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While robust regulatory mechanisms are expected to control the production of splice variants that confer distinct functions, a low level of stochasticity may be tolerated. To investigate stringency of regulation, we followed changes in the splicing of 192 alternative cassette exons after growth of cancer-derived HCT116 cells and embryonic colonocytes. In both cell lines approximately 15% of alternative splicing events changed by more than 10 percentage points over a 42-day period. We then carried out a cycle of transient depletions targeting RNPS1, a splicing regulator implicated in genomic stability. For alternative splicing units not regulated by RNPS1, the level of splicing changes was similar to the stochastic value obtained after normal growth. However, the frequency of perdurable changes was at least twice that value for splicing events regulated by RNPS1. A swap allele assay performed on four RNPS1-responsive units that underwent splicing changes indicated the presence of mutations mediating this effect. Specifically, a T to C mutation in a RNPS1-responsive exon of ADARB1 confered exon skipping. Our results suggest that fluctuations in the level of a splicing regulator preferentially impact the integrity of genes encoding transcripts that are regulated by this splicing factor to produce perdurable changes in alternative splicing. We discuss the potential implication of this process in human evolution.

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for real-time PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.