Targeted Modulation of Eukaryotic Release Factor 1 (OseRF1) by an RNA Aptamer Enhances Drought Adaptation in Rice

Abstract

Drought stress severely restricts rice productivity, necessitating strategies to enhance stress resilience. Here, we identify translation termination as a tunable regulatory node for stress adaptation. Using 25 rounds of SELEX, we isolated RTAR, an RNA aptamer that binds the rice eukaryotic release factor 1 (OseRF1) with nanomolar affinity. Structural and functional analyses revealed that RTAR targets the OseRF1 N-domain through a conserved pocket centered on Arg46, inhibiting stop-codon recognition and promoting translational readthrough in rice cell extracts. Under drought stress, RTAR co-localized with OseRF1 in rice protoplasts and accelerated its depletion, thereby reprogramming termination fidelity. Ribosome profiling (Ribo-seq) identified candidate genes, including OsDR8, with increased ribosome occupancy downstream of annotated stop codons, indicating a transcriptome-wide effect on translation termination. RTAR expression significantly enhanced drought tolerance in rice seedlings, as evidenced by increased survival, chlorophyll retention and reduced membrane injury. These findings were further validated in stable transgenic lines subjected to soil-based drought stress, where RTAR-expressing plants exhibited enhanced whole-plant drought resilience. This phenotype was accompanied by upregulation of drought-responsive genes and was abolished in OseRF1-R46A mutants, demonstrating the functional importance of the Arg46-centered binding pocket. Furthermore, GFP-tagging assays revealed higher-molecular-weight OsDR8 isoforms consistent with C-terminal extension, providing direct evidence of translational readthrough in vivo. Together, these results establish RTAR as a precise and reversible modulator of translation termination and identify OseRF1 as a promising target for engineering drought adaptation in crops.