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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 of MBNL truncations with PTB. (A) Schematic representation of Venus-tagged MBNL1 fusion proteins with the amino acid boundaries indicated (upper). Black boxes represent the four ZF domains, the grey shaded box the C-terminus and the yellow dot the Venus tag. Schematic representation of mCherry-tagged PTB (lower); white numbered boxes represent the RRM domains and ‘L’ denotes inter-RRM linkers. (B) Western blot of input (left panel, 10% of immunoprecipitation) and anti-flag immunoprecipitation (right panel) of Venus–MBNL1 co-expressed with FLAG–PTB4 in formaldehyde cross-linked 293 T extracts. All lanes had FLAG–PTB4 co-expressed with Venus-tagged proteins; vector (lanes 1 and 10), MBNL1-2–382 (lanes 2 and 11), MBNL1-2–253 (lanes 3 and 12), MBNL1-2–183 (lanes 4 and 13), MBNL1-2–115 (lanes 5 and 14), MBNL1-2–88 (lanes 6 and 15), MBNL1-2–104 (lanes 7 and 16), MBNL1-2–72 (lanes 8 and 17) and MBNL1-239–382 (lanes 9 and 18). Top panel, anti-PTB western, lower panel, anti-GFP western. (C) FLIM–FRET analysis of MBNL1 interaction with PTB. MBNL1 and PTB were fused to Venus as the FRET donor or mCherry as the acceptor and expressed in HeLa cells as indicated. MBNL fusion constructs are indicated with the N-terminal ZFs shown in black, and the C-terminal region shaded. First two rows show the Venus fluorescence and the corresponding fluorescence lifetime images of the indicated MBNL1 constructs in absence or presence of full-length mCherry–PTB, respectively. Third row shows the corresponding histograms (red line = donor only control; blue line = double transfection FRET). An energy transfer and, therefore, a reduction of the fluorescence lifetime was observed for N- (a) and C-terminal (b) labelled full-length MBNL1 (Venus–MBNL-2–382 and MBNL-2–382–Venus, respectively) in presence of mCherry-tagged PTB. Furthermore, the N-terminus of MBNL1 was sufficient to establish this interaction (c), whereas the C-terminal end alone did not show a FRET (d). (D) Statistical analysis of (C). Error bars are SEM of at least five independent fields of view with approximately four cells per image. Scale bar = 10 μm.
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gkt168-F5: Interaction of MBNL truncations with PTB. (A) Schematic representation of Venus-tagged MBNL1 fusion proteins with the amino acid boundaries indicated (upper). Black boxes represent the four ZF domains, the grey shaded box the C-terminus and the yellow dot the Venus tag. Schematic representation of mCherry-tagged PTB (lower); white numbered boxes represent the RRM domains and ‘L’ denotes inter-RRM linkers. (B) Western blot of input (left panel, 10% of immunoprecipitation) and anti-flag immunoprecipitation (right panel) of Venus–MBNL1 co-expressed with FLAG–PTB4 in formaldehyde cross-linked 293 T extracts. All lanes had FLAG–PTB4 co-expressed with Venus-tagged proteins; vector (lanes 1 and 10), MBNL1-2–382 (lanes 2 and 11), MBNL1-2–253 (lanes 3 and 12), MBNL1-2–183 (lanes 4 and 13), MBNL1-2–115 (lanes 5 and 14), MBNL1-2–88 (lanes 6 and 15), MBNL1-2–104 (lanes 7 and 16), MBNL1-2–72 (lanes 8 and 17) and MBNL1-239–382 (lanes 9 and 18). Top panel, anti-PTB western, lower panel, anti-GFP western. (C) FLIM–FRET analysis of MBNL1 interaction with PTB. MBNL1 and PTB were fused to Venus as the FRET donor or mCherry as the acceptor and expressed in HeLa cells as indicated. MBNL fusion constructs are indicated with the N-terminal ZFs shown in black, and the C-terminal region shaded. First two rows show the Venus fluorescence and the corresponding fluorescence lifetime images of the indicated MBNL1 constructs in absence or presence of full-length mCherry–PTB, respectively. Third row shows the corresponding histograms (red line = donor only control; blue line = double transfection FRET). An energy transfer and, therefore, a reduction of the fluorescence lifetime was observed for N- (a) and C-terminal (b) labelled full-length MBNL1 (Venus–MBNL-2–382 and MBNL-2–382–Venus, respectively) in presence of mCherry-tagged PTB. Furthermore, the N-terminus of MBNL1 was sufficient to establish this interaction (c), whereas the C-terminal end alone did not show a FRET (d). (D) Statistical analysis of (C). Error bars are SEM of at least five independent fields of view with approximately four cells per image. Scale bar = 10 μm.

Mentions: The U and D regulatory elements are both located adjacent to pyrimidine tracts that can bind 2 or 3 PTB molecules, which repress splicing (18,20). Moreover, the TIRF analyses indicated that the U and D elements increased the apparent affinity of PTB for Tpm1 RNAs (Table 1). Although both proteins have been studied extensively, there have been no previous reports of a direct interaction between them. Co-immunoprecipitation experiments using various epitope-tagged PTB and MBNL1 combinations produced negative results (data not shown). To address whether PTB and MBNL1 interact in a manner that is unstable during cell lysis and immunoprecipitation, we co-transfected HeLa cells with FLAG–PTB and Venus–MBNL and then treated cells with formaldehyde to induce protein–protein cross-links. After anti-FLAG immunoprecipitation and cross-link–reversal, western blots were probed with PTB and GFP antibodies (Figure 5B). Full-length Venus–MBNL and all members of a C-terminal deletion series were pulled down along with FLAG–PTB (Figure 5B, lanes 11–17); indeed, the mutants lacking the C-terminal region were pulled down with greater efficiency. In contrast, the C-terminal fragment of MBNL1 (amino acids 239–382), which lacks the ZnF domains (lanes 1–9), showed background levels of pull-down (lane 18). These data suggest that MBNL1 and PTB are associated together in the cell. To pursue this further, we analysed cells transfected with mCherry-tagged PTB and Venus-tagged MBNL1 by fluorescence lifetime imaging Förster resonance energy transfer (FLIM–FRET, Figure 5C and D). Transfection of either Venus–MBNL or mCherry–PTB showed both proteins to be nuclear localized with additional cytoplasmic signal for Venus–MBNL (Figure 5C). If the two proteins interact, energy transfer from Venus to mCherry should reduce the fluorescence lifetime of Venus. Indeed, the lifetime of nuclear, but not cytoplasmic, Venus–MBNL1 or MBNL1–Venus was reduced on co-transfection with mCherry–PTB (Figure 5C, panels a and b), indicating a direct interaction between MBNL1 and PTB. The reduction in fluorescence lifetime was also observed with the N-terminal but not the C-terminal region of MBNL1 (Figure 5C, panels c and d), consistent with the pull-down data (Figure 5B). Thus, the FLIM–FRET data indicated that the N-terminal region (amino acids 2–253) of MBNL1 is involved in interaction with PTB, whereas the cross-link–immunoprecipitation (IP) suggested amino acids 2–72 may be sufficient.Figure 5.


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 of MBNL truncations with PTB. (A) Schematic representation of Venus-tagged MBNL1 fusion proteins with the amino acid boundaries indicated (upper). Black boxes represent the four ZF domains, the grey shaded box the C-terminus and the yellow dot the Venus tag. Schematic representation of mCherry-tagged PTB (lower); white numbered boxes represent the RRM domains and ‘L’ denotes inter-RRM linkers. (B) Western blot of input (left panel, 10% of immunoprecipitation) and anti-flag immunoprecipitation (right panel) of Venus–MBNL1 co-expressed with FLAG–PTB4 in formaldehyde cross-linked 293 T extracts. All lanes had FLAG–PTB4 co-expressed with Venus-tagged proteins; vector (lanes 1 and 10), MBNL1-2–382 (lanes 2 and 11), MBNL1-2–253 (lanes 3 and 12), MBNL1-2–183 (lanes 4 and 13), MBNL1-2–115 (lanes 5 and 14), MBNL1-2–88 (lanes 6 and 15), MBNL1-2–104 (lanes 7 and 16), MBNL1-2–72 (lanes 8 and 17) and MBNL1-239–382 (lanes 9 and 18). Top panel, anti-PTB western, lower panel, anti-GFP western. (C) FLIM–FRET analysis of MBNL1 interaction with PTB. MBNL1 and PTB were fused to Venus as the FRET donor or mCherry as the acceptor and expressed in HeLa cells as indicated. MBNL fusion constructs are indicated with the N-terminal ZFs shown in black, and the C-terminal region shaded. First two rows show the Venus fluorescence and the corresponding fluorescence lifetime images of the indicated MBNL1 constructs in absence or presence of full-length mCherry–PTB, respectively. Third row shows the corresponding histograms (red line = donor only control; blue line = double transfection FRET). An energy transfer and, therefore, a reduction of the fluorescence lifetime was observed for N- (a) and C-terminal (b) labelled full-length MBNL1 (Venus–MBNL-2–382 and MBNL-2–382–Venus, respectively) in presence of mCherry-tagged PTB. Furthermore, the N-terminus of MBNL1 was sufficient to establish this interaction (c), whereas the C-terminal end alone did not show a FRET (d). (D) Statistical analysis of (C). Error bars are SEM of at least five independent fields of view with approximately four cells per image. Scale bar = 10 μm.
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Related In: Results  -  Collection

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Show All Figures
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gkt168-F5: Interaction of MBNL truncations with PTB. (A) Schematic representation of Venus-tagged MBNL1 fusion proteins with the amino acid boundaries indicated (upper). Black boxes represent the four ZF domains, the grey shaded box the C-terminus and the yellow dot the Venus tag. Schematic representation of mCherry-tagged PTB (lower); white numbered boxes represent the RRM domains and ‘L’ denotes inter-RRM linkers. (B) Western blot of input (left panel, 10% of immunoprecipitation) and anti-flag immunoprecipitation (right panel) of Venus–MBNL1 co-expressed with FLAG–PTB4 in formaldehyde cross-linked 293 T extracts. All lanes had FLAG–PTB4 co-expressed with Venus-tagged proteins; vector (lanes 1 and 10), MBNL1-2–382 (lanes 2 and 11), MBNL1-2–253 (lanes 3 and 12), MBNL1-2–183 (lanes 4 and 13), MBNL1-2–115 (lanes 5 and 14), MBNL1-2–88 (lanes 6 and 15), MBNL1-2–104 (lanes 7 and 16), MBNL1-2–72 (lanes 8 and 17) and MBNL1-239–382 (lanes 9 and 18). Top panel, anti-PTB western, lower panel, anti-GFP western. (C) FLIM–FRET analysis of MBNL1 interaction with PTB. MBNL1 and PTB were fused to Venus as the FRET donor or mCherry as the acceptor and expressed in HeLa cells as indicated. MBNL fusion constructs are indicated with the N-terminal ZFs shown in black, and the C-terminal region shaded. First two rows show the Venus fluorescence and the corresponding fluorescence lifetime images of the indicated MBNL1 constructs in absence or presence of full-length mCherry–PTB, respectively. Third row shows the corresponding histograms (red line = donor only control; blue line = double transfection FRET). An energy transfer and, therefore, a reduction of the fluorescence lifetime was observed for N- (a) and C-terminal (b) labelled full-length MBNL1 (Venus–MBNL-2–382 and MBNL-2–382–Venus, respectively) in presence of mCherry-tagged PTB. Furthermore, the N-terminus of MBNL1 was sufficient to establish this interaction (c), whereas the C-terminal end alone did not show a FRET (d). (D) Statistical analysis of (C). Error bars are SEM of at least five independent fields of view with approximately four cells per image. Scale bar = 10 μm.
Mentions: The U and D regulatory elements are both located adjacent to pyrimidine tracts that can bind 2 or 3 PTB molecules, which repress splicing (18,20). Moreover, the TIRF analyses indicated that the U and D elements increased the apparent affinity of PTB for Tpm1 RNAs (Table 1). Although both proteins have been studied extensively, there have been no previous reports of a direct interaction between them. Co-immunoprecipitation experiments using various epitope-tagged PTB and MBNL1 combinations produced negative results (data not shown). To address whether PTB and MBNL1 interact in a manner that is unstable during cell lysis and immunoprecipitation, we co-transfected HeLa cells with FLAG–PTB and Venus–MBNL and then treated cells with formaldehyde to induce protein–protein cross-links. After anti-FLAG immunoprecipitation and cross-link–reversal, western blots were probed with PTB and GFP antibodies (Figure 5B). Full-length Venus–MBNL and all members of a C-terminal deletion series were pulled down along with FLAG–PTB (Figure 5B, lanes 11–17); indeed, the mutants lacking the C-terminal region were pulled down with greater efficiency. In contrast, the C-terminal fragment of MBNL1 (amino acids 239–382), which lacks the ZnF domains (lanes 1–9), showed background levels of pull-down (lane 18). These data suggest that MBNL1 and PTB are associated together in the cell. To pursue this further, we analysed cells transfected with mCherry-tagged PTB and Venus-tagged MBNL1 by fluorescence lifetime imaging Förster resonance energy transfer (FLIM–FRET, Figure 5C and D). Transfection of either Venus–MBNL or mCherry–PTB showed both proteins to be nuclear localized with additional cytoplasmic signal for Venus–MBNL (Figure 5C). If the two proteins interact, energy transfer from Venus to mCherry should reduce the fluorescence lifetime of Venus. Indeed, the lifetime of nuclear, but not cytoplasmic, Venus–MBNL1 or MBNL1–Venus was reduced on co-transfection with mCherry–PTB (Figure 5C, panels a and b), indicating a direct interaction between MBNL1 and PTB. The reduction in fluorescence lifetime was also observed with the N-terminal but not the C-terminal region of MBNL1 (Figure 5C, panels c and d), consistent with the pull-down data (Figure 5B). Thus, the FLIM–FRET data indicated that the N-terminal region (amino acids 2–253) of MBNL1 is involved in interaction with PTB, whereas the cross-link–immunoprecipitation (IP) suggested amino acids 2–72 may be sufficient.Figure 5.

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