Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis (original) (raw)

  1. Maria Anokhina1,
  2. Andrius Krasauskas2,
  3. Monika M. Golas2,4,
  4. Bjoern Sander2,5,
  5. Cindy L. Will1,
  6. Henning Urlaub3,
  7. Holger Stark2 and
  8. Reinhard Lührmann1
  9. 1Department of Cellular Biochemistry, MPI of Biophysical Chemistry, D-37077 Göttingen, Germany
  10. 2Research Group of 3D Electron Cryomicroscopy, MPI of Biophysical Chemistry, D-37077 Göttingen, Germany
  11. 3Bioanalytical Mass Spectrometry Group, MPI of Biophysical Chemistry, D-37077 Göttingen, Germany

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 Bact 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 Bact and Bact to C transitions, and comparisons with the Saccharomyces cerevisiae Bact 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 Bact and Bact to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human Bact 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 Bact complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.

Footnotes