J. indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1. INTRODUCTION Alternative splicing (AS) affects more than 90% of human genes (1). It allows individual genes to produce structurally and functionally distinct mRNAs and their relative protein isoforms, explaining the discrepancy between the estimated 24,000 protein-coding genes in the human genome and the 100,000 different proteins synthesized (2). Changes in AS may have a major impact on cell survival, exposure of novel antigenic epitopes, alteration of surface location of antigens and post-translational modifications (3C5). There is a growing interest in the role of AS in several autoimmune diseases (6,7), but nearly nothing is known on its role in pancreatic beta cell function and its dysfunction and apoptosis in diabetes. We have recently shown that beta cell exposure to pro-inflammatory cytokines modifies AS of more than 3000 expressed genes, including genes involved in chemotaxis and apoptosis, and Nonivamide of more than 50 splicing-regulating proteins (8,9). Our group has recently clarified the cross-talk between the diabetes candidate gene providing an important proof of concept for the role of AS in diabetes (9). Regulation of splicing involves the cooperation between serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNP) and several other tissue-specific regulators (10). Nova1 has been defined as a neuron-specific splicing factor (11C13) but we recently reported that Nova1 is also expressed in the GDF2 Nonivamide beta cells of the human and rat islets of Langerhans (8). Nova1 binds to YCAY-rich elements (14C17) and it is a central regulator of neuronal AS (16,18C24). By combining proteinCRNA crosslinking and Nova-immunoprecipitation, Darnell and colleagues identified 200 Nova RNA targets in the mouse brain (13). These transcripts regulate the development of brain synapses (19). Some of the known target genes in brain are also expressed in beta cells, including and family members, and binding partners (8) but the global role of in pancreatic beta cell function and survival remains unknown. High-throughput mRNA sequencing (RNA-seq) provides the opportunity to study AS in an unbiased manner and on a genome-wide scale. RNA-seq allows the discovery of novel transcripts and the estimation of their abundance. This is extremely useful when dealing with poorly annotated transcriptomes (25) and is a powerful method to identify splicing networks regulated by different splicing factors (26). In this study, we have combined siRNA technology, RNA-seq and functional studies to characterize the role of in the regulation of beta cell mRNA splicing and its implication in the function and viability of these cells. This combined approach showed that is a master regulator of AS in beta cells, controlling key pathways involved in beta cell function and survival. These findings unveil a novel role for lipid reagent (Invitrogen) as previously described (35,36). Allstars Negative Control siRNA (Qiagen, Venlo, The Netherlands) was used as negative control (siCTL). This siCTL does not affect beta cell gene expression or insulin release, as compared with non-transfected cells (35). After 16 h of transfection, cells were cultured for a 24 h or 48 h recovery period before exposure to cytokines. Assessment of cell viability The percentage of viable, apoptotic and necrotic cells was determined after 15-min incubation with DNA-binding dyes propidium iodide (5 g/ml; Sigma-Aldrich, Poole, UK) and Hoechst dye 33342 (5 g/ml; Sigma-Aldrich) by two independent researchers, Nonivamide one of them unaware of samples identity. A minimum of 600 cells were counted for each experimental condition. In some experiments, apoptosis was also confirmed by caspase-3 and -9 cleavage and KD of Bim, a key pro-apoptotic BH3-only protein in beta cells (37). RNA-sequencing Three preparations of FACS-purified rat primary beta cells were used for.