Periodic Reporting for period 1 - devUTRs (Uncovering the roles of 5′UTRs in translational control during early zebrafish development)
Reporting period: 2021-09-01 to 2023-08-31
The overall goal of this work was to uncover the 5′ untranslated region (5′ UTR) in vivo rules for mRNA translational regulation during zebrafish embryogenesis. In eukaryotes, translation typically starts with the recruitment of the ribosome to the 5′ cap of the transcript. The ribosome then scans the 5′ UTR of the mRNA until it reaches a suitable start codon to initiate protein synthesis. Thus, the 5′ UTR serves as a point of control for selective mRNA translation and can be rate limiting for protein synthesis. But what is the regulatory information contained within 5′ UTR sequences? Do 5′ UTRs control the dynamics of protein synthesis during vertebrate embryogenesis, and how? To start answering these questions, one needs to systematically determine the regulatory capacity of endogenous 5′ UTR sequences as the embryo develops.
This work provides the first quantitative overview of the regulatory capacity of endogenous 5′ UTR sequences to translation initiation control during zebrafish embryogenesis at large scale. I developed an in vivo 5′ UTR massively parallel reporter assay (MPRA) during zebrafish embryogenesis to capture the regulatory potential of 5′ UTRs. By coupling the 5′ UTR MPRA to polysome profiling, I determined the regulatory capacity of ~18,000 5′ UTR sequences, independently of other mRNA features. I found that the 5′ UTR sequence alone is sufficient to regulate the amplitude and temporal dynamics of translation initiation during early zebrafish embryogenesis. Motif enrichment analysis identified short sequence motifs enriched in 5′ UTRs with distinct translational behaviors. I hypothesize that these short motifs enable the binding of RNA-binding proteins (RBPs) to regulate translation initiation throughout embryogenesis. RBPs and their sequence specificities are highly conserved, and many developmental diseases arise from dysregulated RBP activity. This work lays the foundation to identity RBPs with developmental roles and dissect their molecular mechanisms of action. I anticipate that such findings will generate far-reaching insights into how translation control shapes cell fate acquisition in development and disease.
In the last years, we have entered an era of mRNA therapeutics. The engineering of the 5′ UTR sequence of therapeutic mRNAs offers an opportunity to control their translatability and stability. The utilization of experimental data, such as the one generated in this project, to train deep learning models promises to enable generating de novo sequences with desired translational efficiencies in specific cellular contexts.