Small translated ORFs in the 3'UTR enhance translation in vertebrates

Project Summary
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.

Grant Number: 5R01GM136849-05
NIH Institute/Center: NIH
Principal Investigator: Ariel Bazzini