Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing

Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N6-methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA. This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skodowska-Curie grant agreement no. 713673. This work was supported by the Australian Research Council (DP180103571 to E.M.N.) and the MEIC (PGC2018-098152-A-100 to E.M.N.). We acknowledge the support of the MEIC to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya.