Characterizing Modified Nucleosides in RNA by LC/UV/MS

<p>Posttranscriptional nucleoside modification is an important characteristic in all types of ribonucleic acids (RNA) and increases the functional diversity of RNA, exceeding that of the four canonical bases. In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications enhance RNA structure and contribute to the accuracy and efficiency of protein translation. These posttranscriptional modifications are dynamic and function cooperatively within the cell and also exert regulatory influences. The goal of this dissertation is to identify and quantify modified nucleosides in RNA using optimized LC/MS methods of analysis. This method was shown to be sufficient for characterizing changes in the abundance of modified nucleosides as a function of varying cellular conditions and can be used to explore the dynamics of RNA modification. These methods were optimized and characterized, then used to compare the global modification status of tRNA and rRNA under normal and heat shock conditions.</p><p>It was also demonstrated how this LC/UV/MS method can be used to further understand the functional significance and cellular dynamics of RNA modification by applying these methods as an assay for functional characterization of modifying enzymes. A survey and comparison of RNA modifications present in unfractionated tRNAs from bacterial and archaeal organisms is also presented in this dissertation. Studying modification differences among organisms, especially extremophiles that live in harsh conditions, provides clues to how modifications function to enhance structural stability of RNA. RNA modification guides the decoding process in tRNA and organisms of different kingdoms use slightly different modifications for decoding. A new nucleoside, agmatidine, was structurally characterized using LC/UV/MS methods. This nucleoside modification, found only in Archaea, is responsible for specific decoding properties of a rare tRNA and present in very low abundance. </p><p>The methods presented in this dissertation can be used for quantitative comparison of changes in modified nucleoside composition. By establishing the overall method and sample variability for measuring modified nucleosides from tRNA and rRNA, without resorting to stable isotope labeling methods, we are now able to define the statistical significance of changes in modification status when comparing biological systems of varying environmental or physiological cellular conditions, which should enable future studies aimed at measuring relative changes in modification profiles under these experimental conditions.</p>

Name thesis:degree_name

PhD

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Arts and Sciences: Chemistry

Grantor dc:publisher

University of Cincinnati

Year dc:date

2012

Author dc:creator

• Russell, Susan P.

Contributors dc:contributor

• Limbach, Patrick

• Analytical Chemistry
• RNA modification
• posttranscriptional modification
• rRNA
• tRNA
• LCMS

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=ucin1353951985

OAI identifier oai:identifier

oai:etd.ohiolink.edu:ucin1353951985