Caffeine Regulates Alternative Splicing in a Subset of Cancer-Associated Genes: a Role for SC35

Abstract

Alternative splicing of pre-mRNA contributes significantly to human proteomic complexity, playing a key role in development, gene expression and, when aberrant, human disease onset. Many of the factors involved in alternative splicing have been identified, but little is known about their regulation. Here we report that caffeine regulates alternative splicing of a subset of cancer-associated genes, including the tumor suppressor KLF6. This regulation is at the level of splice site selection, occurs rapidly and reversibly, and is concentration dependent. We have recapitulated caffeine-induced alternative splicing of KLF6 using a cell-based minigene assay and identified a “caffeine response element” within the KLF6 intronic sequence. Significantly, a chimeric minigene splicing assay demonstrated that this caffeine response element is functional in a heterologous context; similar elements exist within close proximity to caffeine-regulated exons of other genes in the subset. Furthermore, the SR splicing factor, SC35, was shown to be required for induction of the alternatively spliced KLF6 transcript. Importantly, SC35 is markedly induced by caffeine, and overexpression of SC35 is sufficient to mimic the effect of caffeine on KLF6 alternative splicing. Taken together, our data implicate SC35 as a key player in caffeine-mediated splicing regulation. This novel effect of caffeine provides a valuable tool for dissecting the regulation of alternative splicing of a large gene subset and may have implications with respect to splice variants associated with disease states.

ACKNOWLEDGMENTS

We thank Cyril F. Bourgeois and James Stevenin at Institut de Génétique et de Biologie Moléculaire et Cellulaire, France, for generously providing SC35 monoclonal antibody. We thank Douglas Black (UCLA) for generously providing the DUP4-1 clone. We acknowledge Todd Martinsky (ArrayIT, Inc.), Eric Olson (Genesifter), and Jonathan Bingham and Subha Srinivasan (Jivan Biologics Inc.) for facilitating the microarray data analyses and validation. We are grateful to Rebecca Cimildoro and Julia Pimkina for preliminary studies on caffeine-mediated KLF6 splicing and to other members of the Scotto laboratory as well as our CINJ colleagues for insightful discussions.

This work was supported by NIH-CA06927 (FCCC), NIH-P30-CA072720 (Cancer Institute of New Jersey), NCI-R01 CA 122573 (K.W.S.), and the UMDNJ Foundation grant program (J.S.).