The prevailing doctrine that messenger RNAs (mRNAs) in higher organisms encode for a single protein has undergone a dramatic revision in recent years. Ribosome and proteomic profiling have revealed a large number of small translated open reading frames (ORF) within previously described ?untranslated regions? (UTRs) and long non-coding RNAs. Indeed, some of the peptides derived from small ORFs have been implicated in various fundamental processes (e.g., development). Translation of small ORFs in the 5?UTR, known as upstream-ORFs (uORFs), has been shown to have a profound regulatory effect on gene regulation, independent of the encoded peptide. Further, translation of uORFs vary under pathologic conditions such as cancer, and mutations affecting uORFs are associated with various human diseases. We and others have also indicated the existence of translated small ORFs in the 3?UTR known as downstream open reading frames (dORFs) in human cells and zebrafish embryos. However, contrary to uORFs, there has been no systematic study of dORF functions, and their relationship to human health and disease remains untested. Further, given their location in the 3?UTR, the molecular mechanism by which dORFs engage the translational machinery remain completely unknown. Our long-term goal is to understand how post-transcriptional regulation (mRNA half-life and translation) shapes gene expression in vertebrates, and its impact on human disease. The central hypothesis of this application is that translation of dORFs regulates gene expression. Our preliminary data strongly indicate that, contrary to uORFs, dORFs strongly enhance translation of the canonical ORF and emerge as an uncharacterized and potent regulatory mechanism across vertebrates. The objectives are to: 1) Identify factors involved in enhancing translation of the main ORF. 2) Dissect the regulatory information driving dORF translation, and 3) Characterize the biological impacts of dORF-mediated regulation. The rationale for the proposed research is to gain a mechanistic understanding of dORF-mediated regulation in order to assess the possible biological importance of dORF dysregulation under stress or disease conditions. This proposal is conceptually innovative as it is based on the exploration of a novel, yet widespread and potent translation regulatory mechanism conserved across vertebrates. Technically, this proposal will combine genomic profiles (RNA-seq, Ribosome profiling); reporter (cytometry); biochemistry tools: RNA pulldowns follow by proteomics, CRISPR-Cas-9 and - 12a (to edit) and our novel Cas13d tool (knock-down in embryos); combining human cell and zebrafish embryos. The outcomes from this project will help understand how dORFs are translated, shape gene expression and generate phenotypes. This novel function of the ribosome adds to the recently emerging regulatory effects of translation on gene expression (e.g. uORF, codon optimality). Understanding dORF biology will provide an entry point and perhaps even a diagnostic tool to associate mutations with human diseases. Identifying the molecular machinery involved in this pathway might provide targets for therapeutic interventions.
Non-traditional gene expression regulatory mechanisms continue to emerge that challenge the dogmatic understanding of how genes are transcribed and translated into functional proteins. The results of the studies detailed herein will be a critical contribution to our understanding of how translation of small coding regions in the 3? untranslated region (3?UTR) enhance translation of the canonical coding region in higher organisms. This is a novel, widespread and potent post-transcriptional regulatory mechanism. Understanding the regulatory functions of small ORFs in the 3?UTR will provide an entry point and perhaps even a diagnostic tool to associate mutations with human diseases.