METTL3 facilitates the translation of CircSIK2 during chicken myogenesis in an m6A dependent manner.

Circular RNAs (circRNAs) are generally considered a new class of non-coding RNA (ncRNA) that frequently appears in the eukaryotic transcriptome. In principle, circRNAs may encode proteins, as some of them are generated from exons and possess elements for internal ribosome entry. Circular RNAs have the potential to serve as an unexplored reservoir for the generation of novel proteins, yet the identification of coding-circRNAs is a daunting task. In this study, we developed a specialized strategy for the discovery of coding-circRNA by combining RNA sequencing, ribosome profiling, and mass spectrometry to find a multitude of circRNAs translated in vivo. A total of 40,084 circRNAs were found in chicken myoblasts and myotubes, and 15,332 circRNAs had a predicted open reading frame (ORF). Via ribosome footprints, we discovered that a group of circRNAs (4,069) was associated with translating ribosomes (ribo-circRNAs). Moreover, a total of 3,927 circRNAs with an infinite ORF were discovered, and 860 of them were associated with translating ribosome (ribo-no-stop-codon circRNAs). Mass spectrometry found 5 specific peptides spectra spanning a back-splice junction of circRNAs. circSIK2, one of the ribo-circRNAs, could be methylated by METTL3 and translated into SIK2-176aa, thus promoting the proliferation and differentiation of myoblasts and muscle hypertrophy. Our results suggest that many circRNAs were translating during chicken myogenesis, and METTL3 could enhance the translation of circSIK2. To the best of our knowledge, only two circRNAs translation events have been reported to be mediated by m6A. Our research would represent the third such event, and the first documented instance of a translatable circRNA in poultry.