title: Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing.
abstract: Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States.
authors.forename: Somsakul Pop
authors.initials: SP
authors.lastname: Wongpalee
authors.affiliation: Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States.
authors.forename: Ajay
authors.initials: A
authors.lastname: Vashisht
authors.affiliation: Department of Basic Medical Sciences, University of Arizona, Phoenix, United States.
authors.forename: Shalini
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States.
authors.forename: Darryl
authors.initials: D
authors.lastname: Chui
authors.affiliation: Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States.
authors.forename: James A
authors.initials: JA
authors.lastname: Wohlschlegel
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States.
authors.forename: Douglas L
authors.identifier: 0000-0002-2705-8187
authors.initials: DL
authors.lastname: Black
mesh_terms: D005091:Exons; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics* / Q000378:metabolism*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry* / Q000378:metabolism*; D000072668:Splicing Factor U2AF / Q000378:metabolism
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; C450006:PTBP1 protein, human; D012342:RNA, Small Nuclear; D000072668:Splicing Factor U2AF; C405483:U1 small nuclear RNA; C405484:U2 small nuclear RNA; D038941:Polypyrimidine Tract-Binding Protein
keywords: RNA binding protein; biochemistry; evolutionary biology; gene regulation; genomics; human; ribonucleoprotein; splicing