The role of heme in the oxidative modification of mRNA

PROJECT ABSTRACT RNA oxidation is a hallmark of neurodegeneration, and may contribute to the altered gene expression seen in neurodegenerative disease. The major oxidative modification is 8-hydroxyguanosine (8-OHG), which impairs translation and other aspects of RNA function. Reactive oxygen species (ROS), generated by mitochondria through cellular respiration, are the drivers of RNA oxidation. Although ROS is generally thought to nonspecifically modify nucleic acids, microarray analysis of 8-OHG-containing mRNA from Alzheimer's patient samples found that only certain mRNAs underwent oxidation. However, it is currently unclear how ROS can selectively modify specific mRNA. To understand the basis for selective RNA oxidation, I performed an 8-OHG mapping study using antibodies that bind 8-OHG. I found that these antibodies bind to regions which appear to correspond to G-quadruplexes. In vitro studies previously showed that G-quadruplexes can bind heme, converting heme into a highly efficient ROS generator. Based on these prior studies, I asked if heme can bind to cellular mRNA. I developed Heme-seq, a method for mapping heme-binding sites in mRNA, and found that heme-binding sequences are found throughout the transcriptome and correspond to G-quadruplex regions. Additionally, I developed 8-OHG-seq, a new single-nucleotide 8-OHG mapping method, which shows 8-OHG enrichment in G-rich regions consistent with G-quadruplexes. My 8-OHG mapping data supports the idea that 8-OHG is a selective modification. Furthermore, my data suggest that heme binds to G-quadruplex sequences in cellular mRNA, leading to localized ROS generation and RNA oxidation. In order to test this hypothesis, the aims of this project are: Aim 1: To determine the role of heme-binding events on 8-OHG formation in mRNA. I will use small molecules that displace heme from G-quadruplexes to determine if heme must directly contact G-quadruplexes in RNA to catalyze 8-OHG formation. I found that cell stress leads to increased heme binding. I will determine if this increased binding occurs at G-quadruplexes and is associated with formation of new 8- OHG sites. These experiments will address whether heme-binding to G-quadruplexes is the mechanism of specificity for RNA oxidation. Aim 2: To determine the role of cell stress on the folding of heme-binding sites. Cell stress markedly increases the folding of G-quadruplexes in cells. I will map the location of endogenous heme-binding sites induced by serum deprivation and determine if these sequences are G-quadruplexes that fold in response to stress. These aims will provide insights into the role of heme-binding to RNA as a mechanism in RNA oxidation. These studies will also allow me to achieve training goals related to acquiring knowledge and skills in RNA biology, molecular mechanisms related to neurodegeneration, and developing bioinformatic skills, which will help me develop the skills to become an independent investigator.