Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing

Oguzhan Begik, Morghan C. Lucas, Leszek P. Pryszcz, Jose Miguel Ramirez, Rebeca Medina, Ivan Milenkovic, Sonia Cruciani, Huanle Liu, Helaine Graziele Santos Vieira, Aldema Sas-Chen, John S. Mattick, Schraga Schwartz, Eva Maria Novoa

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)1278-1291
Number of pages14
JournalNature biotechnology
Issue number10
Early online date13 May 2021
StatePublished - Oct 2021

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering


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