PUS7 mRNA pseudouridylation is driven by RNA sequence, structure, and cell-type-specific features

PUS7 is a major mRNA pseudouridine synthase that influences gene expression and is dysregulated in neurodevelopmental disorders and cancer. PUS7 recognizes a prevalent and degenerate UNUAR sequence, but the mechanisms underlying PUS7's specificity remain unknown. We developed Nano-Mod-Amp, a targeted Nanopore high-throughput pseudouridine detection method, to interrogate PUS7 regulatory features. We established that USUAG, accessibility of the target uridine, and RNA structure are drivers of mRNA modification by PUS7. Perturbing structure through mutations or antisense oligos modulates pseudouridine levels. In cells, pseudouridines are responsive to PUS7 levels, demonstrating the regulatory potential of varying PUS7 levels across cell states. Conversely, PUS7 activity varies across cell types independently of expression levels, suggesting a potential regulatory role for RNA-binding proteins, RNA structure, or other cellular factors. We uncovered principles guiding PUS7 activity, enabling site-specific modulation of pseudouridines. These epitranscriptomic mechanisms provide molecular insight into the regulation and dysregulation of PUS7.