RNA quality control against oxidative damage

Li, Zhongwei

Abstract

The goal of this project is to provide research training to students in the area of RNA damage and quality control, an understudied problem that is potentially related to many human diseases. RNA is central to gene expression by directing and regulating protein synthesis, as well as by constituting the machinery that makes proteins from amino acids. The robustness of gene expression depends on the ability of correcting errors in RNA that may affect RNA function. The present grant application aims at studying RNA damage by reactive oxygen species (ROS), which occurs in everyday life and increases under conditions such as oxidative stress (OS). Although our understanding of RNA oxidation is at its infancy, it has been demonstrated that oxidation renders mRNA and rRNA dysfunctional during translation, causing the synthesis of aberrant proteins. Accumulation of oxidized RNA is presumably detrimental to cells and organisms. Our project is designed to uncover molecular mechanisms that specifically remove oxidized RNA and prevent the deleterious effect of RNA oxidation on cell viability. We hypothesize that a major mechanism for removing oxidized RNA may follow three steps: (1) RNA molecules containing oxidized residues are sequestered in a process that possibly employs factors that recognize oxidized RNA;(2) the sequestered oxidized RNA molecules are efficiently degraded to mononucleotides by RNA degradation activities;and (3) the resulting oxidized mononucleotides are removed or prevented from being reincorporated into RNA during transcription. In this project, we propose to test two specific aims using Escherichia coli, a bacterium for which a large body of information in RNA metabolism is available.
Aim 1 : To test the hypothesis that RNA containing the highly toxic oxidized nucleotide 8-oxo-G is removed from functional sites and is selectively degraded. We will examine the levels of 8-oxo-G in various RNA fractions and define the pathway that 8-oxo-G is eliminated from functional RNA to degradation products. We will analyze the roles of RNA degradation and 8-oxo-G binding activities in specific recognition and removal of 8-oxo-G RNA in the pathway.
Aim 2 : To determine the specific roles of polynucleotide phosphorylase (PNPase) in recognizing and degrading 8-oxo-G containing RNA. We will analyze the structural determinants of the 8-oxo-G binding activity and the RNA degradation activity, and study their independent functions in RNA quality control under OS. This training project will last for 3 years, involving 6 to 8 participants at high school, undergraduate and pre-doctoral levels with 0 to 3 years of prior research experiences. The participants are expected to learn and carry out the experimental research in the laboratory of the principal investigator. The trainees are highly encouraged to publish meaningful results as part of the outcome of the project.

Public Health Relevance

Project Narrative This research training project will provide opportunity for the trainees to test the hypothesis that living organisms invest in specific mechanisms for correcting errors in RNA, the molecules through which our genes work. We will study the way cells handle RNA errors that are caused by the unavoidable oxidative byproducts of our daily life. The outcome of this project will enhance public awareness of the potential human health effect of RNA damage and will shed light on how we may prevent related problems.