Programmable Enhancement of IRES-Mediated Translation via RNA-Guided eIF4G Recruitment.

Internal ribosome entry sites (IRESs) enable cap-independent translation but often exhibit low and variable efficiency, limiting their use in synthetic gene circuits. Here, we present a programmable strategy to enhance IRES-dependent translation by recruiting a truncated eIF4G scaffold via engineered RNA-binding protein fusions. A λN-eIF4G fusion selectively increased EMCV IRES-mediated translation without affecting cap-dependent expression, achieving up to 7.7-fold enhancement. Translation output was tunable over a broad dynamic range through effector dosage and linker design, revealing key design parameters for controlling activity. The system functioned across multiple mammalian cell lines and in tricistronic constructs, enabling coordinated regulation of polycistronic expression. Computational modeling was consistent with preferential interaction between λN and the EMCV IRES. These results establish a modular framework for the programmable control of translation, define design principles for RNA-guided translational control, and expand the synthetic biology toolkit for post-transcriptional gene regulation.