S. Baylis H.A. Sattelle D.B. Davies K.E. Hum. Mol. Genet. 1999; 8: 2133-2143Crossref PubMed Scopus (103) Google Scholar). Loss of the SMN homologue in S. pombe also shows a lethal phenotype (30Owen N. Doe C.L. Mellor J. Davies K.E. Hum. Mol. Genet. 2000; 9: 675-684Crossref PubMed Scopus (54) Google Scholar, 31Hannus S. Buhler D. Romano M. Seraphin B. Fischer U. Hum. Mol. Genet. 2000; 9: 663-674Crossref PubMed Scopus (63) Google Scholar, 32Paushkin S. Charroux B. Abel L. Perkinson R.A. Pellizzoni L. Dreyfuss G. J. Biol. Chem. 2000; 275: 23841-23846Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Therefore, SMN is required for fundamental cellular processes that are conserved from fungi to mammals. Although the mice models for SMA generated recently (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar, 34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar, 40Frugier T. Tiziano F.D. Cifuentes-Diaz C. Miniou P. Roblot N. Dierich A. Le Meur M. Melki J. Hum. Mol. Genet. 2000; 9: 849-858Crossref PubMed Scopus (210) Google Scholar) may be very useful in elucidating the pathology of SMA, the cell-based genetic system we describe here provides a setting in which the function of SMN can be studied more directly, and it offers several unique advantages. First, the SMN level in these cell lines can be modulated precisely and over a broad range,i.e. from none to 3–4-fold overexpression. Second, the homogeneity of a cell line should greatly facilitate characterization of phenotypes at the molecular level. More importantly, expression of huSMN protein in cSMN-depleted cells completely rescued the lethal phenotype, indicating that huSMN performs the same function as cSMN does, at least at the level of supporting cell viability and proliferation. Therefore, the function of cSMN defined by characterization of the phenotypes of cSMN-depleted S5 cells directly reflects the function of huSMN in vivo.The interaction of Gemin2 and SMN has been well characterized (14Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). First, Gemin2 directly interacts with SMN in vitro. Second, Gemin2 can be coimmunoprecipitated with an SMN antibody, and vice versa. Finally, SMN and Gemin2 are colocalized in cells. Here we observed a significant decrease of the Gemin2 level concomitant with cSMN depletion. This confirms and extends to an in vivosetting the biochemically defined interaction between SMN and Gemin2 and further suggests that components of the SMN-Gemin2 complex are stabilized by forming this complex. The effect of the SMN level on the stability of Gemin2 is reminiscent of the rapid degradation of the constituents of TFIID and SAGA complexes upon inactivation of a single TATA box-binding protein-associated factor subunit (41Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 42Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 43Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 44Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 45Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). TFIID and SAGA are well characterized complexes that function in gene transcription. It will be interesting to test whether the level of Gemin2 is also reduced in Type I SMA patients. Additional proteins that are associated with the SMN-Gemin2 complex have been reported (19Charroux B. Pellizzoni L. Perkinson R.A. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 1999; 147: 1181-1194Crossref PubMed Scopus (222) Google Scholar, 20Charroux B. Pellizzoni L. Perkinson R.A. Yong J. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 2000; 148: 1177-1186Crossref PubMed Scopus (201) Google Scholar), and it will also be of interest to study what happens to the amounts of these proteins following SMN depletion.The molecular basis of SMA is a reduction in the level of SMN protein. In the most severe type I SMA patients, significant decrease of SMN protein can be seen in all tissues examined (12Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (854) Google Scholar, 13Coovert D.D. Le T.T. McAndrew P.E. Strasswimmer J. Crawford T.O. Mendell J.R. Coulson S.E. Androphy E.J. Prior T.W. Burghes A.H. Hum. Mol. Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar). However, motor neurons appear to be the only cells that are affected. Here we show that S5 cells that express low levels of cSMN (∼30% of the protein in wt DT40 cells) display slow growth. Considering that DT40 is a pre-B cell line, this result indicates that cells other than motor neurons are also dependent on SMN and are sensitive to SMN levels. Overall growth defects can be seen in SMA mouse models (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar, 34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar). Mice that express low levels of SMN protein and display SMA symptoms are smaller than their normal littermates after birth. Thus, it appears that human cells normally express much more SMN protein than they need, and a fraction of the SMN pool may actually be sufficient to support normal growth of cells except for motor neurons. The S5 cell line, whose growth can be specifically modulated by cSMN levels, provides a powerful system to search for potential SMA therapies. When maintained to express low levels of cSMN, S5 cells should be useful for high throughput screening for molecules that may be able to increase cell growth, presumably by enhancing the activity of cSMN, increasing its production, or slowing its turnover. Furthermore, cSMN-depleted S5 cells should be valuable for high throughput screening for compounds (if such exist) that can completely substitute for cSMN function. Given the high conservation of the SMN function, the chemical compounds that we search for will probably exert the same effect on huSMN and will, therefore, be potential therapeutic drugs for SMA. It should also be possible to carry out such screening on cSMN-depleted S5 cells whose growth is supported only by (low levels of) huSMN. Such compounds should also be useful reagents for further understanding of the normal function of SMN and the pathology of SMA. By generating a cell line with a knockout of the SMNgene and conditional expression of the SMN protein, we have demonstrated directly that SMN is required for cell viability. This is consistent with genetic analysis of the SMN gene in organisms. Disruption of SMN expression in mice and C. elegans results in early embryonic lethality, which indicates a requirement of SMN for cell viability in embryos (28Schrank B. Gotz R. Gunnersen J.M. Ure J.M. Toyka K.V. Smith A.G. Sendtner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9920-9925Crossref PubMed Scopus (542) Google Scholar, 29Miguel-Aliaga I. Culetto E. Walker D.S. Baylis H.A. Sattelle D.B. Davies K.E. Hum. Mol. Genet. 1999; 8: 2133-2143Crossref PubMed Scopus (103) Google Scholar). Loss of the SMN homologue in S. pombe also shows a lethal phenotype (30Owen N. Doe C.L. Mellor J. Davies K.E. Hum. Mol. Genet. 2000; 9: 675-684Crossref PubMed Scopus (54) Google Scholar, 31Hannus S. Buhler D. Romano M. Seraphin B. Fischer U. Hum. Mol. Genet. 2000; 9: 663-674Crossref PubMed Scopus (63) Google Scholar, 32Paushkin S. Charroux B. Abel L. Perkinson R.A. Pellizzoni L. Dreyfuss G. J. Biol. Chem. 2000; 275: 23841-23846Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Therefore, SMN is required for fundamental cellular processes that are conserved from fungi to mammals. Although the mice models for SMA generated recently (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar, 34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar, 40Frugier T. Tiziano F.D. Cifuentes-Diaz C. Miniou P. Roblot N. Dierich A. Le Meur M. Melki J. Hum. Mol. Genet. 2000; 9: 849-858Crossref PubMed Scopus (210) Google Scholar) may be very useful in elucidating the pathology of SMA, the cell-based genetic system we describe here provides a setting in which the function of SMN can be studied more directly, and it offers several unique advantages. First, the SMN level in these cell lines can be modulated precisely and over a broad range,i.e. from none to 3–4-fold overexpression. Second, the homogeneity of a cell line should greatly facilitate characterization of phenotypes at the molecular level. More importantly, expression of huSMN protein in cSMN-depleted cells completely rescued the lethal phenotype, indicating that huSMN performs the same function as cSMN does, at least at the level of supporting cell viability and proliferation. Therefore, the function of cSMN defined by characterization of the phenotypes of cSMN-depleted S5 cells directly reflects the function of huSMN in vivo. The interaction of Gemin2 and SMN has been well characterized (14Liu Q. Fischer U. Wang F. Dreyfuss G. Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). First, Gemin2 directly interacts with SMN in vitro. Second, Gemin2 can be coimmunoprecipitated with an SMN antibody, and vice versa. Finally, SMN and Gemin2 are colocalized in cells. Here we observed a significant decrease of the Gemin2 level concomitant with cSMN depletion. This confirms and extends to an in vivosetting the biochemically defined interaction between SMN and Gemin2 and further suggests that components of the SMN-Gemin2 complex are stabilized by forming this complex. The effect of the SMN level on the stability of Gemin2 is reminiscent of the rapid degradation of the constituents of TFIID and SAGA complexes upon inactivation of a single TATA box-binding protein-associated factor subunit (41Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 42Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 43Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 44Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 45Sanders S.L. Klebanow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). TFIID and SAGA are well characterized complexes that function in gene transcription. It will be interesting to test whether the level of Gemin2 is also reduced in Type I SMA patients. Additional proteins that are associated with the SMN-Gemin2 complex have been reported (19Charroux B. Pellizzoni L. Perkinson R.A. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 1999; 147: 1181-1194Crossref PubMed Scopus (222) Google Scholar, 20Charroux B. Pellizzoni L. Perkinson R.A. Yong J. Shevchenko A. Mann M. Dreyfuss G. J. Cell Biol. 2000; 148: 1177-1186Crossref PubMed Scopus (201) Google Scholar), and it will also be of interest to study what happens to the amounts of these proteins following SMN depletion. The molecular basis of SMA is a reduction in the level of SMN protein. In the most severe type I SMA patients, significant decrease of SMN protein can be seen in all tissues examined (12Lefebvre S. Burlet P. Liu Q. Bertrandy S. Clermont O. Munnich A. Dreyfuss G. Melki J. Nat. Genet. 1997; 16: 265-269Crossref PubMed Scopus (854) Google Scholar, 13Coovert D.D. Le T.T. McAndrew P.E. Strasswimmer J. Crawford T.O. Mendell J.R. Coulson S.E. Androphy E.J. Prior T.W. Burghes A.H. Hum. Mol. Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar). However, motor neurons appear to be the only cells that are affected. Here we show that S5 cells that express low levels of cSMN (∼30% of the protein in wt DT40 cells) display slow growth. Considering that DT40 is a pre-B cell line, this result indicates that cells other than motor neurons are also dependent on SMN and are sensitive to SMN levels. Overall growth defects can be seen in SMA mouse models (33Hsieh-Li H.M. Chang J.G. Jong Y.J. Wu M.H. Wang N.M. Tsai C.H. Li H. Nat. Genet. 2000; 24: 66-70Crossref PubMed Scopus (587) Google Scholar, 34Monani U.R. Sendtner M. Coovert D.D. Parsons D.W. Andreassi C. Le T.T. Jablonka S. Schrank B. Rossol W. Prior T.W. Morris G.E. Burghes A.H. Hum. Mol. Genet. 2000; 9: 333-339Crossref PubMed Scopus (622) Google Scholar). Mice that express low levels of SMN protein and display SMA symptoms are smaller than their normal littermates after birth. Thus, it appears that human cells normally express much more SMN protein than they need, and a fraction of the SMN pool may actually be sufficient to support normal growth of cells except for motor neurons. The S5 cell line, whose growth can be specifically modulated by cSMN levels, provides a powerful system to search for potential SMA therapies. When maintained to express low levels of cSMN, S5 cells should be useful for high throughput screening for molecules that may be able to increase cell growth, presumably by enhancing the activity of cSMN, increasing its production, or slowing its turnover. Furthermore, cSMN-depleted S5 cells should be valuable for high throughput screening for compounds (if such exist) that can completely substitute for cSMN function. Given the high conservation of the SMN function, the chemical compounds that we search for will probably exert the same effect on huSMN and will, therefore, be potential therapeutic drugs for SMA. It should also be possible to carry out such screening on cSMN-depleted S5 cells whose growth is supported only by (low levels of) huSMN. Such compounds should also be useful reagents for further understanding of the normal function of SMN and the pathology of SMA. We thank Linda Abel and Robert Perkinson for producing mouse anti-cSMN polysera, Lili Wan for sharing the anti-hnRNP A2 monoclonal antibody, and members of our laboratory for helpful discussions. We thank Drs. Zissimos Morelatos, Westley Freisen, Livio Pellizzoni, and Amelie Gubitz for comments on this manuscript. We are grateful to Dr. Paul Bates for providing us with the retroviral expression system.
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