8681 Preoteogenomic Identification Of Novel Translated And Regulatory Open Reading Frames In Human Beta-Cells

Abstract Disclosure: K. Walters: None. R.C. Guiterrez: None. S. Sakhar: None. A. Baldwin: None. E.S. Nakayasu: None. H. Russ: None. N. Mukherjee: None. Preoteogenomic Identification of Novel Translated and Regulatory Open Reading Frames in Human Beta-cells The discovery of novel autoreactive T cells that target β cells during type 1 diabetes (T1D) has been challenging due to our limited knowledge in autoreactive β cell antigens. Most of these antigens are derived from canonical open reading frames (ORFs) of protein coding genes, such as preproinsulin and chromogranin A. However, a defective ribosomal product (DRiP) was recently identified from the insulin mRNA (INS-DRiP). This antigen is the result of translation from a novel or unannotated open reading frames (nuORFs). nuORFs have a range of functions from regulating translation to encoding novel small bioactive peptides to producing neoantigens. In this study, we use a transcriptome-wide approach to identify nuORFs in human β cells in an unbiased manner. We performed ribosome profiling (ribo-seq) in stem cell derived β cells (sBCs). Human pluripotent stem cells were differentiated into sBCs expressing a flag-tagged ribosomal protein (RPL22-Flag) driven by the rat insulin promoter. We performed IP-ribo-seq on sBCs enriching for RPL22-Flag containing ribosomes. These data exhibited high-resolution 3-nucleotide periodicity consistent with translating codons and identified sequencing reads consistent with translation of the INS-DRiP. We performed de novo ORF-calling using two software using complementary statistical approaches (RiboCode and ORFQuant). We identified over 12,000 canonical annotated ORFs of which ∼85% were had protein level support from matched proteomics confirming the high specificity of our approach. Of nearly 1000 nuORFs identified in sBCs, over half had protein level support in sBCs and cadaveric human islets. We found that 149 nuORFs were associated with Type-1 Diabetes (T1D) genes, including a novel upstream ORF (uORF) in TYK2, which had the strongest association with T1D and is a promising therapeutic target to halt T1D progression. Using reporter assay, we show that the novel uORF in TYK2 is a translational activator and warrants further investigation. Finally, our data represents a valuable database for the search of novel autoreactive antigens in β cells and accelerate the discovery of novel autoreactive T cells implicated in T1D pathogenesis. Presentation: 6/2/2024