Purification of native survival of motor neurons complexes and identification of Gemin6 as a novel component.
The survival of motor neurons (SMN) protein, the product of the gene responsible for the motor neuron degenerative disease spinal muscular atrophy (SMA), is part of a large macromolecular complex. The SMN complex is localized in both the cytoplasm and the nucleus and contains SMN, Gemin2, Gemin3, Gemin4, Gemin5, and a few not yet identified proteins. The SMN complex plays a key role in the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and other ribonucleoprotein particles. As a step toward the complete characterization of the components of the SMN complex, we generated stable cell lines that express FLAG-tagged SMN or Gemin2 under the control of a tetracycline-inducible promoter. Native SMN complexes of identical protein composition to those isolated by immunoprecipitation with anti-SMN antibodies were purified by affinity chromatography from extracts of both cell lines. Here we report the identification by mass spectrometry of a novel protein component of the SMN complex termed Gemin6. Co-immunoprecipitation, immunolocalization, and in vitro binding experiments demonstrate that Gemin6 is a component of the SMN complex that localizes to gems and interacts with several Sm proteins of the spliceosomal snRNPs.
Pellizzoni
Livio
L
Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6148, USA.
Baccon
Jennifer
J
Rappsilber
Juri
J
Mann
Matthias
M
Dreyfuss
Gideon
G
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009046:Motor Neurons / Q000166:cytology*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism
C450836:GEMIN6 protein, human; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; D055532:SMN Complex Proteins; D004247:DNA