Nano-Mod-Amp reveals RNA sequence, structural and cell type specific features of pseudouridylation by PUS7

Pseudouridines are abundant mRNA modifications that can impact splicing, translation, and stability to tune gene expression. PUS7 is one of the major mRNA pseudouridine synthase whose dysregulation leads to neurodevelopmental disorders and cancer, underscoring the critical function of PUS7-dependent pseudouridines. Beyond a short and degenerate consensus sequence, the molecular mechanisms underlying PUS7-mediated pseudouridylation remain unknown. A lack of targeted, high-throughput pseudouridine detection methods limits simultaneous interrogation of PUS7 regulatory features across many experimental conditions. We developed novel Nanopore sequencing tools, including Nano-Mod-Amp, to reveal pseudouridine stoichiometry, its RNA structural context, and dependence on PUS7 levels at specific sites across biological conditions. We identified a novel RNA structural signature that is associated with more efficient mRNA modification by PUS7. Pseudouridines are largely responsive to modulations in PUS7 protein levels, demonstrating the regulatory potential of varying PUS7 levels across cellular conditions. Conversely, PUS7 activity is also regulated in a cell-type specific manner, independent of PUS7 expression levels in a manner consistent with regulation by RNA structure and RNA binding proteins. Together, we developed Nanopore sequencing tools and uncovered new mechanisms of PUS7 regulation with a framework that can be applied to other RNA-modifying enzymes to query the regulation of the epitranscriptome. HighlightsO_LINanopore direct RNA sequencing identifies PUS7-dependent pseudouridines with stoichiometry. C_LIO_LINano-Mod-Amp quantifies PUS7-dependent pseudouridines at hundreds of sites in high-throughput. C_LIO_LIMPRAs define RNA sequence and structural features associated with modification by PUS7. C_LIO_LIIndividual PUS7 target pseudouridines are substoichiometric and poised for regulation. C_LIO_LIPUS7 activity is regulated by cell type in the absence of differences in PUS7 protein levels. C_LI