Alternative splicing (AS) is a tightly regulated mechanism whereby pre-mRNA transcripts are processed to generate structurally distinct mRNA and protein isoforms. AS within the β cell has been proposed as a potential mechanism that may exacerbate autoimmunity and unmask novel immunogenic epitopes in type 1 diabetes (T1D). Here, we employed a computational strategy to catalog AS events in human islets treated with IL-1β + IFN-γ, as an ex vivo model of T1D. In total, 970 events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. To identify potentially pathogenic AS events, we utilized ExonImpact, which is a validated machine learning based method, that derives 31 curated features of protein structure from RNA sequencing results. ExonImpact identified 129 AS events predicted to impact protein structure; differentially spliced mRNAs were enriched in gene ontology pathways that included “actin filament-based movement” and “post-Golgi vesicle-mediated transport.” Whereas variants in MHC Class II genes are strongly associated with T1D risk, expression of Class II genes in the β-cell has been controversial. Notably, we identified 98 AS events in MHC Class II genes. Targeted qPCR was used to validate reduced inclusion of Exon5 in HLA-DMB, which is a Class II gene involved in shaping the immunopeptidome. RNA-FISH demonstrated increased abundance of the splice variant in pancreatic sections from human T1D donors. Finally, a k-mer based approach was used to identify RNA binding proteins associated with skipped-exon events. Serine and Arginine Rich Splicing Factor 2 exhibited increased expression in response to IL-1β + IFN-γ and was implicated in 37.2% of potentially pathogenic events, including HLA-DMB Exon5 exclusion. Together, these findings suggest that stimulus specific isoform expression may control how β-cells respond to inflammatory signals in T1D and potentially contribute to amplification of the autoimmune attack against the β-cells.
W. Wu: None. F. Syed: None. E. Simpson: None. C. Lee: None. D.L. Eizirik: None. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. Y. Liu: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb.
National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, UC4-DK-104166); U.S. Department of Veterans Affairs (I01BX001733); Sigma Beta Sorority; Ball Brothers Foundation; George and Frances Ball Foundation; Indiana Diabetes Research Center (P30DK097512)