mRNA psi profiling using nanopore DRS reveals cell type-specific pseudouridylation.

Pseudouridine (psi) is one of the most abundant human mRNA modifications generated via psi synthases, including and . Nanopore direct RNA sequencing combined with our recently developed tool, Mod- ID, enables psi mapping, transcriptome-wide, without chemical derivatization of the input RNA and/or conversion to cDNA. This method is sensitive for detecting differences in the positional occupancy of psi across cell types, which can inform our understanding of the impact of psi on gene expression. We sequenced, mapped, and compared the positional psi occupancy across six immortalized human cell lines derived from diverse tissue types. We found that lung-derived cells have the highest proportion of psi, while liver-derived cells have the lowest. Further, we find that conserved psi positions on mRNAs produce higher levels of protein than expected, suggesting a role in translation regulation. Interestingly, we identify cell type-specific sites of psi modification in ubiquitously expressed genes. Finally, we characterize transcripts with multiple psi modifications and found that these psi sites can be conserved or cell type-specific, including examples of multiple psi modifications within the same motif. Our data suggest that psi modifications contribute to regulating translation and that cell type-specific transacting factors play a major role in driving pseudouridylation.