ABSTRACT
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.
Subject(s)
Chromatin/chemistry , Chromatography, Affinity/methods , Histones/metabolism , Protein Interaction Mapping/methods , Protein Processing, Post-Translational , Proteome/isolation & purification , Animals , Cell Line , Histones/chemistry , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Isotope Labeling , Methylation , Mice , Peptide Fragments/chemistry , Protein Binding , Proteolysis , Proteome/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Tandem Mass SpectrometryABSTRACT
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.
Subject(s)
Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Spliceosomes/chemistry , Spliceosomes/metabolism , Catalysis , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/isolation & purification , Cell Cycle Proteins/metabolism , Chromatography, Affinity , Enzyme Activation , HeLa Cells , Humans , Microscopy, Electron , Models, Biological , Multiprotein Complexes/isolation & purification , Multiprotein Complexes/ultrastructure , Phosphoproteins/chemistry , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , Phosphorylation , Protein Conformation , Protein Kinases/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , RNA Precursors/chemistry , RNA Precursors/metabolism , RNA Splicing Factors , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U2 Small Nuclear/chemistry , Ribonucleoprotein, U2 Small Nuclear/isolation & purification , Ribonucleoprotein, U2 Small Nuclear/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Spliceosomes/ultrastructure , Structure-Activity RelationshipABSTRACT
Obtaining phase information for the solution of macromolecular structures is still one of the bottlenecks in X-ray crystallography. 5-Amino-2,4,6-triiodoisophthalic acid (I3C), in which three covalently bound iodines form an equilateral triangle, was incorporated into proteins in order to obtain phases by single-wavelength anomalous dispersion (SAD). An improved binding capability compared with simple heavy-metal ions, ready availability, improved recognition of potential heavy-atom sites and low toxicity make I3C particularly suitable for experimental phasing.