<b>Proteogenomic discovery of novel open reading frames with HLA immune presentation on human beta cells</b>

<p dir="ltr">Ribosome profiling measures translational regulation and reveals novel or unannotated open reading frames (nuORFs) otherwise difficult to identify. Recent reports demonstrate that nuORFs regulate gene expression and immune recognition, highlighting their emerging biological roles. Pancreatic beta cells are critical for maintaining euglycemic conditions and beta-cell impairment contributes to diabetes development. Identification of nuORF and protein/peptide products in human beta cells could reveal novel mechanisms that regulate beta-cell function during homeostatic and disease conditions. Here, we applied a proteogenomics approach to human beta cells to define previously unknown protein/peptide products. First, we applied cell-type-specific Ribo-seq to map the translatome of human stem cell-derived beta cells (sBC). Pathways crucial for beta cell function and antigen presentation were subject to translational regulation. We detected a recently described immunogenic neoantigen, INS-DRiP, presumably originating from an downstream start site in INS mRNA. Moreover, our analysis revealed 965 novel nuORFs in sBCs, with the majority showing protein-level support. Comparison with primary human islets further validated nuORF translation and highlighted beta cell specificity. We identified a novel, primate-specific regulatory upstream ORF within TYK2, which is crucial for beta cell function and interferon response and has many variants strongly associated with type 1 diabetes. Finally, we used immunopeptidomics, HLA binding prediction models, and T cell co-culture assays to validate the presentation and immunogenicity of preproinsulin peptides and nuORFs. Our findings underscore the importance of translational regulation in beta cell function and provide an important resource to the diabetes research community.</p>