title: Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses.
abstract: Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of alpha-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Cherny
authors.forename: Clare
authors.initials: C
authors.lastname: Gooding
authors.forename: Giles E
authors.initials: GE
authors.lastname: Eperon
authors.forename: Miguel B
authors.initials: MB
authors.lastname: Coelho
authors.forename: Clive R
authors.initials: CR
authors.lastname: Bagshaw
authors.forename: Christopher W J
authors.initials: CW
authors.lastname: Smith
authors.forename: Ian C
authors.initials: IC
authors.lastname: Eperon
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002466:Cell Nucleolus / Q000378:metabolism / Q000648:ultrastructure; D005091:Exons; D008957:Models, Genetic; D008969:Molecular Sequence Data; D038761:Photobleaching; D038941:Polypyrimidine Tract-Binding Protein / Q000032:analysis* / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D014335:Tropomyosin / Q000235:genetics
chemical_list: D012333:RNA, Messenger; C490773:Tpm1 protein, rat; D014335:Tropomyosin; D038941:Polypyrimidine Tract-Binding Protein