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

Pseudouridine (psi) is one of the most abundant human mRNA modifications generated from the isomerization of uridine via psi synthases, including TRUB1 and PUS7 . Nanopore direct RNA sequencing combined with our recent tool, Mod- p ID, enables psi mapping, transcriptome-wide, without chemical derivatization of the input RNA and/or conversion to cDNA. This method is sensitive for detecting changes in positional psi occupancies across cell types, which can inform our understanding of the impact 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, among a list of highly conserved sites across cell types, most are TRUB1 substrates and fall within the coding sequence. We find that these conserved psi positions correspond to higher levels of protein expression than expected, suggesting translation regulation. Interestingly, we identify cell type-specific sites of psi modification in ubiquitously expressed genes. We validate these sites by ruling out single-nucleotide variants, analyzing current traces, and performing enzymatic knockdowns of psi synthases. Finally, we characterize sites with multiple psi modifications on the same transcript (hypermodification type II) and found that these can be conserved or cell type specific. Among these, we discovered examples of multiple psi modifications within the same k-mer for the first time and analyzed the effect on current distribution. Our data support the hypothesis that motif sequence and the presence of psi synthase are insufficient to drive modifications, that psi modifications contribute to regulating translation and that cell type-specific trans-acting factors play a major role in driving pseudouridylation.