Wu, T.-Y.; Li, Y.-R.; Chang, K.-J.; Urano, D.; Liu, M.-J.

mRNA translation relies on identifying translation initiation sites (TISs) in mRNAs. Alternative TISs are prevalent across plant transcriptomes, but the mechanisms for their recognition are unclear. Using ribosome profiling and machine learning, we developed models for predicting alternative TISs in Arabidopsis thaliana and tomato (Solanum lycopersicum). Distinct feature sets were predictive of AUG and non-AUG TISs in 5\' untranslated regions and coding sequences, including a novel CU-rich sequence that promoted plant TIS activity, a translational enhancer found across dicots and monocots and also in humans and viruses. Our results elucidate the mechanistic and evolutionary basis of TIS recognition, whereby cis-regulatory RNA signatures affect start site selection. The TIS prediction model provides global estimates of TISs to discover neglected protein-coding genes across plant genomes. The prevalence of cis-regulatory signatures across eukaryotes and viruses suggests their broad, critical roles in reprogramming the translational landscape in the plant-virus arms race.