SummaryAmong the targets of the repressive splicing regulator, polypyrimidine tract binding protein (PTB) is its own pre-mRNA, where PTB-induced exon 11 skipping produces an RNA substrate for nonsense-mediated decay (NMD). To identify additional PTB-regulated alternative splicing events, we used quantitative proteomic analysis of HeLa cells after knockdown of PTB. Apart from loss of PTB, the only change was upregulation of the neuronally restricted nPTB, resulting from decreased skipping of nPTB exon 10, a splicing event that leads to NMD of nPTB mRNA. Compared with knockdown of PTB alone, simultaneous knockdown of PTB and nPTB led to larger changes in alternative splicing of known and newly identified PTB-regulated splicing events. Strikingly, the hematopoietic PTB paralog ROD1 also switched from a nonproductive splicing pathway upon PTB/nPTB knockdown. Our data indicate crossregulation between PTB and its paralogs via nonproductive alternative splicing and a large degree of functional overlap between PTB and nPTB.

To gain global insights into the role of the well-known repressive splicing regulator PTB we analyzed the consequences of PTB knockdown in HeLa cells using high-density oligonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB repressed and activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons, but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTB-activated to a PTB-repressed exon. Our results demonstrate that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.

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PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.

Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by ␣ subunits of the heterotrimeric G proteins G 13 and G q . Recently, we showed that rPLD1 is modified by Ser/Thr phosphorylation and palmitoylation. In this study, we first investigated the roles of these posttranslational modifications on the activation of rPLD1 by constitutively active G␣ 13 Q226L and G␣ q Q209L. Mutations of Cys 240 and Cys 241 of rPLD1, which abolish both post-translational modifications, did not affect the ability of either G␣ 13 Q226L or G␣ q Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by G␣ 13 Q226L, although these mutant enzymes responded to phorbol ester and G␣ q Q209L. On the contrary, the PKC-insensitive mutant rPLD1(⌬N168), which lacks the first 168 amino acids of rPLD1, responded to G␣ 13 Q226L but not to G␣ q Q209L. In addition, we found that rPLD2 was strongly activated by G␣ q Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by G␣ 13 Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of G␣ q Q209L or G␣ 13 Q226L. The suppressive effect of G␣ 13 Q226L on rPLD2 was reversed by dominant negative N19RhoA and the C3 exoenzyme of Clostridium botulinum, further supporting a role for RhoA. In summary, G␣ 13 activation of rPLD1 in COS-7 cells is mediated by Rho, while G␣ q activation requires PKC. rPLD2 is activated by G␣ q , but is inhibited by G␣ 13 . Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.

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