An emerging concept in gene expression regulation is that a diverse set of modified nucleotides is found internally within mRNA, and these modifications markedly influence the fate of mRNAs in cells. The goal of this project is to understand the function and regulation of 8-oxoguanosine (8oxoG), a nucleotide modification that can occur in RNA as a result of guanosine oxidation by reactive oxygen species (ROS). ROS is a major mediator of disease pathology, but the mechanisms by which ROS influences cells is poorly understood. ROS is known to induce the formation of 8oxoG in DNA, but it also induces 8oxoG in RNA. Indeed, Alzheimer's disease brains show marked increases in 8oxoG, especially in certain transcripts. However, the prevalence and function of 8oxoG in the transcriptome is poorly understood. I have developed a technique called 8oxoG- iCLIP, which maps 8oxoG sites throughout the transcriptome. I find remarkably specific enrichment of 8oxoG in certain mRNAs, and often in 5'UTRs. One region that appears to be particularly susceptible to 8oxoG formation is G-quadruplex structures in RNA. Based on my new mapping technology, I am in an ideal position to address key questions related to this novel mechanism of disease-relevant RNA modification: What is the source of 8oxoG-inducing ROS in the cell? What RNAs are oxidized and specifically where in the transcript body? How does 8oxoG regulate mRNA function? To address these questions, the goals of this proposal are to identify the pathways that control the formation of 8oxoG in the transcriptome by using my transcriptome- wide 8oxoG mapping method in conjunction with pharmacologic inhibitors of subcellular ROS sources and agonists of ROS-generating signaling pathway. These experiments will provide a transcriptome-wide analysis of how 8oxoG formation is regulated by specific subcellular sources of ROS and will identify signaling pathways responsible for transcript-specific RNA oxidation. I will also determine the effect of 8oxoG on mRNA translation. I will use circular dichroism spectroscopy to test if regions of mRNAs that accumulate 8oxoG can fold into specific structures, termed G-quadruplexes. Furthermore, I will test if 8oxoG formation in G residues of G-quadruplexes disrupts these structures. Lastly, I will use luciferase reporters with 5'UTRs containing G- quadruplexes to determine if ROS affects translation and if these effects are dependent on the G-quadruplex. Together, these experiments take advantage of my new 8oxoG mapping technology to reveal fundamental new insights into the regulation and function of an RNA modification that may have major roles in mediating the effects of ROS on cells.
In cancer, neurodegenerative diseases, and other conditions, cells produce excess reactive oxygen species that oxidize cellular components. I have developed the first method to map oxidation of nucleotides in RNA, and have found that specific guanosine residues in specific mRNAs are targeted by endogenous reactive oxygen species. In this training grant proposal, I will test important questions related to how mRNAs are targeted for oxidation and how mRNA oxidation affects that translation of mRNAs in cells, thereby providing new insights into disease processes mediated by reactive oxygen species.
