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Post-Transcriptional Modification Characterizing and Mapping of Archaea Trnas Using Liquid Chromatography with Tandem Mass Spectrometry

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Post-Transcriptional Modification Characterizing and Mapping of Archaea Trnas Using Liquid Chromatography with Tandem Mass Spectrometry Post-transcriptional Modification Characterizing and Mapping of Archaea tRNAs Using Liquid Chromatography with Tandem Mass Spectrometry A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in the Department of Chemistry of the McMicken College of Arts and Sciences by Ningxi Yu M. Sc. Chemistry, Central China Normal University, 2012 B. Eng. Wuhan Institute of Technology, 2009 October 2018 Committee Chair: Patrick A. Limbach, Ph.D i Abstract This dissertation is focused on exploring the transfer RNA modification profiles in archaea. Transfer RNA (tRNA) plays a key role in decoding the genetic information on messenger RNA (mRNA), and the post-transcriptional modification within tRNAs shape the decoding strategies in different organisms. Bacteria have been extensively studied in term of types and positions of tRNA modifications, and a few eukaryotic organisms have also been investigated. However, our knowledge of tRNA modifications in archaea is still limited. While the modifications in multiple archaeal organisms have been identified, the only sets of tRNA sequences whose modification have been localized to particular tRNAs is from Haloferax volcanii. To improve our understanding of archaeal tRNA modification profiles and decoding strategies, I have used liquid chromatography and tandem mass spectrometry to localize post-transcriptional modifications of selected archaeal organisms. A computational tool has been developed for MS/MS data interpretation and RNA sequence annotation. By using this tool, the modifications were localized to tRNA sequences from five archaeal organisms. Among the five selected organisms, the modifications in the anticodon of Methanocaldococcus jannaschii tRNAs have been fully identified, and the first compilation of modified tRNA sequences of this archaea have been generated. The types of modification in four other archaeal organisms, Methanopyrus kandleri, Methanothermobacter marburgensis, Sulfolobus acidocaldarius, and Thermoproteus tenax, were investigated, and some of these modifications were localized on the tRNA sequences. This study expands our understanding of modification patterns and decoding strategies of archaeal tRNAs, and it also allows us to have a more comprehensive comparison of the tRNA modification landscape among three domains of life. ii iii Acknowledgment This dissertation is dedicated to my family. Your love and support always keep me moving forward. I would like to express my sincere gratitude to my advisor Dr. Patrick A. Limbach for your patience and guidance, his encouragement and help has led me to overcome the challenges and finish my Ph.D. Besides my advisor, I would like to thank the rest of my thesis committee, Dr. Neil Ayres and Dr. Peng Zhang, for their insightful comments and suggestions for my research. I also would like to thank all the members of Limbach group for helping me and bringing me joy, I have been so lucky to work with you. iv Table of Contents Abstract .......................................................................................................................................... ii Acknowledgment .......................................................................................................................... iv Tale of Contents .............................................................................................................................v Chapter 1 Introduction..................................................................................................................1 1.1 Research Goal ........................................................................................................................1 1.2 Post-transcriptional Modifications of tRNA .........................................................................2 1.3 tRNA Modifications and Decoding .......................................................................................4 1.4 Analytical Methods of tRNA Modification Mapping ............................................................9 1.5 tRNA Modifications in Archaea ..........................................................................................13 Chapter 2 RNAModMapper: RNA Modification Mapping Software for Analysis of Liquid Chromatography Tandem Mass Spectrometry Data. ..............................................................19 2.1 Introduction ..........................................................................................................................19 2.2 Experimental ........................................................................................................................20 2.3 Results and Discussion ........................................................................................................23 2.4 Conclusions ..........................................................................................................................51 Chapter 3 Transfer RNA Modification Profiles and Codon Decoding Strategies in Methanocaldococcus jannaschii. .................................................................................................52 3.1 Introduction ..........................................................................................................................52 3.2 Experimental ........................................................................................................................52 3.3 Results and Discussion ........................................................................................................58 3.4 Conclusions ........................................................................................................................130 Chapter 4 The Post-transcriptional Modifications in tRNA from Selected Archaeal Organisms. ..................................................................................................................................131 4.1 Introduction ........................................................................................................................131 4.2 Experimental ......................................................................................................................132 4.3 Results and Discussion ......................................................................................................134 v 4.4 Conclusions ........................................................................................................................159 Chapter 5 Conclusions and Future Work ...............................................................................171 5.1 Conclusions ........................................................................................................................171 5.2 Future Work .......................................................................................................................173 Bibliography ...............................................................................................................................179 Appendices ..................................................................................................................................189 vi vii Chapter 1 Introduction 1.1 Research Goal The goal of this dissertation is to expand our understanding of transfer RNA (tRNA) modification profiles in archaea by using liquid chromatography and tandem mass spectrometry (LC-MS/MS). A new software tool, RNAModMapper, was developed to enable MS/MS spectral data interpretation and modification mapping for RNA (Chapter 2). This tool then was used for mapping the modifications on tRNAs from selected archaeal organisms. I started by analyzing the modification pattern of Methanocaldococcus jannaschii in terms of the types and locations of tRNA modifications, and all the modifications in the anticodon loop were identified, which revealed the decoding coding strategies of this organism. To further explore the tRNA modifications profiles in archaea, four other archaea, Methanopyrus kandleri, Methanothermobacter marburgensis, Sulfolobus acidocaldarius, and Thermoproteus tenax, were studied. This work provides a starting point for understanding tRNA modifications in archaea. Portions of this chapter have previously been published in Analytical Chemistry, 2017, 89 (20), pp 10744-10752. 1 1.2 Post-transcriptional Modifications of tRNA Ribonucleic acid (RNA) is a polymeric biomolecule that plays important roles in many biological processes. The basic composition of RNA is four canonical nucleosides, adenosine (A), guanosine (G), cytidine (C) and uridine (U) as shown in Figure 1.1. After transcription, RNA can be post- transcriptionally modified, and a variety of chemical modifications are enzymatically introduced to the canonical nucleosides within the RNA sequence. There are more than 100 types of naturally- occurring chemical modifications found in RNA, and the modifications vary from simple methylations to complex modifications with multiple function groups1-3. The formation of modifications are catalyzed by enzymes. The simple modifications usually need few enzymes, while the complex modifications require multiple enzymes with many steps. Figure 1.1 The basic structure of RNA and some examples of modifications. Among all types of RNAs, transfer RNA (tRNA) has been found to have the greatest density of modifications. To date, there are more than 100 chemically unique modified nucleosides that have 2 been identified in tRNAs from all three domains of life
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