Analysis of pseudouridines and other RNA modifications using HydraPsiSeq protocol

•Pseudouridine (ψ) residues present in RNAs can be mapped and quantified by HydraPsiSeq.•HydraPsiSeq is based on hydrazine cleavage and coupling to deep sequencing.•Some other RNA modifications are detectable by protection or increased cleavage. Detection of RNA modified nucleotides using deep seque...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2022-07, Vol.203, p.383-391
Main Authors: Marchand, Virginie, Bourguignon-Igel, Valérie, Helm, Mark, Motorin, Yuri
Format: Article
Language:English
Subjects:
Biochemistry, Molecular Biology
Deep sequencing
Epitranscriptome
Genomics
Life Sciences
Molecular biology
Pseudouridylation
Regulation
RNA modification
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Summary:•Pseudouridine (ψ) residues present in RNAs can be mapped and quantified by HydraPsiSeq.•HydraPsiSeq is based on hydrazine cleavage and coupling to deep sequencing.•Some other RNA modifications are detectable by protection or increased cleavage. Detection of RNA modified nucleotides using deep sequencing can be performed by several approaches, including antibody-driven enrichment and natural or chemically induced RT signatures. However, only very few RNA modified nucleotides generate natural RT signatures and antibody-driven enrichment heavily depends on the quality of antibodies used and may be highly biased. Thus, the use of chemically-induced RT signatures is now considered as the most trusted experimental approach. In addition, the use of chemical reagents allows inclusion of simple “mock-treated” controls, to exclude spontaneous RT arrests, SNPs and other misincorporation-prone sites. Hydrazine is a well-known RNA-specific reagent, already extensively used in the past for RNA sequencing and structural probing. Hydrazine is highly reactive to U and shows low reaction rates with ψ residues, allowing their distinction by deep sequencing-based protocols. However, other modified RNA residues also show particular behavior upon hydrazine treatment. Here we present methodological developments allowing to use HydraPsiSeq for precise quantification of RNA pseudouridylation and also detection and quantification of some other RNA modifications, in addition to ψ residues.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2021.08.008