Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N⁶-methyladenosine (m⁶A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m⁶A motifs are enriched in circRNAs and a single m⁶A site is sufficient to drive translation initiation. This m⁶A-driven translation requires initiation factor eIF4G2 and m⁶A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m⁶A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.

The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium Noncoding RNAs: Biology and Applications brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.