Regulation of upstream open reading frames in mRNA stability and translation

PROJECT SUMMARY Upstream open reading frames (uORFs) play important roles in gene expression regulation and are associated with numerous human diseases. Translated uORFs serve two major inhibitory functions: one is reducing mRNA stability, presumably through nonsense-mediated mRNA decay (NMD), and the other is repressing the translation of the downstream main ORFs (mORFs), both leading to decreased protein levels. Despite their significance, our understanding of uORF regulation remains limited. To understand the mechanisms of uORF regulation, we will combine high-throughput sequencing, molecular biology, bioinformatics, and machine- learning models to identify and characterize uORF features and regulation using the tractable model Arabidopsis, which enables in vivo and whole-organism level studies. In the next five years, we will address three important questions about uORFs: (1) Does NMD control uORF-containing mRNAs? Through systematic profiling of translated uORFs, we have made surprising discoveries: NMD appears not to regulate most Arabidopsis uORF mRNAs, even though Arabidopsis uORFs repress mORF translation and reduce protein levels like in other eukaryotes. This unexpected finding raises the possibility that plant uORFs either systematically escape NMD, or trigger NMD but are compensated through alternative mechanisms. Addressing this question is important because aberrant mRNAs in various human diseases evade NMD via unknown mechanisms. (2) How do certain mRNAs overcome uORF repression through 'reinitiation' after uORF translation? Reinitiation is poorly understood due to the lack of a robust in vivo system. A key regulator of reinitiation in Arabidopsis is TARGET OF RAPAMYCIN (TOR), a conserved master regulator of translation and growth. In Arabidopsis, activated TOR leads to the phosphorylation of translation initiation factor eIF3h, promoting the reinitiation of several uORF-containing mRNAs crucial for development and metabolism. We will leverage this TOR-mediated eIF3h phosphorylation system to study genome-wide reinitiation in vivo at the whole-organism level and connect it to potential cis- and trans-regulation. (3) What uORF features control its regulation in mRNA decay, translational repression, and reinitiation? We will integrate the genome-scale datasets generated in this study with machine-learning models to quantify uORF characteristics influencing protein production at multiple levels. Collectively, this research will enable us to 1) determine the relationship between uORF translation and NMD, 2) investigate genome-wide reinitiation regulated by TOR-mediated eIF3h phosphorylation, and 3) identify which uORF features impact mRNA stability and translation. This work will provide a comprehensive understanding of the dual regulation of uORFs. Our long-term vision is to develop Arabidopsis as a model system to identify both the divergent and conserved principles governing gene regulation through uORFs and provide insights into potential innovative therapies for human health.