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 N-6-methyladenosine, we show that other modifications, in particular pseudouridine (psi) and 2 '-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on psi modification sites, we detected known and uncovered previously unreported psi sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent psi 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 psi-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 psi RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA. Nanopore sequencing detects pseudouridine and 2 '-O-methylation modifications in cellular RNAs.

Automated summary

This study demonstrated the potential of nanopore RNA sequencing for detecting and identifying various RNA modifications, including pseudouridine and 2'-O-methylation. By focusing on pseudouridine modification sites, the researchers revealed the distribution and dynamics of these modifications in cellular RNAs. The development of a software tool to estimate modification stoichiometries at each site provides a valuable tool for further research on RNA modifications.