Single-molecule m⁶A detection empowered by endogenous labeling unveils complexities across RNA isoforms

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• N6-methyladenosine (m6A) is a crucial RNA modification, but its distribution across diverse RNA isoforms remains unclear.
• Understanding m6A dynamics is vital for deciphering gene regulation and cellular processes.

Purpose of the Study:

• To develop a high-resolution method for mapping m6A sites across individual RNA molecules and isoforms.
• To investigate the regulatory mechanisms and patterns of m6A modification in different RNA contexts.

Main Methods:

• Endogenous labeling of m6A sites using APOBEC1-YTH-induced C-to-U mutations on Oxford Nanopore Technology (ONT) direct RNA sequencing (DRS) reads.
• Development and application of m6Aiso, a deep residual neural network, for single-read m6A identification and quantification.
• Analysis of m6A patterns across RNA isoforms, including distance-dependent linkages and isoform-specific methylation.

Main Results:

• Generated over 1 million single-read m6A signals, enabling precise mapping.
• m6Aiso accurately identified and quantified m6A sites at single-read resolution.
• Uncovered distance-dependent m6A site linkages and specific methylation on intron-retained isoforms, influenced by exon junction proximity and TARBP2 binding.
• Identified SMAD3 as a promoter of m6A deposition during epithelial-mesenchymal transition, affecting alternative promoter-driven isoforms.

Conclusions:

• m6Aiso provides an effective tool for dissecting the complex landscape of m6A modifications across RNA isoforms.
• The study reveals novel insights into m6A regulation, including isoform-specific methylation and the role of transcription factors like SMAD3.