Finding, folding and characterizing the functions of disease-relevant RNA structures

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PROJECT SUMMARY/ABSTRACT Understanding the basic molecular details behind disease is a critical challenge for science. This proposal aims to approach this by focusing on an understudied aspect of disease: the roles of RNA structure. The overall goal of this project is to optimize a methodological pipeline to discover functional RNA elements in the genomes of humans and their pathogens, deduce their structures, and characterize their functions. The end objective is to gain a better understanding of how RNA structure is implicated in disease and to use this information in novel ways to contribute toward improving human health: for example, by advancing RNA-based therapeutic treatments. Over the course of this project we will optimize this pipeline by focusing on the infection of human B cells by Epstein–Barr virus (EBV). EBV is a widespread human pathogen, which infects over 95% of the adult population, and is implicated in a variety of cancers and autoimmune diseases. The mechanisms behind EBV pathogenicity remain elusive and this proposal aims to focus on the roles of RNA and RNA structure in infection and disease. Over the course of establishing lifelong latent infection, EBV both generates its own RNAs and dysregulates various human RNAs. RNA plays a central role in human and pathogen biology, forming the protein-coding and non-protein-coding molecules that are essential to gene expression. RNA structure plays important regulatory roles: e.g. by mediating interactions or altering accessibility of functional motifs. Thus, knowing the structure of RNA provides a great deal of biological knowledge and can be used to deduce functional sequences from the genome. My previous work combined computational and experimental approaches to discover RNA structures encoded within the EBV genome. This included a novel class of viral RNAs, the stable intronic sequence (sis)RNAs, which I discovered. In this current proposal, we follow up on the functional analyses of EBV-encoded RNAs and expand our focus to include human RNAs. This will be accomplished by revisiting both genomes using innovative new approaches that we developed and novel methods that are currently under development. This will be combined with genome-wide biochemical structure analyses and functional assays performed in vitro (in cell-free systems) and within cultured human B cells. The results of this proposal will be an enhanced basic understanding of how RNA and RNA structure is involved in EBV infection and disease. There will be wider implications beyond EBV and its associated pathologies: for example, part of our pipeline involves the analysis of conservation of RNA structure between pathogens (potentially finding conserved mechanisms). As well, many discovered human motifs affected by EBV will likely have functional roles in cancer and immunity; therefore, this proposal will provide general insights into disease. These functional structures provide attractive targets for emerging RNA-targeting therapies; thus, there is great potential for this proposal to advance human health: e.g. in new treatments for cancer and autoimmune disease.