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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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The upstream UGC element mediates effects of overexpressed MBNL1. (A) RT–PCR analysis of PAC-1 cells transfected with minigenes that are wild-type (WT), lanes 2–4, or have a deletion of upstream UGC element (ΔU) lanes 5–7, a deletion of the downstream UGC element (ΔD) lanes 8–10, point mutations of the UCUU motifs in exon 3 polypyrimidine tract (ΔP3) lanes 11–13 or a deletion of downstream UCUU element (ΔDY) lanes 14–16, were expressed alone or together with GFP–MBNL1 or GFP. Lane 1, mock control. (B) Western blot analysis showing overexpressed GFP–MBNL1 or GFP with actin as a loading control.
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gkt168-F2: The upstream UGC element mediates effects of overexpressed MBNL1. (A) RT–PCR analysis of PAC-1 cells transfected with minigenes that are wild-type (WT), lanes 2–4, or have a deletion of upstream UGC element (ΔU) lanes 5–7, a deletion of the downstream UGC element (ΔD) lanes 8–10, point mutations of the UCUU motifs in exon 3 polypyrimidine tract (ΔP3) lanes 11–13 or a deletion of downstream UCUU element (ΔDY) lanes 14–16, were expressed alone or together with GFP–MBNL1 or GFP. Lane 1, mock control. (B) Western blot analysis showing overexpressed GFP–MBNL1 or GFP with actin as a loading control.

Mentions: We next tested whether the activity of MBNL proteins on Tpm1 exon 3 was mediated by the known silencer elements (14,16–18). We co-transfected GFP–MBNL1 or GFP expression vectors into PAC-1 cells along with reporter constructs with deletions of either the upstream (ΔU) or downstream (ΔD) UGC elements, the downstream pyrimidine tract (ΔDY), or point mutations of the PTB-binding sites in the polypyrimidine tract of exon 3 [ΔP3 (23,44)] (Figure 2). The wild-type construct showed 36% exon skipping, which was elevated to 51% by GFP–MBNL1 but not GFP (Figure 2A, lanes 2–4). As expected, exon skipping was reduced to between 6 and 10% by each of the regulatory element mutations (compare lane 2 with lanes 5, 8, 11 and 14). However, with the exception of the ΔU deletion mutant (lanes 5–7), each of the regulatory element mutants remained responsive to GFP–MBNL1 overexpression. This suggests that the U element is critical for mediating the effects of overexpressed MBNL1.Figure 2.


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)

The upstream UGC element mediates effects of overexpressed MBNL1. (A) RT–PCR analysis of PAC-1 cells transfected with minigenes that are wild-type (WT), lanes 2–4, or have a deletion of upstream UGC element (ΔU) lanes 5–7, a deletion of the downstream UGC element (ΔD) lanes 8–10, point mutations of the UCUU motifs in exon 3 polypyrimidine tract (ΔP3) lanes 11–13 or a deletion of downstream UCUU element (ΔDY) lanes 14–16, were expressed alone or together with GFP–MBNL1 or GFP. Lane 1, mock control. (B) Western blot analysis showing overexpressed GFP–MBNL1 or GFP with actin as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643581&req=5

gkt168-F2: The upstream UGC element mediates effects of overexpressed MBNL1. (A) RT–PCR analysis of PAC-1 cells transfected with minigenes that are wild-type (WT), lanes 2–4, or have a deletion of upstream UGC element (ΔU) lanes 5–7, a deletion of the downstream UGC element (ΔD) lanes 8–10, point mutations of the UCUU motifs in exon 3 polypyrimidine tract (ΔP3) lanes 11–13 or a deletion of downstream UCUU element (ΔDY) lanes 14–16, were expressed alone or together with GFP–MBNL1 or GFP. Lane 1, mock control. (B) Western blot analysis showing overexpressed GFP–MBNL1 or GFP with actin as a loading control.
Mentions: We next tested whether the activity of MBNL proteins on Tpm1 exon 3 was mediated by the known silencer elements (14,16–18). We co-transfected GFP–MBNL1 or GFP expression vectors into PAC-1 cells along with reporter constructs with deletions of either the upstream (ΔU) or downstream (ΔD) UGC elements, the downstream pyrimidine tract (ΔDY), or point mutations of the PTB-binding sites in the polypyrimidine tract of exon 3 [ΔP3 (23,44)] (Figure 2). The wild-type construct showed 36% exon skipping, which was elevated to 51% by GFP–MBNL1 but not GFP (Figure 2A, lanes 2–4). As expected, exon skipping was reduced to between 6 and 10% by each of the regulatory element mutations (compare lane 2 with lanes 5, 8, 11 and 14). However, with the exception of the ΔU deletion mutant (lanes 5–7), each of the regulatory element mutants remained responsive to GFP–MBNL1 overexpression. This suggests that the U element is critical for mediating the effects of overexpressed MBNL1.Figure 2.

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