A Quantitative Sequencing Method for 5-Formylcytosine in RNA.

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• 5-Formylcytosine (f5C) is a known RNA modification found in specific tRNAs.
• The transcriptome-wide distribution of f5C in mammals remains largely uncharacterized.
• Existing methods lack the quantitative resolution to map f5C across the entire RNA landscape.

Purpose of the Study:

• To develop and validate a novel sequencing method for transcriptome-wide mapping of 5-formylcytosine (f5C).
• To investigate the distribution and abundance of f5C modifications in mammalian cells.
• To identify novel f5C-modified RNA species and their potential roles.

Main Methods:

• Development of f5C-seq, a quantitative sequencing technique utilizing pic-borane reduction and C-to-T mutation signature during reverse transcription.
• Validation of f5C-seq using known f5C sites in tRNA and assessing modification levels in ALKBH1-depleted cells.
• Application of f5C-seq to map f5C in total RNA and chromatin-associated RNA (caRNA) from HeLa and mouse embryonic stem cells (mESCs).

Main Results:

• f5C-seq successfully identified and quantified known f5C sites in tRNA, showing significant reduction upon ALKBH1 depletion.
• The study revealed novel, highly modified f5C sites across the transcriptome in both HeLa and mESC lines.
• Application to caRNA identified previously uncharacterized f5C modifications in this RNA fraction.

Conclusions:

• f5C-seq provides a robust and quantitative platform for mapping f5C modifications transcriptome-wide in mammals.
• The findings reveal a broader distribution of f5C in RNA than previously appreciated, including in caRNA.
• This work opens new avenues for studying the functional significance of f5C in gene regulation and cellular processes.