Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• 5-Methylcytosine (m⁵C) is a crucial epitranscriptomic modification in mRNA.
• m⁵C modifications are implicated in the pathogenesis of numerous diseases.
• Existing transcriptome-wide m⁵C mapping assays require improvement in accuracy and comprehensiveness.

Purpose of the Study:

• To develop a novel, antibody-free, and bisulfite-free method for accurate and comprehensive transcriptome-wide m⁵C mapping.
• To enhance the identification of m⁵C loci compared to existing techniques.
• To enable the study of m⁵C modifications using ultralow input RNA.

Main Methods:

• Development of the DRAM (deaminase and reader protein assisted RNA methylation analysis) system.
• Fusion of deaminases (APOBEC1 and TadA-8e) with m⁵C reader proteins (ALYREF and YBX1).
• Identification of m⁵C sites via deamination events neighboring methylation sites.

Main Results:

• The DRAM system provides transcriptome-wide RNA editing regions.
• DRAM-identified regions show high overlap with publicly available bisulfite-sequencing (BS-seq) datasets.
• The method allows for more stable and comprehensive identification of m⁵C loci.
• DRAM supports ultralow input RNA amounts (as low as 10 ng).

Conclusions:

• The DRAM system offers an improved approach for transcriptome-wide m⁵C mapping.
• This antibody-free and bisulfite-free method enhances the accuracy and scope of m⁵C site identification.
• DRAM's ability to work with low RNA input facilitates broader applications in biological research.
• The DRAM system is expected to advance the understanding of m⁵C modification functions in various biological processes and diseases.