Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• 3-Methylcytidine (m3C) is a common modification in transfer RNAs (tRNAs).
• The presence and function of m3C in eukaryotic messenger RNAs (mRNAs) were previously unknown.
• Understanding m3C distribution and formation in mRNAs is crucial for RNA metabolism studies.

Purpose of the Study:

• To develop a novel method for profiling m3C modifications in the transcriptome.
• To investigate the distribution and formation of m3C in eukaryotic mRNAs and long noncoding RNAs (lncRNAs).
• To explore the functional implications of m3C methylation in RNA metabolism.

Main Methods:

• Developed m3C immunoprecipitation and sequencing (m3C-IP-seq) for single-nucleotide resolution profiling.
• Utilized antibody enrichment to capture m3C-modified RNA molecules.
• Analyzed m3C sites in tRNAs, mRNAs, and lncRNAs.

Main Results:

• m3C-IP-seq successfully identified m3C modifications in 12 cytoplasmic and 2 mitochondrial tRNA isoacceptors.
• Comprehensive profiling revealed widespread m3C sites in mRNAs and lncRNAs, dependent on a nuclear METTL8 isoform.
• A significant number of m3C sites were found in mRNA 3' untranslated regions (3' UTRs) and correlated with mRNA degradation.
• m3C methylation levels were observed to be dynamic and responsive to hypoxic conditions.

Conclusions:

• Demonstrated the widespread presence of m3C modification across the human transcriptome.
• Established m3C-IP-seq as a powerful tool for mapping RNA modifications.
• Provided a valuable resource for future functional studies on m3C-mediated RNA metabolism and its regulation.