Mechanism of translation initiation on endogenous eukaryotic circular RNAs

Eukaryotic circular RNAs (circRNAs) perform a wide variety of functions. A subset of circRNAs has been demonstrated to undergo translation in vivo. However, few insights exist on the underlying translation mechanisms. Here, we elucidate the basis of translation initiation in two naturally occurring circular RNAs, circMbl and circSfl. We show that in vitro prepared versions of both circRNAs are translated in eukaryotic cell lysates and cells. Initiation depends on the untranslated region (UTR) and can be reconstituted using only the 43S pre-initiation complex, eukaryotic initiation factors 4G, 4A, 4B, and, for circSfl, the RNA helicase DHX29. Functional assays and structural analysis of the initiation complexes suggest that scanning until recognition of the start codon follows initial ribosome landing occurring on AU-rich, accessible UTR patches upstream of the initiation sites. Together, these results provide key insights into how eukaryotic ribosomes engage with and initiate translation on circular endogenous transcripts.