ABSTRACT
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 PTB-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 exon to a PTB-repressed exon. Our results show that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons.
Subject(s)
Alternative Splicing , Polypyrimidine Tract-Binding Protein/metabolism , Base Sequence , Exons , HeLa Cells , Humans , Introns , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Polypyrimidine Tract-Binding Protein/genetics , Silencer Elements, TranscriptionalABSTRACT
Among 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.
Subject(s)
Alternative Splicing/genetics , Gene Expression Regulation , Polypyrimidine Tract-Binding Protein/genetics , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/genetics , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Exons , HeLa Cells , Humans , Myocytes, Smooth Muscle/metabolism , Neurons/metabolism , Polypyrimidine Tract-Binding Protein/metabolism , RNA, Messenger/chemistry , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Polypyrimidine-tract-binding protein (PTB) is a repressive regulator of alternative splicing. Models for PTB activity have ranged from simple binding competition with splicing factor U2AF(65) at regulated polypyrimidine tracts to looping out of repressed exons by binding of PTB to flanking sites. Structural analysis of PTB bound to RNA suggests how PTB monomers can induce loops, but two recent publications indicate that repression by PTB involves more than just binding to RNA.
Subject(s)
Alternative Splicing/genetics , Exons/genetics , Polypyrimidine Tract-Binding Protein/metabolism , Genes, src/genetics , Models, Genetic , RNA/metabolism , fas Receptor/geneticsABSTRACT
Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by alpha 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 post-translational modifications on the activation of rPLD1 by constitutively active Galpha(13)Q226L and Galpha(q)Q209L. Mutations of Cys(240) and Cys(241) of rPLD1, which abolish both post-translational modifications, did not affect the ability of either Galpha(13)Q226L or Galpha(q)Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by Galpha(13)Q226L, although these mutant enzymes responded to phorbol ester and Galpha(q)Q209L. On the contrary, the PKC-insensitive mutant rPLD1(DeltaN168), which lacks the first 168 amino acids of rPLD1, responded to Galpha(13)Q226L but not to Galpha(q)Q209L. In addition, we found that rPLD2 was strongly activated by Galpha(q)Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by Galpha(13)Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of Galpha(q)Q209L or Galpha(13)Q226L. The suppressive effect of Galpha(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, Galpha(13) activation of rPLD1 in COS-7 cells is mediated by Rho, while Galpha(q) activation requires PKC. rPLD2 is activated by Galpha(q), but is inhibited by Galpha(13). Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.
