HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA.
Fiche publication
Date publication
septembre 2020
Journal
Nucleic acids research
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MOTORINE Iouri, Dr MARCHAND Virginie
Tous les auteurs :
Marchand V, Pichot F, Neybecker P, Ayadi L, Bourguignon-Igel V, Wacheul L, Lafontaine DLJ, Pinzano A, Helm M, Motorin Y
Lien Pubmed
Résumé
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.
Référence
Nucleic Acids Res.. 2020 Sep 25;: