RNA sequence, structure, and cell type specific features drive pseudouridylation by PUS7

Description

Raw images related to the publication. Pseudouridine is an abundant mRNA modification that can tune gene expression. PUS7 is one of the major mRNA pseudouridine synthases whose dysregulation leads to neurodevelopmental disorders and cancer. PUS7 recognizes a prevalent and degenerate UNUAR sequence, but the molecular mechanisms underlying PUS7’s specificity remain unknown. We developed Nano-Mod-Amp, a targeted high-throughput pseudouridine detection method, to interrogate PUS7 regulatory features. We established that USUAG, accessibility of the target uridine, and an RNA structural signature are drivers of mRNA modification by PUS7. Perturbing structure through mutations or antisense oligos modulates pseudouridine levels. In the cell we find that pseudouridines are largely responsive to PUS7 protein levels, demonstrating the regulatory potential of varying PUS7 levels across cell states. Conversely, PUS7 activity also varies across cell types independently of PUS7 expression levels, suggesting a potential regulatory role for RNA binding proteins, RNA structure, or other cellular factors. Together, we uncovered regulatory principles guiding PUS7 activity using Nanopore sequencing tools, enabling site specific modulation of pseudouridines. Our study reveals epitranscriptomic mechanisms that provide molecular insight into the regulation and dysregulation of PUS7 activity.

Institutions

• Stanford University

  California, Stanford

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