Programmable mRNA translation initiation by trans-RNA

The ability to modulate specific gene expression is crucial for interrogating, perturbing, and engineering cellular systems. A wide range of approaches have been developed for gene silencing, but few tools are available to activate individual mRNAs for translation inside cells. 16Guiding ribosomes to specific start codons without altering the original sequence remains a formidable task. Here we design capped trans-RNAs capable of engaging ribosomes to specific initiation sites on individual mRNAs. By positioning the trans-cap near the target start codon, the 19anti-parallel trans-RNA enables the ribosome to select nearby start codons in a simple, specific, robust, and tunable manner. Structural and biochemical data suggest that the capped trans-RNA facilitates ribosome loading and scanning on the target mRNA via a synergistic mechanism. The trans-RNA also acts independently of the original cap, enabling translation of circular RNAs lacking internal ribosome entry sites. We demonstrate that trans-RNAs can be applied in vivo to 24achieve programmable alternative translation of endogenous genes in mouse liver. Finally, we discover the existence of naturally occurring trans-RNAs that differ from antisense RNAs by activating translation of endogenous mRNAs. The discovery and the application of trans-RNAs in programmable mRNA translation holds great potential in advancing fundamental and applied biology. Overall design: Ribo-seq of HEK293 cells transfected with trans-RNAs targeting the main ORF of ATF4; Total and capped small RNA-seq of HEK293 cells; RNA-seq of mouse liver