N⁶-methyladenosine (m⁶A) plays crucial roles in regulating RNA metabolisms. METTL16 identified as a single-component methyltransferase catalyzes m⁶A formation in the nucleus; whether it regulates cytoplasmic RNA fate remains unknown. Here, we detected the dual localization of METTL16 in the nucleus and cytoplasm. METTL16 depletion attenuates protein synthesis, but the methyltransferase activity is not required for its translation-promoting function. Mechanistically, we identified an interactor of METTL16, eIF4E2, which represses translation by acting as a competitor of eIF4E. The METTL16-eIF4E2 interaction impedes the recruitment of eIF4E2 to 5' cap structure, promoting the cap recognition by eIF4E and selective protein synthesis. Depletion of METTL16 suppresses lung tumorigenesis by downregulating the translation of key oncogenes. Collectively, our study reports a role of METTL16 in modulating translation and provides a therapeutic target for lung cancer treatment.

N⁶-methyladenosine (m⁶A) has emerged as a crucial epitranscriptomic mark which regulates a broad spectrum of physiological processes including stem cell differentiation. m⁶A-binding YTHDF proteins have recently been proposed to mediate differentiation of leukemia cell in a redundant manner. However, whether these proteins play semblable roles in pluripotent stem cell remain largely unknown. Here, we showed the differential functions of YTHDF1 and YTHDF3 in controlling the differentiation of embryonic stem cells (ESCs). Depletion of YTHDF3 in ESCs resulted in loss of pluripotency with accelerated expressions of marker genes involved in formation of three germ layers. Phenotypic and transcriptomic analyses revealed that loss of YTHDF1 led to dramatic impairment of cardiomyocytes (CMs) differentiation, accompanied by downregulated CM-specific genes. While, knockdown of YTHDF3 accelerated differentiation through facilitating the expressions of CM-specific gene. Notably, YTHDF3 appears to modulate cellular differentiation partially through suppression of YTHDF1, supporting the distinguishable but interrelated roles of YTHDF1 and YTHDF3 in cell fate determination.

N⁶-Methyladenosine (m⁶A) is the most abundant chemical modification occurring on eukaryotic mRNAs, and has been reported to be involved in almost all stages of mRNA metabolism. The distribution of m⁶A sites is notably asymmetric along mRNAs, with a strong preference toward the 3′ terminus of the transcript. How m⁶A regional preference is shaped remains incompletely understood. In this study, by performing m⁶A-seq on chromatin-associated RNAs, we found that m⁶A regional preference arises during transcription. Nucleosome occupancy is remarkedly increased in the region downstream of m⁶A sites, suggesting an intricate interplay between m⁶A methylation and nucleosome-mediated transcriptional dynamics. Notably, we found a remarkable slowdown of Pol-II movement around m⁶A sites. In addition, inhibiting Pol-II movement increases nearby m⁶A methylation levels. By analyzing massively parallel assays for m⁶A, we found that RNA secondary structures inhibit m⁶A methylation. Remarkably, the m⁶A sites associated with Pol-II pausing tend to be embedded within RNA secondary structures. These results suggest that Pol-II pausing could affect the accessibility of m⁶A motifs to the methyltransferase complex and subsequent m⁶A methylation by mediating RNA secondary structure. Overall, our study reveals a crucial role of transcriptional dynamics in the formation of m⁶A regional preference.