title: MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3.
abstract: Exon 3 of the rat α-tropomyosin (Tpm1) gene is repressed in smooth muscle cells, allowing inclusion of the mutually exclusive partner exon 2. Two key types of elements affect repression of exon 3 splicing: binding sites for polypyrimidine tract-binding protein (PTB) and additional negative regulatory elements consisting of clusters of UGC or CUG motifs. Here, we show that the UGC clusters are bound by muscleblind-like proteins (MBNL), which act as repressors of Tpm1 exon 3. We show that the N-terminal region of MBNL1, containing its four CCCH zinc-finger domains, is sufficient to mediate repression. The same region of MBNL1 can make a direct protein-to-protein interaction with PTB, and RNA binding by MBNL promotes this interaction, apparently by inducing a conformational change in MBNL. Moreover, single molecule analysis showed that MBNL-binding sites increase the binding of PTB to its own sites. Our data suggest that the smooth muscle splicing of Tpm1 is mediated by allosteric assembly of an RNA-protein complex minimally comprising PTB, MBNL and their cognate RNA-binding sites.
authors.affiliation: Department of Biochemistry, University of Cambridge, CB2 1QW, UK.
authors.forename: Clare
authors.initials: C
authors.lastname: Gooding
authors.forename: Christopher
authors.initials: C
authors.lastname: Edge
authors.forename: Mike
authors.initials: M
authors.lastname: Lorenz
authors.forename: Miguel B
authors.initials: MB
authors.lastname: Coelho
authors.forename: Mikael
authors.initials: M
authors.lastname: Winters
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
authors.forename: Dmitry
authors.initials: D
authors.lastname: Cherny
authors.forename: Ian C
authors.initials: IC
authors.lastname: Eperon
authors.forename: Christopher W J
authors.initials: CW
authors.lastname: Smith
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D005091:Exons; D006801:Humans; D009130:Muscle, Smooth / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D038621:Regulatory Sequences, Ribonucleic Acid; D014335:Tropomyosin / Q000235:genetics* / Q000378:metabolism
chemical_list: C493663:MBNL1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; C490773:Tpm1 protein, rat; D014335:Tropomyosin; D038941:Polypyrimidine Tract-Binding Protein