Begik, OguzhanLucas, Morghan C.Pryszcz, Leszek Piotr, 1985-Ramirez, Jose MiguelMedina, RebecaMilenkovic, IvanCruciani, SoniaLiu, HuanleSantos Vieira, Helaine GrazieleSas Chen, AldemaMattick, John S.Schwartz, SchragaNovoa, Eva Maria2021-07-162021Begik O, Lucas MC, Pryszcz LP, Ramirez JM, Medina R, Milenkovic I, Cruciani S, Liu H, Vieira HGS, Sas-Chen A, Mattick JS, Schwartz S, Novoa EM. Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing. Nat Biotechnol. 2021;39:1278-91. DOI: 10.1038/s41587-021-00915-61087-0156http://hdl.handle.net/10230/48205Nanopore 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.application/pdfeng© Springer Nature Publishing AG Begik O, Lucas MC, Pryszcz LP, Ramirez JM, Medina R, Milenkovic I, Cruciani S, Liu H, Vieira HGS, Sas-Chen A, Mattick JS, Schwartz S, Novoa EM. Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing. Nat Biotechnol. 2021;39:1278-91. DOI: 10.1038/s41587-021-00915-6 [http://dx.doi.org/10.1038/s41587-021-00915-6]info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41587-021-00915-6RNA modificationRNA sequencingSoftwareinfo:eu-repo/semantics/openAccess