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.

Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 59 of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 39 splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition.[Keywords: spliceostatin A; U2 snRNP; branch point; splicing fidelity; alternative splicing] Supplemental material is available for this article.Received November 18, 2010; revised version accepted January 10, 2011.Excision of intronic sequences from pre-mRNAs (premRNA splicing) is an obligatory step for the expression of the majority of higher eukaryotic genes and requires the function of a complex molecular machinery, the spliceosome, which is composed of five small nuclear ribonucleoprotein (snRNP) complexes and >200 proteins (for review, see Wahl et al. 2009). The function of the spliceosome relies on an intricate network of protein-protein, protein-RNA, and RNA-RNA interactions that undergo significant conformational and compositional rearrangements to facilitate intron excision (for review, see Smith et al. 2008;Wahl et al. 2009). These interactions can be modulated to allow the generation of alternative patterns of splicing, a phenomenon reported to occur in the majority of human genes (for review, see Chen and Manley 2009;Nilsen and Graveley 2010). Alternative splicing plays important roles in the development of multicellular organisms and in numerous pathologies, including cancer (for review, see Cooper et al. 2009;.The efficiency and precision required for intron removal is in contrast to the limited sequence consensus at intron/exon boundaries, a feature likely connected to the establishment of mechanisms of splice site regulation mediated by a diverse array of auxiliary sequences and cognate factors (for review, see Wang and Burge 2008;Barash et al. 2010). In higher eukaryotes, the 39 end of the intron is characterized by a polypyrim...

Alternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number, and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells. These patterns are predicted to be mainly controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1. We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in nontumorigenic mammary epithelial cells. Our study uncovers novel splicing networks that potentially contribute to cancer development and progression.