Functional Characterization of the Pseudouridine Synthase PUS10

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PROJECT SUMMARY Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract affecting millions of individuals worldwide. The majority of patients afflicted with IBD exhibit chronically active disease symptoms, representing a clear unmet need to advance our understanding of IBD pathology and identify new therapeutic targets. Multiple genome-wide association studies have identified pseudouridine synthase 10 (PUS10) as a susceptibility gene for IBD. PUS10 encodes an enzyme responsible for installing the modified nucleotide pseudouridine (Ψ) in a subset of human tRNAs and is likely to target mRNA as well. Individuals with IBD-associated PUS10 alleles exhibit decreased expression, suggesting that loss of PUS10-dependent Ψ in one or more of its target RNAs underlies its role in IBD. However, the functional consequences of PUS10- dependent pseudouridylation are unknown. Notably, my preliminary analysis demonstrates that loss of PUS10 activity initiates an epithelial-to-mesenchymal transition (EMT), a pro-fibrotic cellular process that is dysregulated in IBD and known to contribute to the pathogenesis of other chronic inflammatory disorders. My work seeks to elucidate the molecular function of PUS10-dependent pseudouridylation and pinpoint its roles in EMT initiation. Ψ has profound impacts in both coding and non-coding RNA, affecting RNA processing, decay, and translation—establishing a broad potential for PUS10 to regulate gene expression. I will begin to characterize the role of PUS10 by comprehensively mapping and quantifying PUS10-dependent Ψ transcriptome-wide, using augmented Ψ profiling methods to ensure possible disease-relevant substrates are identified (Aim 1). With PUS10 target sites defined, I will systematically profile the effects of PUS10-dependent Ψ on tRNA function and gene expression, using engineered cell lines with temporal control of PUS10 activity to discriminate between the direct effects of tRNA and mRNA pseudouridylation (Aim 2). I will then relate these molecular perturbations to IBD-relevant cellular physiology—identifying the precise aspects of EMT directly regulated by PUS10’s pseudouridylation activity (Aim 3). Completion of the proposed work will significantly advance our understanding of the poorly characterized IBD risk gene PUS10. These studies will comprehensively determine the molecular consequences of PUS10- dependent pseudouridylation and delineate its role in the regulation of EMT—a cellular process of significant clinical relevance to IBD pathology.