Systematic screen of RNA binding proteins that enhance circular RNA translation

Translatable circular RNAs (circRNAs) have emerged as a promising alternative to conventional mRNA therapy due to their improved stability that results in a prolonged expression window. The translation of circRNAs is mainly driven by internal ribosome entry site (IRES) or IRES-like elements, which is tightly regulated by various trans-acting RNA binding proteins (RBPs). Here we develop a cell-based system to systematically screen RBPs that enhance translation of circRNAs, identifying 68 proteins as putative activators of noncanonical translation. These translation activators are involved in a broad range of biological processes including RNA processing, ribosomal biogenesis, and translation initiation. We further design a machine learning algorithm to extract common sequence features of these activators and predict more potential RBPs with translation activator activities. The newly identified and predicted activators are subsequently validated to promote the IRES-mediated circRNA translation in a context-dependent fashion. Overall, this study provides new insights into IRES-related trans-acting factors and expands the toolbox for engineered RBPs in RNA synthetic biology.