Type 1 diabetes mellitus (T1D) is a chronic disease resulting from the autoimmune destruction of insulin- producing pancreatic b cells. Although T1D is primarily associated with a dysfunctional immune response, recent genome-wide association studies (GWAS) have determined that a large number of T1D associated genes are expressed in pancreatic b cells. Currently, there are many ongoing studies to identify the underlying molecular mechanisms that impact b cell dysfunction in T1D, with hopes that this knowledge can be used to develop novel biomarkers and preventive disease therapies. We hypothesize that altered RNA splicing events contribute to b cell dysfunction and potentially contribute to the formation of immunogenic protein isoforms to trigger T1D. Recently, our ability to perform genome wide RNA sequencing has revealed the complexity of the transcriptome and has provided significant insight into the transcriptomic modifications that occur during physiological and pathophysiological processes. In particular, there is a growing appreciation for the role of alternative splicing in many biological processes. The biological importance of alternative splicing has been further emphasized by the large number of human diseases caused by mutations that affect the splicing program. In the pancreas, a recent study showed considerable dysregulation of RNA binding proteins (RBPs) and aberrant mRNA splicing in islets derived from type 2 diabetic patients compared to healthy controls. While these studies have illustrated the existence of alternative splicing networks in the pancreas, and identified some of the enzymes involved in the regulation of alternative splicing, there has been relatively little demonstration of the functional consequences of alternative splicing events and how they may contribute to T1D pathogenesis. We propose studies to investigate the physiological relevance of alternative splicing events in b cells. Importantly, given the heterogeneous nature of b cell destruction in T1D, we will assess cell specific altered RNA splicing products at the single cell sequencing level, and in situ at a single cell and near single cell resolution to fully understand the potentially cell-specific functional consequences of altered spliced variants. Furthermore, we will identify potentially novel protein products that arise due to the alternative splicing events. To accomplish our goals, we propose the following specific aims: Aim 1. Use novel RNA sequencing technologies to identify conserved alternatively spliced transcripts in mouse and human T1D islet samples; Aim 2. Determine the localization of candidate RNA isoforms to individual human islet cells using fliFISH as a high accuracy smFISH approach that localizes individual transcripts with 20-30 nm resolution using STORM microscopy; Aim 3. Use advanced proteomics and innovative computational approaches to identify candidate novel protein products generated from alternative spliced transcripts in T1D ...