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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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Interaction between MBNL and PTB N-terminal domains. (A) Schematic representation of Venus–MBNL and Cherry–PTB proteins. (B) Western blot of input (left panel) and GFP Trap (right panel) of Venus-tagged MBNL1 co-expressed with FLAG–PTB4 RRM domains in formaldehyde cross-linked 293 T extracts; markers (lane 1), Venus vector alone (lane 2 and 8), full-length FLAG–PTB4 (lanes 3 and 9). Lanes 4–7 and 10–13 all have full-length Venus–MBNL1 together with full-length PTB4 (lanes 4 and 10), FLAG–PTB4-12 L (lanes 5 and 11), FLAG–PTB4-2 L (lanes 6 and 12) and FLAG–PTB4-34 (lanes 7 and 13). (C) For each panel the top image shows the Venus–MBNL1 fluorescence, and the lower image shows the corresponding fluorescence lifetime in absence (a) or presence of the indicated PTB constructs (b–d). Full-length PTB and the first 2 RRMs plus the following linker bind to MBNL1, as indicated by reduced fluorescence lifetime (b,c) On the other hand the C-terminus of PTB containing RRMs 3 and 4 (d) cannot bind to MBNL1 and no energy transfer occurred with Cherry fused to either terminus of PTB-34. Scale bar = 10 μm. (D) Schematic presentation of tagged PTB RRMs and the statistical analysis of (B). Error bars are SEM of at least five independent fields of view with approximately four cells per image.
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gkt168-F6: Interaction between MBNL and PTB N-terminal domains. (A) Schematic representation of Venus–MBNL and Cherry–PTB proteins. (B) Western blot of input (left panel) and GFP Trap (right panel) of Venus-tagged MBNL1 co-expressed with FLAG–PTB4 RRM domains in formaldehyde cross-linked 293 T extracts; markers (lane 1), Venus vector alone (lane 2 and 8), full-length FLAG–PTB4 (lanes 3 and 9). Lanes 4–7 and 10–13 all have full-length Venus–MBNL1 together with full-length PTB4 (lanes 4 and 10), FLAG–PTB4-12 L (lanes 5 and 11), FLAG–PTB4-2 L (lanes 6 and 12) and FLAG–PTB4-34 (lanes 7 and 13). (C) For each panel the top image shows the Venus–MBNL1 fluorescence, and the lower image shows the corresponding fluorescence lifetime in absence (a) or presence of the indicated PTB constructs (b–d). Full-length PTB and the first 2 RRMs plus the following linker bind to MBNL1, as indicated by reduced fluorescence lifetime (b,c) On the other hand the C-terminus of PTB containing RRMs 3 and 4 (d) cannot bind to MBNL1 and no energy transfer occurred with Cherry fused to either terminus of PTB-34. Scale bar = 10 μm. (D) Schematic presentation of tagged PTB RRMs and the statistical analysis of (B). Error bars are SEM of at least five independent fields of view with approximately four cells per image.

Mentions: We next asked which region(s) of PTB were involved in the interaction (Figure 6) by the cross-link co-immunopreciptation approach using full-length Venus–MBNL1 and FLAG-tagged PTB deletion mutants. Pull-down was carried out with GFP–TRAP beads and western blots with anti-GFP and anti-FLAG (Figure 6B). Venus–MBNL1 pulled down full-length PTB (Figure 6B, lane 10), as well as the N-terminal part of PTB containing RRM domains 1 and 2 and following linker (lane 11). However, RRM2 and the following linker (2L, lane 12) or RRMs 3 and 4 (lane 13) were not pulled down. Similar results were seen by FLIM–FRET (Figure 6C and D); the fluorescence lifetime of nuclear Venus–MBNL1 was reduced by mCherry–PTB or Cherry–PTB-12L, but not by mCherry–PTB-34 (Figure 6C and D).Figure 6.


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)

Interaction between MBNL and PTB N-terminal domains. (A) Schematic representation of Venus–MBNL and Cherry–PTB proteins. (B) Western blot of input (left panel) and GFP Trap (right panel) of Venus-tagged MBNL1 co-expressed with FLAG–PTB4 RRM domains in formaldehyde cross-linked 293 T extracts; markers (lane 1), Venus vector alone (lane 2 and 8), full-length FLAG–PTB4 (lanes 3 and 9). Lanes 4–7 and 10–13 all have full-length Venus–MBNL1 together with full-length PTB4 (lanes 4 and 10), FLAG–PTB4-12 L (lanes 5 and 11), FLAG–PTB4-2 L (lanes 6 and 12) and FLAG–PTB4-34 (lanes 7 and 13). (C) For each panel the top image shows the Venus–MBNL1 fluorescence, and the lower image shows the corresponding fluorescence lifetime in absence (a) or presence of the indicated PTB constructs (b–d). Full-length PTB and the first 2 RRMs plus the following linker bind to MBNL1, as indicated by reduced fluorescence lifetime (b,c) On the other hand the C-terminus of PTB containing RRMs 3 and 4 (d) cannot bind to MBNL1 and no energy transfer occurred with Cherry fused to either terminus of PTB-34. Scale bar = 10 μm. (D) Schematic presentation of tagged PTB RRMs and the statistical analysis of (B). Error bars are SEM of at least five independent fields of view with approximately four cells per image.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt168-F6: Interaction between MBNL and PTB N-terminal domains. (A) Schematic representation of Venus–MBNL and Cherry–PTB proteins. (B) Western blot of input (left panel) and GFP Trap (right panel) of Venus-tagged MBNL1 co-expressed with FLAG–PTB4 RRM domains in formaldehyde cross-linked 293 T extracts; markers (lane 1), Venus vector alone (lane 2 and 8), full-length FLAG–PTB4 (lanes 3 and 9). Lanes 4–7 and 10–13 all have full-length Venus–MBNL1 together with full-length PTB4 (lanes 4 and 10), FLAG–PTB4-12 L (lanes 5 and 11), FLAG–PTB4-2 L (lanes 6 and 12) and FLAG–PTB4-34 (lanes 7 and 13). (C) For each panel the top image shows the Venus–MBNL1 fluorescence, and the lower image shows the corresponding fluorescence lifetime in absence (a) or presence of the indicated PTB constructs (b–d). Full-length PTB and the first 2 RRMs plus the following linker bind to MBNL1, as indicated by reduced fluorescence lifetime (b,c) On the other hand the C-terminus of PTB containing RRMs 3 and 4 (d) cannot bind to MBNL1 and no energy transfer occurred with Cherry fused to either terminus of PTB-34. Scale bar = 10 μm. (D) Schematic presentation of tagged PTB RRMs and the statistical analysis of (B). Error bars are SEM of at least five independent fields of view with approximately four cells per image.
Mentions: We next asked which region(s) of PTB were involved in the interaction (Figure 6) by the cross-link co-immunopreciptation approach using full-length Venus–MBNL1 and FLAG-tagged PTB deletion mutants. Pull-down was carried out with GFP–TRAP beads and western blots with anti-GFP and anti-FLAG (Figure 6B). Venus–MBNL1 pulled down full-length PTB (Figure 6B, lane 10), as well as the N-terminal part of PTB containing RRM domains 1 and 2 and following linker (lane 11). However, RRM2 and the following linker (2L, lane 12) or RRMs 3 and 4 (lane 13) were not pulled down. Similar results were seen by FLIM–FRET (Figure 6C and D); the fluorescence lifetime of nuclear Venus–MBNL1 was reduced by mCherry–PTB or Cherry–PTB-12L, but not by mCherry–PTB-34 (Figure 6C and D).Figure 6.

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
Related in: MedlinePlus