Poster abstracts
Poster number 10 submitted by Mary Brintha Croos Anburaj
Impact of guanine oxidation on RNA structure and function
Mary Brintha Croos (Chemistry and Biochemistry), Soumitra Basu (Chemistry and Biochemistry)
Abstract:
RNA oxidation is a hallmark of major neurological diseases, including, Alzheimer and Parkinson’s. Guanine (G) has a lower redox potential out of the four bases, which makes it more susceptible to oxidation to form 8-hydroxoguanine (8-OHG). Although barely characterized, conversion of G to 8-OHG in RNAs could impact their structures, thus affecting their function. To locate and study the impact of 8-OHG on RNA structure, we used the structurally well characterized P4-P6 molecule, an independently folding domain of Tetrahymena group I intron. Based on our RNaseT1 structural mapping data, nearly 40% of the Gs in P4-P6 RNA get oxidized under oxidative microenvironment. Among them nearly 17% of the Gs are significantly oxidized (fold change in RNaseT1 protection ≥ 5). Interestingly all of these Gs (G147, G180-G181, G188, G141, G163-G164) are located in the p5abc subdomain of P4-P6 and are known to be involved in tertiary interactions and divalent metal ion coordination. Furthermore, we identified that the oxidation in P4-P6 domain disrupts its secondary structure and promotes unfolding. Since the highly oxidized Gs are present within p5abc, we oxidized p5abc subdomain alone, and proceeded to determine its stability by using UV melting. The UV melting data showed that the oxidation weakens the stability of the p5abc subdomain. Furthermore, we observed by gel mobility shift assay that when oxidized p5abc RNA was incubated with Δ-p5abc RNA in trans a less stable structure was formed, but in comparison an unoxidized p5abc formed a more stable structure in conjunction with the Δ-p5abc in trans. smFRET studies on the same system indicate weakening of the p5abc - Δ-p5abc complex, corroborating the data obtained from bulk studies. Further studies with site specific 8-OHG incorporation and other smFRET analysis of our model are underway and potentially could reveal the structural and functional impacts on RNAs when specific Gs’ are oxidized under oxidative microenvironments.
Keywords: RNA oxidation, Tetrahymena group I intron, 8-OHG