Abstract 5412: Nuclear AMPKα2 is a repressor of alternative splicing of a subset of cancer-associated genes

Alternative splicing of pre-mRNA is a fundamental cellular process that selectively joins alternate exons together to produce isoforms that differ in sequence, thereby significantly contributing to proteomic complexity as well as to post-transcriptional gene regulations. More than 90% of human genes, including oncogenes and tumor suppressor genes, have been predicted to undergo alternative splicing. Alternatively spliced isoforms function differently, sometimes antagonistically, and often exist in a delicate equilibrium that is perturbed during tumorigenesis. Previously, using caffeine as a tool, we have revealed a mechanism for alternative splicing regulation of a subset of cancer-associated genes, and have shown that this regulation is mediated, at least in part, by splicing factor SFRS2 (sc35) [Shi et al., MCB 28(2):883-895,2008]. In the present study, we found that the nucleoside adenosine, which is structurally similar to caffeine, can inhibit the caffeine-mediated and sc35-induced alternative splicing. This inhibition requires the function of adenosine transporters, suggesting an intracellular adenosine-mediated mechanism rather than adenosine receptor-mediated mechanisms. In addition, Iodotubercidin (Itu), an inhibitor of adenosine kinase, was found to inhibit the caffeine-mediated splicing as well. Since both caffeine and adenosine are known for roles in metabolic homeostasis, we specifically examined AMPKα, the catalytic subunit of the heterotrimeric AMPK (5’AMP-activated protein kinase) that is encoded by two genes: α1 and α2. Surprisingly, while RNAi-knockdown of AMPKα1 slightly reduced the basal-level as well as the caffeine-induced alternative splicing, AMPKα2-knockdown significantly increased alternative splicing of a reporter minigene. Importantly, neither caffeine nor adenosine significantly changed the level of AMPKα1 and α2. However, immunofluorescence studies revealed that AMPKα2 was predominantly localized in the nucleus in the presence of adenosine while largely cytoplasmic following caffeine treatment. Taken together, we propose that nuclear AMPKα2 plays a repressive role in the regulation of alternative splicing. Cancer is being increasingly recognized as a metabolic disease. AMPK is a “master switch” that senses cellular energy levels to rewire cellular metabolism accordingly. A great number of cancer-specific splice variants have been observed in both the laboratory and the clinic, and some of these splice variants are believed to