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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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RNA binding by MBNL1 enhances the interaction with PTB. (A) Silver stain gel of GST pull-down in HeLa nuclear extract with GST-MBNL-2–116, 2–116 m, 2–72 and GST alone (lanes 2–6). The doublet identified by mass spectrometry as PTB is marked by an asterisk. This doublet was not observed if extract was treated with RNase. (B) Western blot of pull-down from A probing with anti-PTB, anti-hnRNP C, anti-hnRNP H and anti-hnRNP L (right panel) with a titration of the input 0.01, 0.1, 1, 10 and 100% (left panel). (C) Schematic diagram of Tpm1 RNA species used in panels D, F and G. (D) Western blots using anti-PTB (upper panel) and anti-GST (lower panel). GST–MBNL-2–116 pull-down in HeLa nuclear extract with increasing amounts of RNA. No RNA added (lane 1), RNA a (lanes 2–5, Figure 3), MBNL SELEX RNA (lanes 6–8), PTB SELEX RNA (lanes 9–11), MBNL and PTB SELEX RNAs together (lanes 12–14). (E) UV X-linking of recombinant pQE–PTB4, left panel, or GST–MBNL1 amino acids 2–253, right panel, to either the MBNL SELEX RNA, lanes 1 and 3, or the PTB SELEX RNA, lanes 2 and 4. (F) GST pull-down with recombinant GST–MBNL1 amino acids 2–253 and pQE–PTB4 with a titration of RNA. Markers (lane 1), recombinant GST protein bound to beads minus and plus recombinant pQE–PTB4 (lanes 2 and 3), recombinant GST–MBNL1-2–253 bound to beads (lane 4, input), recombinant pQE–PTB4 (lane 5, 15% of input), GST–MBNL beads plus pQE–PTB4 (lanes 6–21) with no RNA added (lanes 6, 10, 14 and 18), titration of RNA a (20, 2 and 0.2 pmol, lanes 7–9), titration of MBNL SELEX (20, 2 and 0.2 pmol, lanes 11–13), titration of PTB SELEX (20, 2 and 0.2 pmol, lanes 15–17) and titration of RNA f (20, 2 and 0.2 pmol, lanes 19–21). (G) GST pull-down with recombinant GST–MBNL1 C-terminal truncations indicated and pQE–PTB4 minus RNA (lanes 1, 3, 5, 7, 9 and 11) and plus 40 pmol RNA f (lanes 2, 4, 6, 8, 10 and 12).
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gkt168-F7: RNA binding by MBNL1 enhances the interaction with PTB. (A) Silver stain gel of GST pull-down in HeLa nuclear extract with GST-MBNL-2–116, 2–116 m, 2–72 and GST alone (lanes 2–6). The doublet identified by mass spectrometry as PTB is marked by an asterisk. This doublet was not observed if extract was treated with RNase. (B) Western blot of pull-down from A probing with anti-PTB, anti-hnRNP C, anti-hnRNP H and anti-hnRNP L (right panel) with a titration of the input 0.01, 0.1, 1, 10 and 100% (left panel). (C) Schematic diagram of Tpm1 RNA species used in panels D, F and G. (D) Western blots using anti-PTB (upper panel) and anti-GST (lower panel). GST–MBNL-2–116 pull-down in HeLa nuclear extract with increasing amounts of RNA. No RNA added (lane 1), RNA a (lanes 2–5, Figure 3), MBNL SELEX RNA (lanes 6–8), PTB SELEX RNA (lanes 9–11), MBNL and PTB SELEX RNAs together (lanes 12–14). (E) UV X-linking of recombinant pQE–PTB4, left panel, or GST–MBNL1 amino acids 2–253, right panel, to either the MBNL SELEX RNA, lanes 1 and 3, or the PTB SELEX RNA, lanes 2 and 4. (F) GST pull-down with recombinant GST–MBNL1 amino acids 2–253 and pQE–PTB4 with a titration of RNA. Markers (lane 1), recombinant GST protein bound to beads minus and plus recombinant pQE–PTB4 (lanes 2 and 3), recombinant GST–MBNL1-2–253 bound to beads (lane 4, input), recombinant pQE–PTB4 (lane 5, 15% of input), GST–MBNL beads plus pQE–PTB4 (lanes 6–21) with no RNA added (lanes 6, 10, 14 and 18), titration of RNA a (20, 2 and 0.2 pmol, lanes 7–9), titration of MBNL SELEX (20, 2 and 0.2 pmol, lanes 11–13), titration of PTB SELEX (20, 2 and 0.2 pmol, lanes 15–17) and titration of RNA f (20, 2 and 0.2 pmol, lanes 19–21). (G) GST pull-down with recombinant GST–MBNL1 C-terminal truncations indicated and pQE–PTB4 minus RNA (lanes 1, 3, 5, 7, 9 and 11) and plus 40 pmol RNA f (lanes 2, 4, 6, 8, 10 and 12).

Mentions: To further investigate the role of RNA in the PTB–MBNL1 interaction, we purified recombinant GST fusion proteins containing MBNL1 amino acids 2–116, 2–91 and 2–72, and also a 2–116-m protein with point mutations that impair RNA binding by ZnF1 and 2 and also abolish its activity in an MS2 tethering assay (C. Edge et al. in preparation). These fragments were chosen on the basis that 2–116 maintained high levels of activity in HeLa cells when tethered at the U or D position, whereas 2–72 had little residual activity (Figure 4). The GST–MBNL fusion proteins were bound to glutathione sepharose beads and then incubated in HeLa cell nuclear extract in the presence or absence of RNases. Beads were washed, and bound proteins were eluted in SDS loading buffer. A prominent 57/59 kDa doublet was pulled down by 2–116 in the absence of RNase, but not with the 2–72 construct or after treatment with RNase (Figure 7A). The 57/59 kDa doublet is the characteristic size of PTB, and its identity was confirmed by mass spectrometry (data not shown). Western blotting further confirmed that PTB was pulled down by 2–116 in the absence of RNase, but not by the 2–116 -m RNA-binding mutant or the 2–72 proteins (Figure 7B). RNase sensitive interactions between RNA-binding proteins might be explained by independent binding of each protein to a common ‘bridging’ RNA, and they are often dismissed as non-specific. Nevertheless, the RNA-dependent interaction between PTB and MBNL1 2–116 showed some specificity because other abundant hnRNP proteins (hnRNPs C, H, L) were not pulled down (Figure 7B).Figure 7.


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)

RNA binding by MBNL1 enhances the interaction with PTB. (A) Silver stain gel of GST pull-down in HeLa nuclear extract with GST-MBNL-2–116, 2–116 m, 2–72 and GST alone (lanes 2–6). The doublet identified by mass spectrometry as PTB is marked by an asterisk. This doublet was not observed if extract was treated with RNase. (B) Western blot of pull-down from A probing with anti-PTB, anti-hnRNP C, anti-hnRNP H and anti-hnRNP L (right panel) with a titration of the input 0.01, 0.1, 1, 10 and 100% (left panel). (C) Schematic diagram of Tpm1 RNA species used in panels D, F and G. (D) Western blots using anti-PTB (upper panel) and anti-GST (lower panel). GST–MBNL-2–116 pull-down in HeLa nuclear extract with increasing amounts of RNA. No RNA added (lane 1), RNA a (lanes 2–5, Figure 3), MBNL SELEX RNA (lanes 6–8), PTB SELEX RNA (lanes 9–11), MBNL and PTB SELEX RNAs together (lanes 12–14). (E) UV X-linking of recombinant pQE–PTB4, left panel, or GST–MBNL1 amino acids 2–253, right panel, to either the MBNL SELEX RNA, lanes 1 and 3, or the PTB SELEX RNA, lanes 2 and 4. (F) GST pull-down with recombinant GST–MBNL1 amino acids 2–253 and pQE–PTB4 with a titration of RNA. Markers (lane 1), recombinant GST protein bound to beads minus and plus recombinant pQE–PTB4 (lanes 2 and 3), recombinant GST–MBNL1-2–253 bound to beads (lane 4, input), recombinant pQE–PTB4 (lane 5, 15% of input), GST–MBNL beads plus pQE–PTB4 (lanes 6–21) with no RNA added (lanes 6, 10, 14 and 18), titration of RNA a (20, 2 and 0.2 pmol, lanes 7–9), titration of MBNL SELEX (20, 2 and 0.2 pmol, lanes 11–13), titration of PTB SELEX (20, 2 and 0.2 pmol, lanes 15–17) and titration of RNA f (20, 2 and 0.2 pmol, lanes 19–21). (G) GST pull-down with recombinant GST–MBNL1 C-terminal truncations indicated and pQE–PTB4 minus RNA (lanes 1, 3, 5, 7, 9 and 11) and plus 40 pmol RNA f (lanes 2, 4, 6, 8, 10 and 12).
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gkt168-F7: RNA binding by MBNL1 enhances the interaction with PTB. (A) Silver stain gel of GST pull-down in HeLa nuclear extract with GST-MBNL-2–116, 2–116 m, 2–72 and GST alone (lanes 2–6). The doublet identified by mass spectrometry as PTB is marked by an asterisk. This doublet was not observed if extract was treated with RNase. (B) Western blot of pull-down from A probing with anti-PTB, anti-hnRNP C, anti-hnRNP H and anti-hnRNP L (right panel) with a titration of the input 0.01, 0.1, 1, 10 and 100% (left panel). (C) Schematic diagram of Tpm1 RNA species used in panels D, F and G. (D) Western blots using anti-PTB (upper panel) and anti-GST (lower panel). GST–MBNL-2–116 pull-down in HeLa nuclear extract with increasing amounts of RNA. No RNA added (lane 1), RNA a (lanes 2–5, Figure 3), MBNL SELEX RNA (lanes 6–8), PTB SELEX RNA (lanes 9–11), MBNL and PTB SELEX RNAs together (lanes 12–14). (E) UV X-linking of recombinant pQE–PTB4, left panel, or GST–MBNL1 amino acids 2–253, right panel, to either the MBNL SELEX RNA, lanes 1 and 3, or the PTB SELEX RNA, lanes 2 and 4. (F) GST pull-down with recombinant GST–MBNL1 amino acids 2–253 and pQE–PTB4 with a titration of RNA. Markers (lane 1), recombinant GST protein bound to beads minus and plus recombinant pQE–PTB4 (lanes 2 and 3), recombinant GST–MBNL1-2–253 bound to beads (lane 4, input), recombinant pQE–PTB4 (lane 5, 15% of input), GST–MBNL beads plus pQE–PTB4 (lanes 6–21) with no RNA added (lanes 6, 10, 14 and 18), titration of RNA a (20, 2 and 0.2 pmol, lanes 7–9), titration of MBNL SELEX (20, 2 and 0.2 pmol, lanes 11–13), titration of PTB SELEX (20, 2 and 0.2 pmol, lanes 15–17) and titration of RNA f (20, 2 and 0.2 pmol, lanes 19–21). (G) GST pull-down with recombinant GST–MBNL1 C-terminal truncations indicated and pQE–PTB4 minus RNA (lanes 1, 3, 5, 7, 9 and 11) and plus 40 pmol RNA f (lanes 2, 4, 6, 8, 10 and 12).
Mentions: To further investigate the role of RNA in the PTB–MBNL1 interaction, we purified recombinant GST fusion proteins containing MBNL1 amino acids 2–116, 2–91 and 2–72, and also a 2–116-m protein with point mutations that impair RNA binding by ZnF1 and 2 and also abolish its activity in an MS2 tethering assay (C. Edge et al. in preparation). These fragments were chosen on the basis that 2–116 maintained high levels of activity in HeLa cells when tethered at the U or D position, whereas 2–72 had little residual activity (Figure 4). The GST–MBNL fusion proteins were bound to glutathione sepharose beads and then incubated in HeLa cell nuclear extract in the presence or absence of RNases. Beads were washed, and bound proteins were eluted in SDS loading buffer. A prominent 57/59 kDa doublet was pulled down by 2–116 in the absence of RNase, but not with the 2–72 construct or after treatment with RNase (Figure 7A). The 57/59 kDa doublet is the characteristic size of PTB, and its identity was confirmed by mass spectrometry (data not shown). Western blotting further confirmed that PTB was pulled down by 2–116 in the absence of RNase, but not by the 2–116 -m RNA-binding mutant or the 2–72 proteins (Figure 7B). RNase sensitive interactions between RNA-binding proteins might be explained by independent binding of each protein to a common ‘bridging’ RNA, and they are often dismissed as non-specific. Nevertheless, the RNA-dependent interaction between PTB and MBNL1 2–116 showed some specificity because other abundant hnRNP proteins (hnRNPs C, H, L) were not pulled down (Figure 7B).Figure 7.

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