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MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3.

Gooding C, Edge C, Lorenz M, Coelho MB, Winters M, Kaminski CF, Cherny D, Eperon IC, Smith CW - Nucleic Acids Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, CB2 1QW, UK.

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.

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MBNL binds to the upstream and downstream UGC elements. (A) Schematic representation of RNAs a–g containing Tpm1 exon 3 and flanking regulatory elements. RNA a has all the cis-elements, RNA b has the UGC motifs deleted and RNA c has the UCUU motifs mutated in P3 or deleted in DY. RNAs d–g contain the individual regulatory elements as indicated. (B) In vitro transcribed RNAs a–c were UV cross-linked with either recombinant pCGT7–MBNL1 (rMBNL1, lanes 1–3) or PAC-1 nuclear extract (NE, lanes 4–6). RNAs a–c were UV cross-linked with rMBNL1 or PAC-1 nuclear extract and then immunoprecipitated with either α-MBNL1 (lanes 7–12) or UV cross-linked with PAC-1 nuclear extracts and immunoprecipitated with α-PTB antibody (lanes 13–15). (C) In vitro transcribed RNA a or the individual cis-elements, d–g, were UV cross-linked with rMBNL1 (lanes 1–5) or recombinant pQE–PTB4 (rPTB4, lanes 6–10).
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gkt168-F3: MBNL binds to the upstream and downstream UGC elements. (A) Schematic representation of RNAs a–g containing Tpm1 exon 3 and flanking regulatory elements. RNA a has all the cis-elements, RNA b has the UGC motifs deleted and RNA c has the UCUU motifs mutated in P3 or deleted in DY. RNAs d–g contain the individual regulatory elements as indicated. (B) In vitro transcribed RNAs a–c were UV cross-linked with either recombinant pCGT7–MBNL1 (rMBNL1, lanes 1–3) or PAC-1 nuclear extract (NE, lanes 4–6). RNAs a–c were UV cross-linked with rMBNL1 or PAC-1 nuclear extract and then immunoprecipitated with either α-MBNL1 (lanes 7–12) or UV cross-linked with PAC-1 nuclear extracts and immunoprecipitated with α-PTB antibody (lanes 13–15). (C) In vitro transcribed RNA a or the individual cis-elements, d–g, were UV cross-linked with rMBNL1 (lanes 1–5) or recombinant pQE–PTB4 (rPTB4, lanes 6–10).

Mentions: To address whether MBNL1 affects Tpm1 splicing directly we carried out UV cross-linking of in vitro transcribed Tpm1 RNA to recombinant MBNL1 and to proteins in PAC1 nuclear extract. Wild-type RNA, with all four cis-regulatory elements intact, cross-linked to rMBNL1 (Figure 3B lane 1, RNA ‘a’). In PAC1 NE, it cross-linked to proteins of ∼59, 57, 45 and 35 kDa. The 59/57 kDa doublet was shown by immunoprecipitation to be PTB (Figure 3B, lane 13), whereas the ∼44 kDa band was MBNL1 (lane 10). As expected, deletion of the DY element together with point mutations of UCUU motifs in P3 (RNA ‘c’) led to reduced PTB cross-linking (lanes 6 and 15) (17,18,20), but it had no effect on MBNL1 cross-linking (lane 3, 6, 9 and 12). Conversely, combined deletion of the U and D elements (RNA ‘b’) substantially reduced cross-linking of both recombinant (lanes 2 and 8) and NE MBNL1 (lanes 5 and 11), indicating that these are the major sites of MBNL1 interaction. The cross-linking of PTB also seemed to be slightly reduced by the combined U/D element mutations in RNA (lanes 5 and 14, see later in the text). To test whether the U or D elements are sufficient for MBNL1 binding, we used RNAs e and f for UV cross-linking (Figure 3A). MBNL1 cross-linked to both the U and D elements (Figure 3C, lanes 3 and 4). In contrast, recombinant PTB bound to the pyrimidine tract elements (RNAs d and g, lanes 7 and 10) but not to the UGC elements (lanes 8 and 9). Although only the U element was essential for mediating the influence of overexpressed MBNL1 (Figure 2), both the U and D elements can bind MBNL1 (Figure 3C). Indeed, the D element cross-linked more efficiently than the U element (Figure 3C, lanes 3 and 4).Figure 3.


MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3.

Gooding C, Edge C, Lorenz M, Coelho MB, Winters M, Kaminski CF, Cherny D, Eperon IC, Smith CW - Nucleic Acids Res. (2013)

MBNL binds to the upstream and downstream UGC elements. (A) Schematic representation of RNAs a–g containing Tpm1 exon 3 and flanking regulatory elements. RNA a has all the cis-elements, RNA b has the UGC motifs deleted and RNA c has the UCUU motifs mutated in P3 or deleted in DY. RNAs d–g contain the individual regulatory elements as indicated. (B) In vitro transcribed RNAs a–c were UV cross-linked with either recombinant pCGT7–MBNL1 (rMBNL1, lanes 1–3) or PAC-1 nuclear extract (NE, lanes 4–6). RNAs a–c were UV cross-linked with rMBNL1 or PAC-1 nuclear extract and then immunoprecipitated with either α-MBNL1 (lanes 7–12) or UV cross-linked with PAC-1 nuclear extracts and immunoprecipitated with α-PTB antibody (lanes 13–15). (C) In vitro transcribed RNA a or the individual cis-elements, d–g, were UV cross-linked with rMBNL1 (lanes 1–5) or recombinant pQE–PTB4 (rPTB4, lanes 6–10).
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gkt168-F3: MBNL binds to the upstream and downstream UGC elements. (A) Schematic representation of RNAs a–g containing Tpm1 exon 3 and flanking regulatory elements. RNA a has all the cis-elements, RNA b has the UGC motifs deleted and RNA c has the UCUU motifs mutated in P3 or deleted in DY. RNAs d–g contain the individual regulatory elements as indicated. (B) In vitro transcribed RNAs a–c were UV cross-linked with either recombinant pCGT7–MBNL1 (rMBNL1, lanes 1–3) or PAC-1 nuclear extract (NE, lanes 4–6). RNAs a–c were UV cross-linked with rMBNL1 or PAC-1 nuclear extract and then immunoprecipitated with either α-MBNL1 (lanes 7–12) or UV cross-linked with PAC-1 nuclear extracts and immunoprecipitated with α-PTB antibody (lanes 13–15). (C) In vitro transcribed RNA a or the individual cis-elements, d–g, were UV cross-linked with rMBNL1 (lanes 1–5) or recombinant pQE–PTB4 (rPTB4, lanes 6–10).
Mentions: To address whether MBNL1 affects Tpm1 splicing directly we carried out UV cross-linking of in vitro transcribed Tpm1 RNA to recombinant MBNL1 and to proteins in PAC1 nuclear extract. Wild-type RNA, with all four cis-regulatory elements intact, cross-linked to rMBNL1 (Figure 3B lane 1, RNA ‘a’). In PAC1 NE, it cross-linked to proteins of ∼59, 57, 45 and 35 kDa. The 59/57 kDa doublet was shown by immunoprecipitation to be PTB (Figure 3B, lane 13), whereas the ∼44 kDa band was MBNL1 (lane 10). As expected, deletion of the DY element together with point mutations of UCUU motifs in P3 (RNA ‘c’) led to reduced PTB cross-linking (lanes 6 and 15) (17,18,20), but it had no effect on MBNL1 cross-linking (lane 3, 6, 9 and 12). Conversely, combined deletion of the U and D elements (RNA ‘b’) substantially reduced cross-linking of both recombinant (lanes 2 and 8) and NE MBNL1 (lanes 5 and 11), indicating that these are the major sites of MBNL1 interaction. The cross-linking of PTB also seemed to be slightly reduced by the combined U/D element mutations in RNA (lanes 5 and 14, see later in the text). To test whether the U or D elements are sufficient for MBNL1 binding, we used RNAs e and f for UV cross-linking (Figure 3A). MBNL1 cross-linked to both the U and D elements (Figure 3C, lanes 3 and 4). In contrast, recombinant PTB bound to the pyrimidine tract elements (RNAs d and g, lanes 7 and 10) but not to the UGC elements (lanes 8 and 9). Although only the U element was essential for mediating the influence of overexpressed MBNL1 (Figure 2), both the U and D elements can bind MBNL1 (Figure 3C). Indeed, the D element cross-linked more efficiently than the U element (Figure 3C, lanes 3 and 4).Figure 3.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, CB2 1QW, UK.

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.

Show MeSH