Targeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled...

Area of Science:

• Molecular Biology
• Epigenetics
• Proteomics

Background:

• N6-Methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins are key regulators of mRNA processing.
• The interplay between m6A and other RNA modifications remains largely unexplored.
• Systematic investigation of epitranscriptomic RWE protein crosstalk is needed.

Purpose of the Study:

• To develop and apply an enhanced liquid chromatography-parallel-reaction monitoring (LC-PRM) method for profiling epitranscriptomic RWE proteins.
• To investigate potential crosstalk between m6A and other RNA modifications.
• To quantify RWE proteins in cells with specific gene ablations.

Main Methods:

• Modification of an established LC-PRM method to incorporate stable isotope-labeled (SIL) peptides as internal or surrogate standards.
• Profiling of epitranscriptomic RWE proteins in HEK293T cells, including those with individually ablated m6A eraser (ALKBH5, FTO) and writer (METTL3) genes.
• Analysis of previously published m6A mapping data.

Main Results:

• Reproducible detection of 114 RWE proteins in HEK293T cells.
• Identification of eight proteins, including writers for 5-methylcytidine and pseudouridine, with significant alterations in cells depleted of METTL3 and ALKBH5.
• Confirmation of m6A presence in the corresponding mRNAs for four of these altered proteins.

Conclusions:

• The modified LC-PRM method with SIL peptides enables robust quantification of epitranscriptomic RWE proteins.
• The study provides evidence for potential crosstalk between m6A and other epitranscriptomic modifications.
• The method is suitable for high-throughput profiling of RWE proteins in various cell types and tissues.