No abstract

In higher eukaryotes, the polypyrimidine-tract (Py-tract) adjacent to the 3' splice site is recognized by several proteins, including the essential splicing factor U2AF65, the splicing regulator Sex-lethal (Sxl), and polypyrimidine tract-binding protein (PTB), whose function is unknown. Iterative in vitro genetic selection was used to show that these proteins have distinct sequence preferences. The uridine-rich degenerate sequences selected by U2AF65 are similar to those present in the diverse array of natural metazoan Py-tracts. In contrast, the Sxl-consensus is a highly specific sequence, which can help explain the ability of Sxl to regulate splicing of transformer pre-mRNA and autoregulate splicing of its own pre-mRNA. The PTB-consensus is not a typical Py-tract; it can be found in certain alternatively spliced pre-mRNAs that undergo negative regulation. Here it is shown that PTB can regulate alternative splicing by selectively repressing 3' splice sites that contain a PTB-binding site.

Synonymous mutations change the sequence of a gene without directly altering the sequence of the encoded protein. Here, we present evidence that these "silent" mutations frequently contribute to human cancer. Selection on synonymous mutations in oncogenes is cancer-type specific, and although the functional consequences of cancer-associated synonymous mutations may be diverse, they recurrently alter exonic motifs that regulate splicing and are associated with changes in oncogene splicing in tumors. The p53 tumor suppressor (TP53) also has recurrent synonymous mutations, but, in contrast to those in oncogenes, these are adjacent to splice sites and inactivate them. We estimate that between one in two and one in five silent mutations in oncogenes have been selected, equating to ~6%- 8% of all selected single-nucleotide changes in these genes. In addition, our analyses suggest that dosage-sensitive oncogenes have selected mutations in their 3' UTRs.

No abstract

RBM5, a regulator of alternative splicing of apoptotic genes, and its highly homologous RBM6 and RBM10 are RNA-binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation, and its modulation recapitulates or antagonizes the effects of RBM5, 6, and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation.

Chromatin structure influences transcription, but its role in subsequent RNA processing is unclear. Here we present analyses of high-throughput data that imply a relationship between nucleosome positioning and exon definition. First, we have found stable nucleosome occupancy within human and Caenorhabditis elegans exons that is stronger in exons with weak splice sites. Conversely, we have found that pseudoexons--intronic sequences that are not included in mRNAs but are flanked by strong splice sites--show nucleosome depletion. Second, the ratio between nucleosome occupancy within and upstream from the exons correlates with exon-inclusion levels. Third, nucleosomes are positioned central to exons rather than proximal to splice sites. These exonic nucleosomal patterns are also observed in non-expressed genes, suggesting that nucleosome marking of exons exists in the absence of transcription. Our analysis provides a framework that contributes to the understanding of splicing on the basis of chromatin architecture.

Fas exon 6 can be included or skipped to generate mRNAs encoding, respectively, a membrane bound form of the receptor that promotes apoptosis or a soluble isoform that prevents programmed cell death. We report that the apoptosis-inducing protein TIA-1 promotes U1 snRNP binding to the 5' splice site of intron 6, which in turn facilitates exon definition by enhancing U2AF binding to the 3' splice site of intron 5. The polypyrimidine tract binding protein (PTB) promotes exon skipping by binding to an exonic splicing silencer and inhibiting the association of U2AF and U2 snRNP with the upstream 3' splice site, without affecting recognition of the downstream 5' splice site by U1. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is required both for efficient U2AF binding and for U2AF inhibition by PTB. We propose that TIA-1 and PTB regulate Fas splicing and possibly Fas-mediated apoptosis by targeting molecular events that lead to exon definition.

Several bacterial fermentation products and their synthetic derivatives display antitumour activities and bind tightly to components of the spliceosome, which is the complex molecular machinery involved in the removal of introns from mRNA precursors in eukaryotic cells. The drugs alter gene expression, including alternative splicing, of genes that are important for cancer progression. A flurry of recent reports has revealed that genes encoding splicing factors, including the drug target splicing factor 3B subunit 1 (SF3B1), are among the most highly mutated in various haematological malignancies such as chronic lymphocytic leukaemia and myelodysplastic syndromes. These observations highlight the role of splicing factors in cancer and suggest that an understanding of the molecular effects of drugs targeting these proteins could open new perspectives for studies of the spliceosome and its role in cancer progression, and for the development of novel antitumour therapies.