Liu, X.; Zhang, Q.; Wang, J.; Zhang, Z.; Zhang, L.

Translation efficiency remains a major limitation for RNA therapeutics. Conventional optimization targets the 5'untranslated region (5' UTR), while the 3'UTR is viewed mainly as a stabilizing element. Here, we demonstrate that the 3'UTR can be rationally engineered to actively enhance translation. Using an intracellular directed-evolution platform based on the SINEB2 element, we identified RNA modules P51 and its compact variant P51t3,which markedly increased protein output without affecting mRNA levels. P51t3 consistently boosted expression two- to six-fold across plasmid, in vitro transcribed mRNA, and recombinant AAV systems. Mechanistic studies revealed that P51t3 binds ribosomal protein RPL39, recruiting 60S subunits to the initiation site through the natural closed-loop translation model. By integrating evolutionary selection with 3'UTR design, this work redefines the 3'UTR as an active translational enhancer and provides a broadly applicable regulatory element for next-generation mRNA and gene-delivery therapeutics.