Nanopore signal deviations from pseudouridine modifications in RNA are sequence-specific: quantification requires dedicated synthetic controls.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
28 Sep 2024
28 Sep 2024
Historique:
received:
15
07
2024
accepted:
11
09
2024
medline:
29
9
2024
pubmed:
29
9
2024
entrez:
28
9
2024
Statut:
epublish
Résumé
Chemical modifications to mRNA respond dynamically to environmental cues and are important modulators of gene expression. Nanopore direct RNA sequencing has been applied for assessing the presence of pseudouridine (ψ) modifications through basecalling errors and signal analysis. These approaches strongly depend on the sequence context around the modification, and the occupancies derived from these measurements are not quantitative. In this work, we combine direct RNA sequencing of synthetic RNAs bearing site-specific modifications and supervised machine learning models (ModQuant) to achieve near-analytical, site-specific ψ quantification. Our models demonstrate that the ionic current signal features important for accurate ψ classification are sequence dependent and encompass information extending beyond n + 2 and n - 2 nucleotides from the ψ site. This is contradictory to current models, which assume that accurate ψ classification can be achieved with signal information confined to the 5-nucleotide k-mer window (n + 2 and n - 2 nucleotides from the ψ site). We applied our models to quantitatively profile ψ occupancy in five mRNA sites in datasets from seven human cell lines, demonstrating conserved and variable sites. Our study motivates a wider pipeline that uses ground-truth RNA control sets with site-specific modifications for quantitative profiling of RNA modifications. The ModQuant pipeline and guide are freely available at https://github.com/wanunulab/ModQuant .
Identifiants
pubmed: 39341872
doi: 10.1038/s41598-024-72994-9
pii: 10.1038/s41598-024-72994-9
doi:
Substances chimiques
Pseudouridine
1445-07-4
RNA, Messenger
0
RNA
63231-63-0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
22457Informations de copyright
© 2024. The Author(s).
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