MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

MBNL binds to the upstream and downstream UGC elements. (A) Schematic representation of RNAs a–g containing Tpm1 exon 3 and flanking regulatory elements. RNA a has all the cis-elements, RNA b has the UGC motifs deleted and RNA c has the UCUU motifs mutated in P3 or deleted in DY. RNAs d–g contain the individual regulatory elements as indicated. (B) In vitro transcribed RNAs a–c were UV cross-linked with either recombinant pCGT7–MBNL1 (rMBNL1, lanes 1–3) or PAC-1 nuclear extract (NE, lanes 4–6). RNAs a–c were UV cross-linked with rMBNL1 or PAC-1 nuclear extract and then immunoprecipitated with either α-MBNL1 (lanes 7–12) or UV cross-linked with PAC-1 nuclear extracts and immunoprecipitated with α-PTB antibody (lanes 13–15). (C) In vitro transcribed RNA a or the individual cis-elements, d–g, were UV cross-linked with rMBNL1 (lanes 1–5) or recombinant pQE–PTB4 (rPTB4, lanes 6–10).

Figure 4.

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).

Figure 5.

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.

Figure 6.

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.

Figure 7.

RNA binding by MBNL1 enhances the interaction with PTB. (A) Silver stain gel of GST pull-down in HeLa nuclear extract with GST-MBNL-2–116, 2–116 m, 2–72 and GST alone (lanes 2–6). The doublet identified by mass spectrometry as PTB is marked by an asterisk. This doublet was not observed if extract was treated with RNase. (B) Western blot of pull-down from A probing with anti-PTB, anti-hnRNP C, anti-hnRNP H and anti-hnRNP L (right panel) with a titration of the input 0.01, 0.1, 1, 10 and 100% (left panel). (C) Schematic diagram of Tpm1 RNA species used in panels D, F and G. (D) Western blots using anti-PTB (upper panel) and anti-GST (lower panel). GST–MBNL-2–116 pull-down in HeLa nuclear extract with increasing amounts of RNA. No RNA added (lane 1), RNA a (lanes 2–5, Figure 3), MBNL SELEX RNA (lanes 6–8), PTB SELEX RNA (lanes 9–11), MBNL and PTB SELEX RNAs together (lanes 12–14). (E) UV X-linking of recombinant pQE–PTB4, left panel, or GST–MBNL1 amino acids 2–253, right panel, to either the MBNL SELEX RNA, lanes 1 and 3, or the PTB SELEX RNA, lanes 2 and 4. (F) GST pull-down with recombinant GST–MBNL1 amino acids 2–253 and pQE–PTB4 with a titration of RNA. Markers (lane 1), recombinant GST protein bound to beads minus and plus recombinant pQE–PTB4 (lanes 2 and 3), recombinant GST–MBNL1-2–253 bound to beads (lane 4, input), recombinant pQE–PTB4 (lane 5, 15% of input), GST–MBNL beads plus pQE–PTB4 (lanes 6–21) with no RNA added (lanes 6, 10, 14 and 18), titration of RNA a (20, 2 and 0.2 pmol, lanes 7–9), titration of MBNL SELEX (20, 2 and 0.2 pmol, lanes 11–13), titration of PTB SELEX (20, 2 and 0.2 pmol, lanes 15–17) and titration of RNA f (20, 2 and 0.2 pmol, lanes 19–21). (G) GST pull-down with recombinant GST–MBNL1 C-terminal truncations indicated and pQE–PTB4 minus RNA (lanes 1, 3, 5, 7, 9 and 11) and plus 40 pmol RNA f (lanes 2, 4, 6, 8, 10 and 12).