Fast and faithful translation of the cellular messenger RNAs is a defining feature of the ribosome and the translation factors. High-accuracy protein synthesis ensures that errant proteins, which are more prone to misfold, are not made. On aberrant mRNAs, such as truncated ones and those either containing premature or lacking stop codons, a number of ribosome-based quality control processes ensure that these RNAs are not translated and instead are targeted for degradation. While these mRNA-surveillance mechanisms have received much attention during the past decade, curiously a different class of aberrant mRNAs has received little study. In particular, chemically-damaged RNAs pose a significant hurdle to translational fidelity and efficiency. Of particular interest to this proposa are oxidized and alkylated mRNA. Interestingly, our recent data suggests that oxidized mRNAs stall translation and appear to utilize already described mRNA-surveillance processes. The long-term goal of my laboratory is to expand our understanding of the quality control processes that are responsible for recognizing damaged RNAs and their impact on cellular fitness. In this proposal, we argue for an active role for the ribosome in the pathway in which the recognition process initiates in the decoding center of the ribosome. Changes in decoding during tRNA selection are likely to trigger a signaling cascade taking advantage of existent quality control processes to target the mRNA for degradation. The immediate goal is to study the effect of oxidized and alkylated bases on decoding by the ribosome through the use of a well-defined high-resolution in vitro system (aim 1). This goal is built around a range of pre-steady- state kinetics approaches in the context of mutated translation factors. By establishing a kinetic and thermodynamic framework for the ribosomal response to damaged mRNAs, we hope not only to define the molecular mechanism of the unwanted consequence of damaged RNA on cellular metabolism but also define signaling cues that are likely responsible for downstream quality control processes. To this end, we also plan to investigate how these RNAs are subsequently targeted for degradation in bacteria and eukaryotes and whether the ribosome plays an active role in the process (aim 2). We propose to use unbiased approaches that will allow us to globally assess the landscape of RNA damage in the cell and how the ribosome and mRNA- surveillance factors alter this landscape. We also plan to introduce mRNAs damaged at specific sites to overcome some of the difficulties of studying the fate of damaged mRNAs. Finally, whereas the cellular response to DNA damage has been the subject of many studies, the response to RNA damage has received little to no attention. We have preliminary data that suggests that the bacterial adaptive response is triggered by RNA damage.
In aim 3 we propose to biophysically characterize this activity and study its utility to cellular response. Overall, th experiments described in this proposal address key biological problems that are likely to have a broad impact on our understanding of the cellular response to damaging agents.
Nucleic acids are constantly under assault from both endogenous and exogenous agents that can affect their properties and hence their function. Oxidative damage to RNA appears to be correlated with a number of neurodegenerative diseases and is likely to be a contributing factor to the pathogenicity of these diseases. The exact mechanism by which the cell recognizes the aberrant mRNAs remains elusive.
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