Nucleotide resolution profiling of m3C RNA modification by HAC-seq

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Cellular RNA modifications, known as the epitranscriptome, are crucial for gene expression and function.
• Dysregulation of epitranscriptomic marks is linked to various human diseases.
• Accurate mapping of RNA modifications is essential for understanding their physiological and pathological roles.

Purpose of the Study:

• To develop a method for precise, transcriptome-wide mapping of 3-methylcytidine (m3C) RNA modifications.
• To profile the m3C methylome at single-nucleotide resolution.
• To investigate the prevalence of m3C modifications in different RNA species.

Main Methods:

• Development of a novel m3C-specific sequencing technique: Hydrazine-Aniline Cleavage sequencing (HAC-seq).
• Application of HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs from human cells (MCF7).
• Single-nucleotide resolution profiling of m3C modification sites.

Main Results:

• HAC-seq successfully profiled the m3C methylome at single-nucleotide resolution.
• Transfer RNAs (tRNAs) were identified as the predominant m3C-modified RNA species.
• 17 m3C sites were detected on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors; no significant m3C modification was found on mRNA or other non-coding RNAs.

Conclusions:

• HAC-seq is a novel, unbiased method for transcriptome-wide m3C identification.
• The study provides a comprehensive map of m3C modifications in human tRNAs.
• HAC-seq has broad applicability for studying the m3C methylome in diverse biological contexts.