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

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

Chromatin Affinity Purification and Quantitative Mass Spectrometry Defining the Interactome of Histone Modification Patterns*

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated