title: Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis.
abstract: To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3' splice site and 3' exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human B(act) complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to B(act) and B(act) to C transitions, and comparisons with the Saccharomyces cerevisiae B(act) complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to B(act) and B(act) to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human B(act) complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae B(act) complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.
authors.affiliation: Department of Cellular Biochemistry, MPI of Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Sergey
authors.initials: S
authors.lastname: Bessonov
authors.forename: Maria
authors.initials: M
authors.lastname: Anokhina
authors.forename: Andrius
authors.initials: A
authors.lastname: Krasauskas
authors.forename: Monika M
authors.initials: MM
authors.lastname: Golas
authors.forename: Bjoern
authors.initials: B
authors.lastname: Sander
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Holger
authors.initials: H
authors.lastname: Stark
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D013329:Structure-Activity Relationship
chemical_list: C508357:CDC5L protein, human; D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011743:Pyrimidines; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C030986:pyrimidine