Large-scale RNA-binding protein tethering screen discovers modulators of circular RNA metabolism

Abstract Circular RNAs (circRNAs) are emerging as important regulators in mammalian cells and promising substrates for RNA therapeutics, yet the trans -acting factors governing their metabolism remain poorly defined. Here we systematically evaluate 730 RNA-binding proteins (RBPs) using a tethered reporter assay and identify 281 RBPs that significantly modulate circRNA activity. Follow-up assays implicate these RBPs across key stages of the circRNA life cycle, including biogenesis, nuclear export, degradation and translation. We identify seven RBPs for tethered translation enhancement of therapeutically applicable circRNA vectors and highlight LARP4B as a potent enhancer of exogenous circRNA translation across multiple internal ribosomal entry sites and cell types. We uncover an evolutionarily conserved role for RBM15 in circRNA biogenesis in fly, mouse and human, further demonstrating a role in circRNA nuclear retention, and that perturbing RBM15 sites with dCasRx or antisense oligonucleotides modulates endogenous circRNA expression. Together, our study provides a global view of circRNA trans -regulation and describes practical entry points for therapeutic development.