PPM: Illuminating the Dihydrouridine Epitranscriptome

The goal of this project is to deepen our understanding of chemical modifications on ribonucleic acid (RNA) and their role in biological processes. Cellular RNAs are decorated with a large diversity of enzyme-mediated modifications that are post-transcriptionally installed and can modulate RNA function and corresponding gene expression. There exist major gaps in our understanding of RNA modifications, including which RNA molecules are modified and at what sites, which enzymes mediate modification, and how modifications affect RNA function.

This project seeks to address these knowledge gaps for the RNA modification dihydrouridine (D), which is one of the most abundant and evolutionarily conserved modifications in biology, through the application of innovative approaches integrating chemical and biological disciplines. The project will also provide unique training opportunities for students at the chemical biology interface and facilitate education and outreach aimed at increasing engagement with nucleic acid chemistry and biology.

The research is focused on the study of dihydrouridine (D), an abundant modification (mod) on transfer RNA (tRNA). D is installed by dihydrouridine synthases (DUS) and is commonly found in the eponymous tRNA D-loop as well as in the variable loop in eukaryotes. The function of D mods in tRNA biology and cellular/organismal processes is largely unknown.

This is largely due to the lack of efficient tools to characterize D sites and DUS enzymes (writers), as well as related tRNA mods, and the paucity of targeted functional studies integrating biochemical effects with biological phenotypes. The long-term goal of this project is to characterize D mods and writer enzymes and elucidate their biological role through the application of chemical biology methods in combination with biochemical studies, high-throughput transcriptomic analyses, and cell biological assays.

In Aim 1 is to study the biochemistry and structural biology of DUS proteins; Aim 2 is to characterize D mods with chemical sequencing technology and mass spectrometry; and Aim 3 is to study the role of D in tRNA stability and protein translation. Taken together, this research will reveal insights into the regulation of RNA structure and function through enzymatic modification and its role in gene expression regulation.

This project is jointly funded by the Genetic Mechanisms Program of the Division of Molecular and Cellular Biosciences (MCB) in the Directorate for Biological Sciences (BIO) and by the Chemistry of Life Processes Program of the Division of Chemistry (CHE) in the Directorate for Mathematical and Physical Sciences (MPS).

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.