Investigating the functional roles of RPS3 in RNA and DNA damage

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Abstract Damaging agents, such as UV irradiation and hydrogen peroxide, can compromise functional integrity by damaging intracellular macromolecules. Cells have quality control systems that deal with damaging modifications, and defects in these pathways could lead to chronic diseases such as cancer and neurodegeneration3-6. A considerable amount of work has focused on how cells deal with damaged lipids, proteins, and DNA; however, little is known about how cells deal with damaged RNA. RNA is highly susceptible to damaging agents and such lesions could lead to translation errors and ribosome stalling10-12. Several RNA quality control systems occur at the translational level. No-Go Decay (NGD) targets mRNAs with sequence features that can induce ribosome stalling. NGD is triggered by an endonucleolytic cleavage at the stall site, followed by the dissociation of the ribosomal subunits. The endonuclease responsible for the initial cleavages is currently unknown17-21. Intriguingly, previous in vitro experiments have shown that the small ribosomal subunit S3 (RPS3) cleaves damaged dsDNA substrates22-27. However, the majority of RPS3 is associated with ribosomes, directly interacting with incoming mRNA28-30. Therefore, I propose that RPS3 functions to recognize and cleave damaged RNA molecules as part of a quality control system that targets damaged RNA substrates. To test this, I will first determine whether RPS3 contains endonuclease activity by performing biochemical assays using purified RPS3 and various damaged RNA substrates. I will also assess whether RPS3 contains activity while associated with purified ribosomes. Next, I will determine if RPS3 recognizes and cleaves damaged RNA substrates in vivo. I will attempt to isolate yeast rps3 mutants that are defective at dealing with damaged RNA by generating yeast rps3 variants and employing deep sequencing technologies to monitor fitness levels in the presence or absence of damaging agents. To rule out mutations affecting normal ribosome function, I intend to select mutants that exhibit wild-type growth rates under normal conditions, but increased sensitivity in the presence of damaging agents. Finally, since it was originally proposed that RPS3 functions as a DNA damage repair protein, I will determine if RPS3 plays a role in DNA repair in vivo. I will test whether yeast rps3 mutants genetically interact with other DNA damage repair proteins. In addition, I will perform ChIP assays to determine whether RPS3 physically associates with DNA upon cellular exposure to damaging agents. Overall, this research seeks to elucidate quality control mechanisms that serve to maintain healthy cellular function.