Abstract
We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. 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. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.
Highlights
The minimum length of the RNA circles used in this study was set as 129 nt, based on the previous finding that a circular RNA of 126 nt with multiple FLAG-coding sequences was well translated in an E. coli cell-free system[1]
It was thought that circular mRNA can be translated only if it contains a sequence for internal ribosome entry[24]
In this study, we demonstrated that circular RNA can be translated without such particular sequences, even in living human cells
Summary
We show for the first time that circular RNA synthesised in vitro can be translated in living human cells in the absence of particular elements for internal initiation. Small circular RNAs of 129, 258 and 387 nucleotides, which contain multiple FLAG-coding sequences, were synthesised (Figs 2A and 3 and Supplementary Table S1). The minimum length of the RNA circles used in this study was set as 129 nt, based on the previous finding that a circular RNA of 126 nt with multiple FLAG-coding sequences was well translated in an E. coli cell-free system[1].
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