Genome-wide identification of circRNAs translated by eIF4A3

Exon junction complexes (EJCs) deposited on spliced mRNAs play multifunctional roles in the regulation of gene expression. Whereas the formation and components of EJCs are well characterized, the underlying molecular mechanisms for gene regulation remain poorly understood. Here we find that a eukaryotic translation initiation factor (eIF) 4A3, a core component of EJC directly interacts with eIF3g, a subunit of eIF3 complex. This interaction serves as a linker between the EJC and eIF3 complex, consequently driving an internal ribosomal recruitment. Accordingly, artificially tethered EJC component or cellular EJC deposited on mRNA after splicing promotes internal initiation of translation in a way that is resistant to cellular stress induced by serum starvation. We also demonstrate that translatable endogenous or reporter circular RNAs depend on EJC for their association with polysomes. Our results uncover an internal initiation driven by EJC, expanding the protein-coding potential of human transcriptome including circular RNAs. HeLa cells were either undepleted or depleted of eIF4A3 by synthetic siRNAs. The cells were subjected to polysome fractionation experiments. Polysome or subpolysome (40S/60S/80S) were pooled according to the absorbance at 254 nm, and the RNAs in each pooled sample were extracted using TRIzol reagent. The RNAs were treated with RNase R to remove linear RNAs, followed by cRNA synthesis using Arraystar Super RNA Labeling Kit. After hybridization and scanning, the acquired signal intensities from polysome or subpolysome in each samples were utilized to calculate the relative polysome-subpolysome distribution of circRNAs upon eIF4A3 downregulation.