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Analysis of pseudouridines and other RNA modifications using HydraPsiSeq protocol
Virginie Marchand1, Valérie Bourguignon-Igel2, Mark Helm3
1Université de Lorraine, CNRS, INSERM, UMS2008/US40 IBSLor, EpiRNA-Seq Core facility, F-54000 Nancy, France.
Methods (San Diego, Calif.)
|September 4, 2021
Summary
This study introduces HydraPsiSeq, a chemical method for detecting RNA modifications. It precisely quantifies pseudouridylation and identifies other modifications using deep sequencing, overcoming limitations of previous techniques.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Detecting RNA modifications via deep sequencing traditionally relies on antibody enrichment or reverse transcriptase (RT) signatures.
- Antibody methods can be biased and unreliable, while natural RT signatures are rare for most modified nucleotides.
- Chemically induced RT signatures offer a more robust and controllable approach, enabling the use of mock-treated controls to validate results.
Purpose of the Study:
- To develop and present methodological advancements for the HydraPsiSeq technique.
- To enable precise quantification of RNA pseudouridylation (ψ) using chemical induction of RT signatures.
- To expand the capability of HydraPsiSeq for detecting and quantifying other RNA modifications beyond pseudouridine.
Main Methods:
- Utilized hydrazine, an RNA-specific chemical reagent known for its reactivity towards uracil (U) and differential reaction rates with pseudouridine (ψ).
- Developed deep sequencing-based protocols leveraging chemically induced RT signatures.
- Incorporated mock-treated controls to differentiate true modification signals from spontaneous RT arrests and other sequencing artifacts.
Main Results:
- Demonstrated the precise quantification of RNA pseudouridylation using the refined HydraPsiSeq methodology.
- Successfully detected and quantified additional RNA modifications, in addition to pseudouridine, by analyzing their unique behaviors upon hydrazine treatment.
- Validated the reliability of chemical induction for RNA modification detection, overcoming limitations of antibody-dependent and natural RT signature methods.
Conclusions:
- HydraPsiSeq represents a significant advancement in RNA modification detection, offering precise quantification and broader applicability.
- The method provides a trusted experimental approach for studying RNA pseudouridylation and other modifications.
- This technique enhances the understanding of RNA modifications' roles in biological processes through reliable deep sequencing analysis.
