Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing

• Published in: Nature biotechnology Vol. 39; no. 10; pp. 1278 - 1291
• Main Authors: Begik, Oguzhan, Lucas, Morghan C., Pryszcz, Leszek P., Ramirez, Jose Miguel, Medina, Rebeca, Milenkovic, Ivan, Cruciani, Sonia, Liu, Huanle, Vieira, Helaine Graziele Santos, Sas-Chen, Aldema, Mattick, John S., Schwartz, Schraga, Novoa, Eva Maria
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.10.2021; Nature Publishing Group
• Subjects: 631/114/794; 631/1647/514/1949; 631/337/1645/2570; Agriculture; Algorithms; Bioinformatics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Datasets; Gene sequencing; Genetic aspects; Heat stress; Heat tolerance; Life Sciences; Methods; Methylation; Mitochondria; N6-methyladenosine; Nanoparticles; Nucleoli; Post-translational modification; Pseudouridylation; RNA modification; RNA sequencing; rRNA; Stoichiometry; Tuna; Yeast; Spain
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/s41587-021-00915-6

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 (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. Nanopore sequencing detects pseudouridine and 2′- O -methylation modifications in cellular RNAs.