Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Nanopore sequencing offers a novel approach for detecting RNA modifications.
• Previous work focused on N6-methyladenosine, leaving other modifications underexplored.

Purpose of the Study:

• To investigate the utility of nanopore sequencing for identifying pseudouridine (Ψ) and 2 -O-methylation (Nm) in native RNA.
• To develop methods for quantifying RNA modification stoichiometry.

Main Methods:

• Utilized nanopore sequencing to analyze native RNA.
• Developed the nanoRMS software for analyzing base-calling signatures and current intensity data.
• Applied stress conditions (oxidative, cold, heat) to yeast cells to study RNA modification dynamics.

Main Results:

• Identified characteristic nanopore signatures for Ψ and Nm modifications.
• Detected known and novel Ψ sites across various RNA types (mRNAs, non-coding RNAs, rRNAs).
• Discovered a Pus4-dependent Ψ modification in yeast mitochondrial rRNA and heat-sensitive Ψ sites.

Conclusions:

• Nanopore sequencing effectively detects and distinguishes between different RNA modifications, including Ψ and Nm.
• The nanoRMS software enables quantification of RNA modification stoichiometry from nanopore data.
• This approach provides valuable insights into RNA modification patterns and dynamics in cellular contexts.