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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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MS2 tethering of MBNL1 promotes exon 3 skipping. (A) Schematic representation of FLAG-tagged MBNL1–MS2 fusion proteins. All C-terminal truncations start at amino acid 2 and are full-length (382–MS2), 2–253 (253–MS2), 2–183 (183–MS2), 2–115 (115–MS2), 2–102 (102–MS2), 2–91 (91–MS2) and 2–72 (72–MS2), with a C-terminal fragment amino acids 239–382 (239–382–MS2). The black boxes represent the four ZF domains; the grey shaded box represents the C-terminus. The MS2 coat protein is at the C-terminus and the FLAG tag at the N-terminus. (B) A schematic representation of the Tpm1 minigenes used for recruitment of MS2-fused MBNL1 truncations in PAC-1 cells. Two MS2 hairpins were used to replace the D element (Dms2) and one MS2 hairpin to replace the U element (Ums2). RT–PCR analysis of the MS2 minigene reporters, lanes 2 and 13. Lane 3–11 and 14–22 overexpressed full-length MBNL1–MS2 (382–MS2), amino acids 2–253–MS2 (253–MS2), 2–183–MS2 (183–MS2), 2–115–MS2 (115–MS2), 2–102–MS2 (102–MS2), 2–91–MS2 (91–MS2), 2–72–MS2 (72–MS2) and MS2 only, respectively. Lanes 1 and 12 are mock transfected cells. The ‘% activity’ (white text on black background) was calculated from the difference in percentage of exon skipping between MS2 alone and each construct, normalized to the response of full-length 382–MS2 as 100%. (C) MS2 recruitment in HeLa cells as in (B) using reporter minigene with the branch point mutation (pTΔBP). (D) Western blot analysis using anti-MS2 antibody to detect overexpressed MBNL1 and anti-actin as a loading control in PAC-1 cells (left panel) and HeLa cells (right panel).
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gkt168-F4: MS2 tethering of MBNL1 promotes exon 3 skipping. (A) Schematic representation of FLAG-tagged MBNL1–MS2 fusion proteins. All C-terminal truncations start at amino acid 2 and are full-length (382–MS2), 2–253 (253–MS2), 2–183 (183–MS2), 2–115 (115–MS2), 2–102 (102–MS2), 2–91 (91–MS2) and 2–72 (72–MS2), with a C-terminal fragment amino acids 239–382 (239–382–MS2). The black boxes represent the four ZF domains; the grey shaded box represents the C-terminus. The MS2 coat protein is at the C-terminus and the FLAG tag at the N-terminus. (B) A schematic representation of the Tpm1 minigenes used for recruitment of MS2-fused MBNL1 truncations in PAC-1 cells. Two MS2 hairpins were used to replace the D element (Dms2) and one MS2 hairpin to replace the U element (Ums2). RT–PCR analysis of the MS2 minigene reporters, lanes 2 and 13. Lane 3–11 and 14–22 overexpressed full-length MBNL1–MS2 (382–MS2), amino acids 2–253–MS2 (253–MS2), 2–183–MS2 (183–MS2), 2–115–MS2 (115–MS2), 2–102–MS2 (102–MS2), 2–91–MS2 (91–MS2), 2–72–MS2 (72–MS2) and MS2 only, respectively. Lanes 1 and 12 are mock transfected cells. The ‘% activity’ (white text on black background) was calculated from the difference in percentage of exon skipping between MS2 alone and each construct, normalized to the response of full-length 382–MS2 as 100%. (C) MS2 recruitment in HeLa cells as in (B) using reporter minigene with the branch point mutation (pTΔBP). (D) Western blot analysis using anti-MS2 antibody to detect overexpressed MBNL1 and anti-actin as a loading control in PAC-1 cells (left panel) and HeLa cells (right panel).

Mentions: Replacement of the D element by two MS2 sites led to a reduction of exon skipping in PAC 1 cells to 7% (Figure 4B, lane 2). Full-length MBNL1–MS2 led to an increase in exon skipping to 57%, whereas co-transfection with MS2 was without effect (lanes 3 and 11). Control experiments showed that MBNL1 alone produced a much smaller effect than MBNL1–MS2 (Supplementary Figure S3B, lanes 6 and 10). Thus, artificial recruitment of MBNL1–MS2 compensates for deletion of the D element. We next tested a series of C-terminally truncated MBNL1 mutants fused to MS2, as well as an N-terminal deletion mutant lacking the four ZF domains (Figure 4B), all of which were expressed to similar levels (Figure 4D, left panel). To facilitate comparison of the effects of deletion mutations in different contexts, we calculated the activity of mutants as a percentage of the exon skipping caused by full-length MBNL1–MS2 compared with MS2 alone (numbers with black background in Figure 4B and C). This value is not corrected for the residual activity of untethered MBNL proteins (Supplementary Figure S3), but only the full-length protein and the 2–253 mutant showed any activity in an untethered assay (Figure 1D). The C-terminal deletion mutant containing all four ZFs (amino acids 2–253) retained 83% of full-length activity, whereas ZF1 and 2 and the complete following linker region (amino acids 2–183) retained 78% of full-length activity (lanes 4 and 5). Further deletions removing the linker region between ZF2 and 3 reduced activity progressively (lanes 5–9), with amino acids 2–72 having only 8% activity. An N-terminal deletion mutant lacking all ZFs (amino acids 239–382) had 34% of full-length activity (lane 10).Figure 4.


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)

MS2 tethering of MBNL1 promotes exon 3 skipping. (A) Schematic representation of FLAG-tagged MBNL1–MS2 fusion proteins. All C-terminal truncations start at amino acid 2 and are full-length (382–MS2), 2–253 (253–MS2), 2–183 (183–MS2), 2–115 (115–MS2), 2–102 (102–MS2), 2–91 (91–MS2) and 2–72 (72–MS2), with a C-terminal fragment amino acids 239–382 (239–382–MS2). The black boxes represent the four ZF domains; the grey shaded box represents the C-terminus. The MS2 coat protein is at the C-terminus and the FLAG tag at the N-terminus. (B) A schematic representation of the Tpm1 minigenes used for recruitment of MS2-fused MBNL1 truncations in PAC-1 cells. Two MS2 hairpins were used to replace the D element (Dms2) and one MS2 hairpin to replace the U element (Ums2). RT–PCR analysis of the MS2 minigene reporters, lanes 2 and 13. Lane 3–11 and 14–22 overexpressed full-length MBNL1–MS2 (382–MS2), amino acids 2–253–MS2 (253–MS2), 2–183–MS2 (183–MS2), 2–115–MS2 (115–MS2), 2–102–MS2 (102–MS2), 2–91–MS2 (91–MS2), 2–72–MS2 (72–MS2) and MS2 only, respectively. Lanes 1 and 12 are mock transfected cells. The ‘% activity’ (white text on black background) was calculated from the difference in percentage of exon skipping between MS2 alone and each construct, normalized to the response of full-length 382–MS2 as 100%. (C) MS2 recruitment in HeLa cells as in (B) using reporter minigene with the branch point mutation (pTΔBP). (D) Western blot analysis using anti-MS2 antibody to detect overexpressed MBNL1 and anti-actin as a loading control in PAC-1 cells (left panel) and HeLa cells (right panel).
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gkt168-F4: MS2 tethering of MBNL1 promotes exon 3 skipping. (A) Schematic representation of FLAG-tagged MBNL1–MS2 fusion proteins. All C-terminal truncations start at amino acid 2 and are full-length (382–MS2), 2–253 (253–MS2), 2–183 (183–MS2), 2–115 (115–MS2), 2–102 (102–MS2), 2–91 (91–MS2) and 2–72 (72–MS2), with a C-terminal fragment amino acids 239–382 (239–382–MS2). The black boxes represent the four ZF domains; the grey shaded box represents the C-terminus. The MS2 coat protein is at the C-terminus and the FLAG tag at the N-terminus. (B) A schematic representation of the Tpm1 minigenes used for recruitment of MS2-fused MBNL1 truncations in PAC-1 cells. Two MS2 hairpins were used to replace the D element (Dms2) and one MS2 hairpin to replace the U element (Ums2). RT–PCR analysis of the MS2 minigene reporters, lanes 2 and 13. Lane 3–11 and 14–22 overexpressed full-length MBNL1–MS2 (382–MS2), amino acids 2–253–MS2 (253–MS2), 2–183–MS2 (183–MS2), 2–115–MS2 (115–MS2), 2–102–MS2 (102–MS2), 2–91–MS2 (91–MS2), 2–72–MS2 (72–MS2) and MS2 only, respectively. Lanes 1 and 12 are mock transfected cells. The ‘% activity’ (white text on black background) was calculated from the difference in percentage of exon skipping between MS2 alone and each construct, normalized to the response of full-length 382–MS2 as 100%. (C) MS2 recruitment in HeLa cells as in (B) using reporter minigene with the branch point mutation (pTΔBP). (D) Western blot analysis using anti-MS2 antibody to detect overexpressed MBNL1 and anti-actin as a loading control in PAC-1 cells (left panel) and HeLa cells (right panel).
Mentions: Replacement of the D element by two MS2 sites led to a reduction of exon skipping in PAC 1 cells to 7% (Figure 4B, lane 2). Full-length MBNL1–MS2 led to an increase in exon skipping to 57%, whereas co-transfection with MS2 was without effect (lanes 3 and 11). Control experiments showed that MBNL1 alone produced a much smaller effect than MBNL1–MS2 (Supplementary Figure S3B, lanes 6 and 10). Thus, artificial recruitment of MBNL1–MS2 compensates for deletion of the D element. We next tested a series of C-terminally truncated MBNL1 mutants fused to MS2, as well as an N-terminal deletion mutant lacking the four ZF domains (Figure 4B), all of which were expressed to similar levels (Figure 4D, left panel). To facilitate comparison of the effects of deletion mutations in different contexts, we calculated the activity of mutants as a percentage of the exon skipping caused by full-length MBNL1–MS2 compared with MS2 alone (numbers with black background in Figure 4B and C). This value is not corrected for the residual activity of untethered MBNL proteins (Supplementary Figure S3), but only the full-length protein and the 2–253 mutant showed any activity in an untethered assay (Figure 1D). The C-terminal deletion mutant containing all four ZFs (amino acids 2–253) retained 83% of full-length activity, whereas ZF1 and 2 and the complete following linker region (amino acids 2–183) retained 78% of full-length activity (lanes 4 and 5). Further deletions removing the linker region between ZF2 and 3 reduced activity progressively (lanes 5–9), with amino acids 2–72 having only 8% activity. An N-terminal deletion mutant lacking all ZFs (amino acids 239–382) had 34% of full-length activity (lane 10).Figure 4.

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