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Small Molecule Regulation of Alternative Splicing

Abstract

Alternative splicing aberrations can cause disease or promote its progression, including the development of cancer. Reversal of pathologic splicing events is a potential therapy for those cases. Efficient methods for identification and characterization of small molecule modulators of specific splicing events are required to develop therapeutics. The cardiotonic steroids, long prescribed for congestive heart failure, alter splicing of many exons. Two methods are described to define their mechanism of action. First, an unbiased analysis of transcriptome-wide changes in alternative splicing predicted loss of function of two specific splicing factors, SRp20 and Tra2-beta. Both proteins are depleted by the cardiotonic steroid digitoxin, and their restoration blocks digitoxin induced splicing changes. This general method can be applied to any identified drugs in future screens. Second, a candidate approach was taken to identify which of the known cardiotonic steroid effectors is required for induced splicing changes. An inhibitor to protein phosphatase 1, tautomycin, antagonizes digitoxin induced changes, through a yet to be determined mechanism.

BIN1 exon 12a inclusion is associated with progression of metastatic melanoma. Using a high throughput approach, cDNA expression and small molecule libraries were screened for regulators of exon 12a splicing. Specific RNA-binding proteins including hnRNP-K, hnRNP-LL, Tra2-beta and PCBP1/4 were identified as repressors while hnRNP-R is an enhancer of 12a splicing. Furthermore, expression of cell signaling effectors such as Map3k3 and -11, as well as CLK2 and FAST can modulate 12a inclusion levels. hnRNP-K, which has conflicting roles in the development of cancer, represses 12a splicing through an exonic RNA element. Small molecules that modulate exon 12a include modulators of prostaglandin activity, the antibiotic puromycin, and the psychoactive MAOA inhibitor harmaline. Interaction between these molecules and the identified genetic regulators of 12a splicing could elucidate the mechanism by which BIN1 splicing changes to promote tumorigenesis.

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