The N6-methyladenosine (m6A) post-transcriptional modification is the most prevalent, profoundly impacting mRNA metabolism. The m6A modification can be dynamically added by the methyltransferase complex METTL3/METTL14. Global knockout either Mettl3 or Mettl14 in mice results in early embryonic lethality. Their functions during heart development remain unclear. We found that conditional knockout (cKO) of either METTL3 (MT3cKO) or METTL14 (MT14cKO) in cardiomyocytes of mice displayed neonatal lethality, presenting the same fetal dilated cardiomyopathy (DCM) phenotypes (enlarged hearts, thin compact wall and disorganized sarcomeric structures), demonstrating their cooperative roles in heart development. Transcriptomic analysis of E13.5 hearts revealed that MT3cKO and MT14cKO mutants had reduced transcripts of genes encoding proteins involved in sarcomere assembly and maturation compared to controls. We investigated whether the loss of m6A modification in sarcomere-related transcripts contributed to the altered gene expression patterns. We identified hypomethylated sarcomere transcripts, including Myopalladin (Mypn) and Titin (Ttn), which exhibited multiple hypomethylated peaks in their exonic regions in both KO hearts compared to controls. CUT&RUN m6A RNA enrichment assays confirmed a significant reduction in m6A methylation of Mypn and Ttn transcripts in both KO hearts. To investigate the role of this methyltransferase complex in sarcomere maturation in human cardiomyocytes, we independently knocked down METTL3 using shRNA adenovirus in stages of cardiomyocyte differentiation from human pluripotent stem cells (hPSCs) and found that knockdown after the onset of beating significantly impaired maturation and downregulated sarcomere transcripts including Mypn and Ttn , indicating that methyltransferase primarily affects cardiomyocyte maturation. In total, our findings expose the critical role of METTL3- and METTL14-mediated m6A modification in regulating sarcomere maturation in the developing mouse heart. To our knowledge, this is the first study to reveal a shared mechanism of the methyltransferase complex in heart development, specifically in the post-transcriptional regulation of sarcomere transcripts.
Burke et al. (Fri,) studied this question.