mRNA Pseudouridine Profiling Using Nanopore DRS Reveals Cell Type Specific Pseudouridylation

Pseudouridine (psi) is one of the most abundant mRNA modifications in the human transcriptome arising from the post-transcriptional isomerization of uridine via pseudouridine synthases, including TRUB1 and PUS7. Nanopore direct RNA sequencing combined with a recent tool our group developed enables semi-quantitative psi site maps in whole transcriptomes without the need for chemical derivatization of the input RNA and/or conversion to cDNA. While semi-quantitative, our method is efficient for detecting changes in site-specific psi occupancies across cell conditions. Since the biological significance of psi at specific sites within coding sequences is unknown, cell type-specific analysis can better inform our understanding of the impact on gene expression and disease associations. 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 identify cell type-specific presence or absence of psi modification positions for ubiquitously expressed genes. We validate these sites by ruling out single-nucleotide variants, analyzing current traces, and performing enzymatic knockdowns. Finally, we characterize sites with multiple modifications on the same transcript and found that these can be conserved or cell type-specific. Additionally, we discovered examples of psi within the same k-mer for the first time and analyzed the effect on current distribution. Given the numerous biological processes affected by RNA modification, our analyses guide profiling modifications across cell types, diseased states, and perturbations.