De novo basecalling of m 6 A modifications at single molecule and single nucleotide resolution

作者：Sonia Cruciani, Anna Delgado-Tejedor, Leszek P. Pryszcz, Rebeca Medina, Laia Llovera, Eva Maria Novoa · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2023 · DOI：10.1101/2023.11.13.566801 · 被引用次数：15 · 研究领域：RNA modifications and cancer、HVDC Systems and Fault Protection、RNA Research and Splicing

ABSTRACT RNA modifications hold pivotal roles in shaping the fate and function of RNA molecules. Although nanopore sequencing technologies have proven successful at transcriptome-wide detection of RNA modifications, current algorithms are limited to predicting modifications at a per-site level rather than within individual RNA molecules. Herein, we introduce m 6 ABasecaller , an innovative method enabling direct basecalling of m 6 A modifications from raw nanopore signals within individual RNA molecules. This approach facilitates de novo prediction of m 6 A modifications with precision down to the single nucleotide and single molecule levels, without the need of paired knockout or control conditions. Using the m 6 ABasecaller , we find that the median transcriptome-wide m 6 A modification stoichiometry is ∼10-15% in human, mouse and zebrafish. Furthermore, we show that m 6 A modifications affect polyA tail lengths, exhibit a propensity for co-occurrence within the same RNA molecules, and show relatively consistent stoichiometry levels across isoforms. We further validate the m 6 ABasecaller by treating mESC with increasing concentrations of STM2457, a METTL3 inhibitor as well as in inducible METTL3 knockout systems. Overall, this work demonstrates the feasibility de novo basecalling of m 6 A modifications, opening novel avenues for the application of nanopore sequencing to samples with limited RNA availability and for which control knockout conditions are unavailable, such as...