title: Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method.
abstract: Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly.
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Hartmuth
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Vornlocher
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.forename: Marc
authors.initials: M
authors.lastname: Gentzel
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilm
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D002499:Centrifugation, Density Gradient; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000032:analysis; D006801:Humans; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000032:analysis*; D012322:RNA Precursors / Q000032:analysis; D017411:Ribonucleoproteins, Small Nuclear / Q000032:analysis; D017381:Spliceosomes / Q000737:chemistry*; D014031:Tobramycin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D017411:Ribonucleoproteins, Small Nuclear; D014031:Tobramycin