New insights into the dynamics of m6A epitranscriptome: hybrid-seq identifies novel mRNAs of the m6A writers METTL3/14

<b>Background:</b> N6-methyladenosine (m6A), a prevalent mRNA modification, is dynamically regulated by methyltransferases, including METTL3 and METTL14. <b>Materials &amp; methods:</b> In the current study, we employed a custom hybrid-seq method to identify novel <i>METTL3</i>/<i>14</i> transcripts, explore their protein-coding capacities and predict the putative role of the METTL isoforms. <b>Results:</b> Demultiplexing of the hybrid-seq barcoded datasets unraveled the expression patterns of the newly identified mRNAs in major malignancies as well as in non-malignant cells, providing a deeper understanding of the methylation pathways. Open reading frame query revealed novel METTL3/14 isoforms, broadening our perspective for the structural diversity within METTL family. <b>Conclusion:</b> Our findings offer significant insights into the intricate transcriptional landscape of <i>METTL3</i>/<i>14</i>, shedding light on the regulatory mechanisms underlying methylation in mRNAs. Nanopore sequencing unveils novel alternative <i>METTL3</i> and <i>METTL14</i> mRNAs. In total, eight novel <i>METTL3</i> mRNAs (<i>METTL3</i> v.2–v.9) and eight novel <i>METTL14</i> mRNAs (<i>METTL14</i> v.2–v.9) were identified. Expression profiling of the <i>METTL3/14</i> mRNAs was performed in human malignant and non-cancerous cells. <i>METTL3</i> v.9 exhibits significantly higher expression levels across the investigated human malignancies. <i>METTL14</i> v.5 emerged as the most abundant alternative mRNA in the tested cancer types. <i>METTL3</i> v.2–v.9 and <i>METTL14</i> v.2–v.9 were successfully quantified through variant-specific qPCR assays. Ribosome profiling supports that specific <i>METTL3/14</i> mRNAs are highly promising to encode protein isoforms. <i>In silico</i> structural analysis highlights the methyltransferase activity in most of the described putative novel isoforms.