MFB: Massively parallel identification of translation regulatory sequences in human and viral mRNAs

Messenger RNAs (mRNAs) encode instructions for making proteins, which constitute the fundamental machinery for cellular function. Recent technological advancements have enabled the development of therapeutic mRNAs that can be delivered to humans, notably evident in widely used vaccines for SARS-CoV-2. The success of these therapeutics suggests the potential of new generations of mRNA medicines with applications beyond vaccines, such as anti- cancer therapies and treatment for genetic disorders. Realizing these goals will require the design of mRNAs that optimize protein expression and can be customized for specific tissues and cellular environments. The goal of this project is to leverage a strategy for quantifying the translation functions of synthetic libraries of thousands of RNAs to discover features that modify the timing and quantity of protein production. Insights into these fundamental rules for gene expression will be important building blocks for engineering new classes of mRNA therapeutics to address a broader spectrum of human disease, thus advancing RNA biotechnology. The project will also provide training opportunities for postdoctoral scholars and engage middle and high school students in RNA biology. The quantity of protein synthesized from an mRNA can span more than two orders of magnitude, vary across cell types, and rapidly change in response to cellular signals. The features of mRNAs that determine this range of expression remain largely unknown. Possibilities include sequence motifs recognized by RNA-binding proteins, structured RNA elements and nucleotide modifications that affect the translation process. This project will employ a recently developed massively parallel reporter assay to systematically identify features of thousands of human and viral 5′ UTRs that determine the amount and timing of protein production. Aim 1 will examine the translation functions of diverse classes of nucleotide modifications and identify the molecular mechanisms that recognize them. Aim 2 will quantify protein production from a synthetic library of circular RNAs to identify RNA elements capable of accessing mechanisms for non-canonical translation initiation. Aim 3 will identify features of mRNAs that determine cell-type specific expression and the mechanisms that establish this specificity. These efforts will provide a comprehensive understanding of mRNA- encoded determinants of translation, providing insights into basic determinants of gene expression and guiding the design of new mRNA therapeutics. This project is supported by the Genetic Mechanisms program/Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and by the Division of Chemistry in the Directorate for Mathematical and Physical Sciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.