Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions

Mol Cell Biol. 2006 Jul;26(14):5528-43. doi: 10.1128/MCB.00582-06.

Abstract

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Carrier Proteins / analysis
  • Carrier Proteins / chemistry
  • Cell Cycle Proteins / analysis
  • Cell Cycle Proteins / chemistry
  • Cell Fractionation / methods
  • Chromatography, Affinity
  • DNA Repair Enzymes
  • HeLa Cells
  • Humans
  • Microscopy, Electron
  • Multiprotein Complexes
  • Nuclear Proteins / analysis*
  • RNA Precursors / metabolism
  • RNA Splicing
  • RNA Splicing Factors
  • Spliceosomes / chemistry*
  • Spliceosomes / metabolism
  • Spliceosomes / ultrastructure*
  • Tobramycin

Substances

  • Affinity Labels
  • Carrier Proteins
  • Cell Cycle Proteins
  • Multiprotein Complexes
  • Nuclear Proteins
  • RNA Precursors
  • RNA Splicing Factors
  • DNA Repair Enzymes
  • PRPF19 protein, human
  • Tobramycin