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Alternative splicing of METTL3 explains apparently METTL3-independent m6A modifications in mRNA

Hui Xian Poh1, Aashiq H Mirza1, Brian F Pickering1

  • 1Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, New York, United States of America.

Plos Biology
|July 19, 2022
PubMed

Insights

The methyltransferase METTL3 (N6-methyladenosine methyltransferase like 3) is responsible for most mRNA N6-methyladenosine (m6A) modifications. Previously reported METTL3 knockouts retained m6A due to alternative splicing producing functional METTL3 isoforms.

Area of Science:

  • Molecular Biology
  • Epigenetics
  • RNA Biology

Background:

  • N6-methyladenosine (m6A) is a prevalent mRNA modification impacting gene expression.
  • METTL3 is identified as the primary enzyme catalyzing m6A formation.
  • Previous studies reported residual m6A in METTL3-depleted cells, suggesting other enzymes contribute.

Purpose of the Study:

  • To re-evaluate the source of m6A in the mRNA transcriptome.
  • To investigate the role of METTL3 in m6A synthesis.
  • To clarify discrepancies in previous METTL3 knockout studies.

Main Methods:

  • Characterization of mouse embryonic stem cell lines with Mettl3 knockout.
  • Analysis of alternatively spliced Mettl3 transcript isoforms.
  • Assessment of m6A levels using an exon 4 deletion Mettl3 model.
  • Evaluation of gene dependency datasets for cell proliferation after METTL3 deletion.

Main Results:

  • Mouse embryonic stem cells with Mettl3 knockout expressed alternatively spliced isoforms producing functional METTL3.
  • Reported METTL3 knockout cell lines exhibited altered METTL3 proteins, not complete knockout.
  • Gene dependency data indicated most cell lines require METTL3 for proliferation.
  • An exon 4 deletion of Mettl3 confirmed METTL3's responsibility for >95% of mRNA m6A.

Conclusions:

  • METTL3 is responsible for the vast majority of m6A in the mRNA transcriptome.
  • Residual m6A in previous METTL3 knockout models is attributed to functional METTL3 isoforms bypassing mutations.
  • Accurate assessment of METTL3's role requires accounting for alternative splicing and functional protein expression.

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