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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-like proteins repress Tpm1 exon 3. (A) Schematic representation of the mutually exclusive splicing of Tpm1 exons 2 and 3. The essential negative regulatory elements flanking exon 3 are indicated. The P3 and DY elements bind PTB and are denoted by black and white rectangles, respectively. The U and D elements are indicated by black and white diamonds, respectively, and their sequences are indicated. Matches to MBNL1 consensus sequences are underlined (YGCU(U/G)Y) and overlined (YGCY). (B) Western blot of siRNA knockdown, probed with α-MBNL1, α-MBNL2 or α-actin antibodies with a titration of control (C2) knockdown sample at 20, 40, 60, 80 and 100% (lanes 1–5) compared with knockdown of MBNL1, MBNL2 or MBNL1 plus MBNL2 (lanes 6–8). (C) qRT–PCR analysis of endogenous Tpm1 on knockdown of MBNL1 and MBNL2. The histogram shows the fold change of exon 2 or exon 3 products comparing control siRNA (C2) with knockdown of MBNL1 (1), MBNL2 (2) or MBNL1 plus MBNL2 (1 + 2). (D) Schematic representation of Tpm1 minigene reporter containing exons 1, 3 and 4 on the left, and Venus-tagged MBNL1 on the right. RT–PCR analysis of RNA isolated from PAC-1 cells (left panel) or HeLa cells (right panel) transfected with the Tpm1 minigene reporter (lanes 1 and 11). In HeLa cells, the Tpm1 mingene has a branch point mutation changing the wild-type sequence from GGCUAAC to GGCUGGC. Lanes 2–10, siRNA knockdown of MBNL1 and MBNL2 in PAC-1 cells together with overexpressed Venus–MBNL1 with the siRNA site mutated (lane 3), or various C-terminal truncations of Venus–MBNL1 (lanes 4–9); the yellow dots indicate Venus and the adjacent number represents the position of the C-terminal deletion. Lane 10 is transfected with an N-terminal MBNL1 deletion containing amino acids 239–382. Lanes 12 and 13, siRNA knockdown of MBNL1 in HeLa cells together with overexpressed GFP–MBNL1 with the siRNA site mutated, MBNL1m (lane 13). Right panel: anti-GFP (detects Venus) western blot; lane numbers correspond to those in the left panel.
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gkt168-F1: MBNL-like proteins repress Tpm1 exon 3. (A) Schematic representation of the mutually exclusive splicing of Tpm1 exons 2 and 3. The essential negative regulatory elements flanking exon 3 are indicated. The P3 and DY elements bind PTB and are denoted by black and white rectangles, respectively. The U and D elements are indicated by black and white diamonds, respectively, and their sequences are indicated. Matches to MBNL1 consensus sequences are underlined (YGCU(U/G)Y) and overlined (YGCY). (B) Western blot of siRNA knockdown, probed with α-MBNL1, α-MBNL2 or α-actin antibodies with a titration of control (C2) knockdown sample at 20, 40, 60, 80 and 100% (lanes 1–5) compared with knockdown of MBNL1, MBNL2 or MBNL1 plus MBNL2 (lanes 6–8). (C) qRT–PCR analysis of endogenous Tpm1 on knockdown of MBNL1 and MBNL2. The histogram shows the fold change of exon 2 or exon 3 products comparing control siRNA (C2) with knockdown of MBNL1 (1), MBNL2 (2) or MBNL1 plus MBNL2 (1 + 2). (D) Schematic representation of Tpm1 minigene reporter containing exons 1, 3 and 4 on the left, and Venus-tagged MBNL1 on the right. RT–PCR analysis of RNA isolated from PAC-1 cells (left panel) or HeLa cells (right panel) transfected with the Tpm1 minigene reporter (lanes 1 and 11). In HeLa cells, the Tpm1 mingene has a branch point mutation changing the wild-type sequence from GGCUAAC to GGCUGGC. Lanes 2–10, siRNA knockdown of MBNL1 and MBNL2 in PAC-1 cells together with overexpressed Venus–MBNL1 with the siRNA site mutated (lane 3), or various C-terminal truncations of Venus–MBNL1 (lanes 4–9); the yellow dots indicate Venus and the adjacent number represents the position of the C-terminal deletion. Lane 10 is transfected with an N-terminal MBNL1 deletion containing amino acids 239–382. Lanes 12 and 13, siRNA knockdown of MBNL1 in HeLa cells together with overexpressed GFP–MBNL1 with the siRNA site mutated, MBNL1m (lane 13). Right panel: anti-GFP (detects Venus) western blot; lane numbers correspond to those in the left panel.

Mentions: Mutually exclusive exons 2 and 3 of the rat α-tropomyosin (Tpm1) gene have been used as a model system to investigate mechanisms of smooth muscle-regulated alternative splicing [Figure 1A; reviewed in (9)]. Exon 2 is selected in smooth muscle tissues, such as the vasculature, whereas exon 3 is exclusively selected in skeletal muscle, heart and brain (10,11). This event was singled out as an exemplar ‘switch-event’ in one of the first RNA-Seq–based global analyses of tissue-specific alternative splicing because of the magnitude of variation in the splicing pattern between tissues (1). In most tissue culture cells, transcripts with exon 3 are the default products (12), but in the smooth muscle, PAC1 cell line exon 2 is included (13,14). Default selection of exon 3 is driven by its strong branch point and pyrimidine tract elements (12) (denoted BP and P3 in Figure 1A). The exon 3 branch point is sufficiently close to exon 2 to prevent the two exons from being spliced together, thereby enforcing mutually exclusive behaviour (15). The switch to exon 2 selection involves regulated inhibition of exon 3 (11,14). Silencer elements consisting of two types of sequence motif are located in both introns flanking exon 3 (11,14,16,17). The branch point-associated P3 pyrimidine tract and a second downstream pyrimidine tract (denoted DY) bind the splicing regulator polypyrimidine tract-binding protein (PTB) (Figure 1A) (17–20). Two clusters of UGC motifs also lie adjacent to these pyrimidine tracts closer to the exon and are denoted here as ‘D’ and ‘U’ (previously referred to as Dugc and URE, respectively) (14,16).Figure 1.


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-like proteins repress Tpm1 exon 3. (A) Schematic representation of the mutually exclusive splicing of Tpm1 exons 2 and 3. The essential negative regulatory elements flanking exon 3 are indicated. The P3 and DY elements bind PTB and are denoted by black and white rectangles, respectively. The U and D elements are indicated by black and white diamonds, respectively, and their sequences are indicated. Matches to MBNL1 consensus sequences are underlined (YGCU(U/G)Y) and overlined (YGCY). (B) Western blot of siRNA knockdown, probed with α-MBNL1, α-MBNL2 or α-actin antibodies with a titration of control (C2) knockdown sample at 20, 40, 60, 80 and 100% (lanes 1–5) compared with knockdown of MBNL1, MBNL2 or MBNL1 plus MBNL2 (lanes 6–8). (C) qRT–PCR analysis of endogenous Tpm1 on knockdown of MBNL1 and MBNL2. The histogram shows the fold change of exon 2 or exon 3 products comparing control siRNA (C2) with knockdown of MBNL1 (1), MBNL2 (2) or MBNL1 plus MBNL2 (1 + 2). (D) Schematic representation of Tpm1 minigene reporter containing exons 1, 3 and 4 on the left, and Venus-tagged MBNL1 on the right. RT–PCR analysis of RNA isolated from PAC-1 cells (left panel) or HeLa cells (right panel) transfected with the Tpm1 minigene reporter (lanes 1 and 11). In HeLa cells, the Tpm1 mingene has a branch point mutation changing the wild-type sequence from GGCUAAC to GGCUGGC. Lanes 2–10, siRNA knockdown of MBNL1 and MBNL2 in PAC-1 cells together with overexpressed Venus–MBNL1 with the siRNA site mutated (lane 3), or various C-terminal truncations of Venus–MBNL1 (lanes 4–9); the yellow dots indicate Venus and the adjacent number represents the position of the C-terminal deletion. Lane 10 is transfected with an N-terminal MBNL1 deletion containing amino acids 239–382. Lanes 12 and 13, siRNA knockdown of MBNL1 in HeLa cells together with overexpressed GFP–MBNL1 with the siRNA site mutated, MBNL1m (lane 13). Right panel: anti-GFP (detects Venus) western blot; lane numbers correspond to those in the left panel.
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gkt168-F1: MBNL-like proteins repress Tpm1 exon 3. (A) Schematic representation of the mutually exclusive splicing of Tpm1 exons 2 and 3. The essential negative regulatory elements flanking exon 3 are indicated. The P3 and DY elements bind PTB and are denoted by black and white rectangles, respectively. The U and D elements are indicated by black and white diamonds, respectively, and their sequences are indicated. Matches to MBNL1 consensus sequences are underlined (YGCU(U/G)Y) and overlined (YGCY). (B) Western blot of siRNA knockdown, probed with α-MBNL1, α-MBNL2 or α-actin antibodies with a titration of control (C2) knockdown sample at 20, 40, 60, 80 and 100% (lanes 1–5) compared with knockdown of MBNL1, MBNL2 or MBNL1 plus MBNL2 (lanes 6–8). (C) qRT–PCR analysis of endogenous Tpm1 on knockdown of MBNL1 and MBNL2. The histogram shows the fold change of exon 2 or exon 3 products comparing control siRNA (C2) with knockdown of MBNL1 (1), MBNL2 (2) or MBNL1 plus MBNL2 (1 + 2). (D) Schematic representation of Tpm1 minigene reporter containing exons 1, 3 and 4 on the left, and Venus-tagged MBNL1 on the right. RT–PCR analysis of RNA isolated from PAC-1 cells (left panel) or HeLa cells (right panel) transfected with the Tpm1 minigene reporter (lanes 1 and 11). In HeLa cells, the Tpm1 mingene has a branch point mutation changing the wild-type sequence from GGCUAAC to GGCUGGC. Lanes 2–10, siRNA knockdown of MBNL1 and MBNL2 in PAC-1 cells together with overexpressed Venus–MBNL1 with the siRNA site mutated (lane 3), or various C-terminal truncations of Venus–MBNL1 (lanes 4–9); the yellow dots indicate Venus and the adjacent number represents the position of the C-terminal deletion. Lane 10 is transfected with an N-terminal MBNL1 deletion containing amino acids 239–382. Lanes 12 and 13, siRNA knockdown of MBNL1 in HeLa cells together with overexpressed GFP–MBNL1 with the siRNA site mutated, MBNL1m (lane 13). Right panel: anti-GFP (detects Venus) western blot; lane numbers correspond to those in the left panel.
Mentions: Mutually exclusive exons 2 and 3 of the rat α-tropomyosin (Tpm1) gene have been used as a model system to investigate mechanisms of smooth muscle-regulated alternative splicing [Figure 1A; reviewed in (9)]. Exon 2 is selected in smooth muscle tissues, such as the vasculature, whereas exon 3 is exclusively selected in skeletal muscle, heart and brain (10,11). This event was singled out as an exemplar ‘switch-event’ in one of the first RNA-Seq–based global analyses of tissue-specific alternative splicing because of the magnitude of variation in the splicing pattern between tissues (1). In most tissue culture cells, transcripts with exon 3 are the default products (12), but in the smooth muscle, PAC1 cell line exon 2 is included (13,14). Default selection of exon 3 is driven by its strong branch point and pyrimidine tract elements (12) (denoted BP and P3 in Figure 1A). The exon 3 branch point is sufficiently close to exon 2 to prevent the two exons from being spliced together, thereby enforcing mutually exclusive behaviour (15). The switch to exon 2 selection involves regulated inhibition of exon 3 (11,14). Silencer elements consisting of two types of sequence motif are located in both introns flanking exon 3 (11,14,16,17). The branch point-associated P3 pyrimidine tract and a second downstream pyrimidine tract (denoted DY) bind the splicing regulator polypyrimidine tract-binding protein (PTB) (Figure 1A) (17–20). Two clusters of UGC motifs also lie adjacent to these pyrimidine tracts closer to the exon and are denoted here as ‘D’ and ‘U’ (previously referred to as Dugc and URE, respectively) (14,16).Figure 1.

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