Structured elements drive extensive circular RNA translation

The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs requires internal ribosome entry sites (IRES) if they are to undergo translation without 5' cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify over 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue -specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is down regulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease. Overall design: High-throughput oligo-split-eGFP-circRNA reporter screening assay for obtaining cap-independent translation measurements of 55,000 fully designed sequences in parallel on circRNA using fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq).