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Characterization of oxidation induced damage to Ribosomal RNA through LC-MS

Estevez, Mariana
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659533759720327

Abstract Details

2022, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
Ribosomal RNA (rRNA) is the most abundant RNA of cells. It is part of the ribosome, complex machinery where protein synthesis takes place. rRNA develops important functions as part of translational process, being involved in aminoacyl tRNA selection, catalysis of peptide bond formation and translocation process. Therefore, damage to rRNA can have a detrimental impact on cells’ survival. Oxidative stress can happen due to cell exposure to endogenous and/or exogenous sources, in which Reactive oxygen species (ROS) are generated and lead to oxidative damage to biomolecules. Although many of the harmful effects of oxidative stress on DNA have been documented, RNA has just recently been explored, with few studies conducted on rRNA. This dissertation is focused on characterizing the damaging effects of oxidation on rRNA. The investigations were carried out using E. coli as model system and through employment of liquid chromatography tandem mass spectrometry (LC-MS/MS). These studies characterized the oxidation products generated in rRNA, with differences observed when rRNA is exposed to different stressors, UVA and H2O2 through Fenton Reaction. Altered levels of oxidation products were measured when rRNA is exposed in vitro in the absence of ribosomal proteins (not in the ribosome) or lacking certain rRNA modifications. After improvement of a rRNA modification mapping technique which allowed to increase sequence coverage, gain better confidence in modification placement and in mapping higher number of modifications, the oxidized nucleotides of rRNA were identified, with a few positions being detected more often and considered sites more susceptible to oxidation, labeled “hotspots”, while the majority of oxidation happened randomly and not position-specific. This study also determined that most of the oxidation occurs in the outer shell of ribosomes, far in distance from rRNA functional sites, and potentially affected by proximity with ribosomal proteins. The influence of rRNA modifications absence on cells exposed to oxidative stress was explored, and although the lack of methyltranferases responsible for placing the modifications did not impact cells’ growth and viability under normal conditions, a significant effect was observed for UVA-exposed cells. However, this impact was not necessarily a consequence of higher rRNA oxidative damage neither position-dependent oxidation. These findings characterized rRNA oxidation and determined that types and levels of oxidation products may differ depending on exposure and of rRNA context, and certain rRNA nucleotide positions are more prone to oxidation, although not necessarily located in/near functional sites. This knowledge broadens our understanding of the rRNA oxidative damage and facilitate the elucidation of potential impact on cellular translation.
Balasubrahmanyam Addepalli, Ph.D. (Committee Member)
Ryan White, Ph.D. (Committee Member)
In-Kwon Kim, Ph.D. (Committee Member)
Patrick Limbach, Ph.D. (Committee Member)
144 p.
Chemistry
RNA; oxidation; LC-MS

Recommended Citations

Citations

  • Estevez, M. (2022). Characterization of oxidation induced damage to Ribosomal RNA through LC-MS [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659533759720327

    APA Style (7th edition)

  • Estevez, Mariana. Characterization of oxidation induced damage to Ribosomal RNA through LC-MS. 2022. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659533759720327.

    MLA Style (8th edition)

  • Estevez, Mariana. "Characterization of oxidation induced damage to Ribosomal RNA through LC-MS." Doctoral dissertation, University of Cincinnati, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1659533759720327

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Cincinnati and OhioLINK.