HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

Abstract

AbstractDeveloping methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed ‘HydraPsiSeq’, a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10–50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20–25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

Autoren

Virginie Marchand
Florian Pichot
Paul Neybecker
Lilia Ayadi
Valérie Bourguignon-Igel
Ludivine Wacheul
Denis LJ Lafontaine
Astrid Pinzano
Mark Helm
Yuri Motorin

DOI

10.1093/nar/gkaa769

eISSN

1362-4962

ISSN

0305-1048

Ausgabe der Veröffentlichung

19

Zeitschrift

Nucleic Acids Research

Sprache

en

Online publication date

2020

Paginierung

e110 - e110

Datum der Veröffentlichung

2020

Status

Published

Herausgeber

Oxford University Press (OUP)

Herausgeber URL

http://dx.doi.org/10.1093/nar/gkaa769

Datum der Datenerfassung

2023

Titel

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

Ausgabe der Zeitschrift

48

Data source: Crossref

Abstract

Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

Addresses

Université de Lorraine, CNRS, INSERM, IBSLor (UMS2008/US40), Epitranscriptomics and RNA Sequencing Core Facility, F54000 Nancy, France.

Autoren

Virginie Marchand
Florian Pichot
Paul Neybecker
Lilia Ayadi
Valérie Bourguignon-Igel
Ludivine Wacheul
Denis LJ Lafontaine
Astrid Pinzano
Mark Helm
Yuri Motorin

DOI

10.1093/nar/gkaa769

eISSN

1362-4962

Externe Identifier

PubMed Identifier: 32976574
PubMed Central ID: PMC7641733

Funding acknowledgements

Belgian Fonds de la Recherche Scientifique: R.8015.19
Deutsche Forschungsgemeinschaft: HE3397/17-1
Grand Est Region:
Université Libre de Bruxelles: 1810070
EPITRAN COST: CA16120
Deutsche Forschungsgemeinschaft: HE 3397/13-2
Deutsche Forschungsgemeinschaft: RO 4681/6-1
Belgian Fonds de la Recherche Scientifique: T.0144.20

Open access

true

ISSN

0305-1048

Ausgabe der Veröffentlichung

19

Zeitschrift

Nucleic acids research

Schlüsselwörter

Cells, Cultured
Mesenchymal Stem Cells
Humans
Saccharomyces cerevisiae
RNA, Messenger
RNA, Ribosomal
RNA, Transfer
Pseudouridine
Sequence Analysis, RNA

Sprache

eng

Medium

Print

Open access status

Open Access

Paginierung

e110

Datum der Veröffentlichung

2020

Status

Published

Publisher licence

CC BY-NC

Datum der Datenerfassung

2020

Titel

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA.

Sub types

Research Support, Non-U.S. Gov't
research-article
Journal Article

Ausgabe der Zeitschrift

48

Files

https://academic.oup.com/nar/article-pdf/48/19/e110/34133434/gkaa769.pdf https://europepmc.org/articles/PMC7641733?pdf=render

Data source: Europe PubMed Central

Abstract

Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

Date of acceptance

2020

Autoren

Virginie Marchand
Florian Pichot
Paul Neybecker
Lilia Ayadi
Valérie Bourguignon-Igel
Ludivine Wacheul
Denis LJ Lafontaine
Astrid Pinzano
Mark Helm
Yuri Motorin

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/32976574

DOI

10.1093/nar/gkaa769

eISSN

1362-4962

Externe Identifier

PubMed Central ID: PMC7641733

Ausgabe der Veröffentlichung

19

Zeitschrift

Nucleic Acids Res

Schlüsselwörter

Cells, Cultured
Humans
Mesenchymal Stem Cells
Pseudouridine
RNA, Messenger
RNA, Ribosomal
RNA, Transfer
Saccharomyces cerevisiae
Sequence Analysis, RNA

Sprache

eng

Country

England

Paginierung

e110

PII

5911743

Datum der Veröffentlichung

2020

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2020

Titel

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

48

Data source: PubMed

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

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