The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing

Abstract

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.

Fig. 1. Regulation of TM splicing by artificial recruitment of PTB. (A) The wild-type TM construct (pT2) contains exons 1, 3 and 4 with the four defined regulatory elements flanking exon 3. Deletions of exon 2 and flanking sequences and in the intron between exons 3 and 4 are denoted by the diagonal lines. P3 and DY are pyrimidine tracts (rectangles) containing optimal PTB-binding UCUU motifs (vertical lines). URE and DUGC contain clusters of UGC motifs (diamonds). Construct TM–2MS2 has the DY tract replaced by two MS2 binding sites, while TM–2ΔMS2 has two mutant MS2 sites, lacking the essential bulged A in the stem–loop. (B) The three reporters were co-transfected into PAC-1 SM cells with expression constructs for PTB, PTB–MS2, MS2 or pGEM4Z (4Z) negative control. Spliced RNA was analyzed by RT–PCR. PTB–MS2 was able to restore exon skipping to TM–2MS2 (compare lanes 1 with 5 and 5 with 7). (C) TM–2MS2 reporter was co-transfected with 1 µg pGEM4Z (lane 1), 1 µg PTB expression plasmid (lane 2), or 1, 10, 100, 500 or 1000 ng of PTB–MS2 plasmid (lanes 3–7). PTB–MS2 caused a dose-dependent increase in TM exon 3 skipping.

Fig. 2. Raver1 is expressed in tissues that regulate TM splicing. TM splicing was analyzed by RT–PCR followed by digestion with PvuII, which digests the 134 product at the exon 1:3 junction, but leaves the 124 product intact. The percentage of exon 2 containing product is indicated below each lane. Raver1 and β-actin expression were analyzed by semi-quantitative RT–PCR using RNA from various rat tissues and from the rat PAC-1 smooth muscle cell line. The ratio of raver1/β-actin RT–PCR product levels are shown underneath the β-actin panel, with the exception of PAC-1 cells (ND, not determined) where the raver1 was undetectable with the number of PCR cycles used. Raver1 was widely expressed in tissues including gut, aorta and uterus, where the major TM splicing pattern is exon 2 inclusion.

Fig. 3. Raver regulates TM splicing. (A) The TM splicing reporter TS23D, containing TM exons 1–4, was transfected into HeLa and PAC-1 SM cells along with pGEM4Z, or expression plasmids for raver1 or β-gal. Splicing was analyzed by RT–PCR followed by digestion with PvuII (exon 3 specific, lanes 2, 6 and 10), XhoI (exon 2 specific, lanes 3, 7 and 11) or both enzymes (lanes 4, 8 and 12). Raver1 promoted splicing of exon 2, as indicated by the increase in the 141 bp XhoI product in lanes 7. (B) Increasing amounts of raver1 expression plasmid (1, 10, 100, 500, 1000 ng) were co-transfected into PAC-1 cells with the reporter plasmid pT2, which lacks exon 2 but retains all essential regulatory elements. RNA was analyzed by RT–PCR and PhosphorImager. Raver1 caused a dose-dependent increase in TM exon 3 skipping.

Fig. 4. Raver regulation is mediated by essential PTB binding elements. TM constructs containing mutations in one or more of the four essential regulatory elements (P3, URE, DUGC, DY) were transfected in the presence or absence of a raver1 expression plasmid into PAC-1 cells. RNA was analyzed by RT–PCR and quantified by PhosphorImager. The percentage of exon skipping is mean ± SD for at least three independent experiments. The ‘raver response’ was quantified as the arithmetic difference in the percentage of exon 3 skipping in the presence and absence of raver1, and is illustrated in the histogram below. Raver response was critically dependent upon the PTB binding elements, but not upon the UGC motif elements.

Fig. 5. Raver1 is not antagonized by PTB. The wild-type pT2 TM reporter was co-transfected in the presence or absence of 100 ng raver1 expression plasmid. A titration of PTB1 expression plasmid (1, 10, 100, 500, 1000 ng) was carried out in the presence of the raver plasmid. Splicing was analyzed by RT–PCR and quantified by PhosphorImager.

Fig. 6. Raver1 activity depends upon a PTB-interacting segment. (A) Schematic structure of raver1, with amino acid numbering. The N-terminal part contains the three RRMs and interacts with PTB. The C-terminal region contains a proline-rich region. The two nuclear localization signals are shown as black boxes and the nuclear export signal as a dashed box. (B) The wild-type TM reporter was co-transfected into PAC-1 cells along with constructs expressing the indicated segments of raver1 (lanes 1–4) or empty vector (lane 5). Note that the degree of regulated exon skipping of the reporter construct in this series of experiments (lane 5) was approximately twice that observed in the other experiments shown. Numbers below each lane represent mean ± SD of three experiments. (C) [³⁵S]methionine labeled in vitro translated raver1 proteins corresponding to amino acids 1–748, 1–401 or 1–307 were incubated with GST–PTB1 (lanes 4–6) or GST (lanes 7–9), and then pulled down with glutathione–agarose beads. The ‘input’ lanes contain 20% of the equivalent reactions shown in the pull-down lanes. Lanes ‘M’ contain radiolabeled protein markers (97, 67, 58, 56, 43 and 36 kDa).

Fig. 7. Direct recruitment of raver1 C-terminal domain bypasses PTB binding site requirement. Tropomyosin alternative splicing reporters (500 ng) were co-transfected into PAC-1 cells with expression constructs (60 ng) for various MS2 fusion proteins. Lanes 1–6, wild-type TM reporter (WT); lanes 7–12, TM–2MS2 reporter with DY element replaced by two MS2 binding sites; lanes 13–18, DY→SXL reporter with DY element replaced by SXL binding site (Gooding et al., 1998). Co-transfection was with pGEM4Z negative control (lanes 1, 7 and 13), raver1 1–441–MS2 (lanes 2, 8 and 14), raver1 442–748–MS2 (lanes 3, 9 and 15), full-length raver1 1–748–MS2 (lanes 4, 10 and 16), MS2 alone (lanes 5, 11 and 17) or hnRNPA1-MS2 (A1-MS2; lanes 6, 12 and 18). The band marked with an asterisk is a PCR artifact that does not appear consistently between experiments (compare with Figures 1–5). Numbers below each lane represent the percentage exon skipping in the experiment shown. The results are qualitatively reproducible, but due to variations in the experimental procedures between repeats, SDs are not given.