In principle, the key feature of IRES-driven translation is its 5′-end independence, rather than cap-independence. IRES sequences were first reported in viral RNAs and bind to eukaryotic ribosomes when internal to the RNA. Cap-independent translation is an alternative means of translation initiation in eukaryotes that depends on the presence of particular elements that induce internal initiation, such as an internal ribosome entry site (IRES) 7, 19. Eukaryotic translation initiation is normally cap-dependent, because recognition of the cap is required for assembly of the initiation complex 18. In general, the majority of eukaryotic messenger RNAs (mRNAs) possess a 5′ cap structure and a 3′ poly(A) tail 8. To the best of our knowledge, translation of circular RNA in the absence of any particular element for internal ribosome entry has never been reported in eukaryotic systems 7, 11, 12, 13, 14, 15, 16, 17. In 1979, circular RNA was found to bind to prokaryotic but not eukaryotic ribosomes 9, 10. Synthesis of protein via RCA has not previously been reported in living eukaryotic cells therefore, we posed the question of whether this is possible. In the present study, we applied circular RNA to eukaryotic translation systems, which are more complex than those in prokaryotes 8. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism.

The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. However, whether these circular RNAs can be translated into proteins within cells remains unclear.

Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system.

Kozak consensus sequence