Backward translation of circular RNAs

Despite advancements in understanding protein synthesis, numerous eukaryotic proteins might have been overlooked and remain elusive. RNA translation, proceeding in the 5’ end-to-3’ end direction exclusively, has been recognized as the norm for nearly a century. Here, we report a novel translation mode called ‘backward translation’ (BT) of circular RNAs, in which the triplet codons of circular RNAs can be decoded in the 3’ to 5’ direction to synthesize polypeptides/proteins, e.g., 5’-GUA-3’ as the start codon, instead of the conventional 5’-AUG-3’. Backward translation occurs broadly and conservatively as our comprehensive analysis of available transcriptomes, proteomes and translatomes revealed over 990,000 BT polypeptides/proteins across various tissues and species, including over 90,000 human BT proteins with dual experimental evidence from ribosome profile sequencing and mass spectrometry. Intriguingly, these proteins exhibit unique sequences and structural characteristics that distinguish them from conventional proteins. We studied in-depth BT of human hs.circCAPN15 and Drosophila dm.circSCRIB in vitro and in vivo, respectively. Both knock-out/knock-in and overexpression experiments confirmed the endogenous presence of BT proteins and highlighted their biological functions through pronounced phenotypic changes. Moreover, engineered exogenous circRNAs can be successfully backward-translated into proteins in human cells using synthetic Backward Translation Initiator Sequences (BTISs) derived from humans or Drosophila. In conclusion, our findings offer an unprecedented perspective to the genetic code and RNA translation, further unveiling the hidden proteome and elucidating the molecular diversity in living organisms, and potentially opening up a new frontier for exploring protein synthesis with broad practical implications.