N⁶-methyladenosine (m⁶A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m⁶A-selective ‘reader’ proteins of the YTH family, which incorporate m⁶A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m⁶A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m⁶A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m⁶A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

N⁶-methyladenosine (m⁶A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m⁶A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m⁶A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m⁶A and have increased m⁶A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m⁶A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

N6-Methyladenosine (m⁶A) modification has been implicated in the progression of several cancers. We reveal that during epithelial-mesenchymal transition (EMT), one important step for cancer cell metastasis, m⁶A modification of mRNAs increases in cancer cells. Deletion of methyltransferase-like 3 (METTL3) down-regulates m⁶A, impairs the migration, invasion and EMT of cancer cells both in vitro and in vivo. m⁶A-sequencing and functional studies confirm that Snail, a key transcription factor of EMT, is involved in m⁶A-regulated EMT. m⁶A in Snail CDS, but not 3’UTR, triggers polysome-mediated translation of Snail mRNA in cancer cells. Loss and gain functional studies confirm that YTHDF1 mediates m⁶A-increased translation of Snail mRNA. Moreover, the upregulation of METTL3 and YTHDF1 act as adverse prognosis factors for overall survival (OS) rate of liver cancer patients. Our study highlights the critical roles of m⁶A on regulation of EMT in cancer cells and translation of Snail during this process.

A new transcriptome-wide method produces reliable m ⁶ A maps at single-base resolution and reveals site conservation in mammals.

N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.