UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Characterization of Non-protein Coding Ribonucleic Acids by their Signature Digestion Products and 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 MAHMUD HOSSAIN M.S., Chemistry, Murray State University, KY, 2004 M.S., Biochemistry, University of Dhaka, Bangladesh, 1996 Committee Chair: Patrick A. Limbach, PhD ABSTRACT Transfer RNA (tRNA) and ribosomal RNA (rRNA) are two major non-protein coding ribonucleic acids (ncRNAs) in cell. In addition to their housekeeping roles during protein synthesis, they also participate in other cellular activities. The main objective of this dissertation is to characterize these two classic ncRNAs by their signature digestion products and MALDI mass spectrometry. The separation of biologically active, pure, specific tRNAs is difficult due to the overall similarity in secondary and tertiary structures of different tRNAs. Because prior methods do not facilitate high-resolution separations of the extremely complex mixture represented by a cellular tRNA population, global studies of tRNA characterization are rare. I have found that the enzymatic digestion of an individual tRNA by a ribonuclease will generate digestion products yields a set of unique or signature digestion products that ultimately enable the detection of individual tRNA from a total tRNA pool. The detection is facilitated by MALDI-MS. This facile method enables the individual identification of tRNA isoacceptors without requiring any purification steps. I also developed a new approach including multiple ribonucleases to increase tRNA detection where an RNA mixture is digested separately with three ribonucleases, RNase T1, RNase A, and RNase TA, which generate their own sets of signature digestion products. The digestion conditions of these three ribonucleases with E. coli and B. subtilis were optimized. This signature digestion product-based detection technique has been extended to 16O- and 18O-isotope labeling for RNA quantification. I introduced two equations to overcome the interfering peak-related difficulty and was able to calculate the ion abundance ratios of those 1 Da overlapping peaks. My studies on a mixture of standard tRNAs and total tRNAs of E. coli iii grown in MOPS minimal and EZ rich defined medium found that this approach provided quantitative results from those complex samples at light-to-heavy ratio between 2.5:1 and 1:2.5 with MALDI-MS. Selection of an appropriate product ion in the MALDI spectra is crucial for accurate results. Preliminary work has also been done related to the quantification of large rRNAs of E. coli and to the detection of cytoplasmic and mitochondrial tRNAs in yeast S. cerevisiae. iv v ACKNOWLEDGEMENTS I would like to provide a great deal of thanks for patience, understanding, consideration and encouragement that my mentor Professor Patrick A. Limbach has shown me over the past four and half years. I am really grateful to him. I would also like to thank him for the guidance and endless support throughout this project, and for letting me work and think almost independently. Committee members, Professor Joe Caruso and Professor Albert Bobst, are highly recognized for their constructive suggestions, guidance, and continuous support for my graduate research and dissertation. Very special thanks to Professor William Heineman, Professor Brian Halsall, Professor Apryll Stalcup and Professor Tom Ridgway for all they have done for me. I would also like to thank Department of Chemistry at University of Cincinnati for the financial support and for the valuable teaching experience. Special thanks to Dr. Stephen Macha of Mass Spectrometry Facility and all of the Limbach research group members, from both present and past, for their support, encouragement and help. This is a wonderful research group to work in. Finally, I would like to thank my lovely wife Khodeja Tull Fatema and my Halloween daughter Fariha Fardin for all of their love, endurance and cooperation. All my love also goes to my parents, Md. Amir Hossain and Ms. Fatema Hossain, without them, I could never have accomplished so much, and I am forever grateful for their affection, sacrifice, support and contribution in their only child’s life. vi TABLE OF CONTENTS ABSTRACT…………………………………………………………………………………… iii ACKNOWLEDGEMENTS…………………………………………………………………... vi LIST OF FIGURES……………………………………………………………………………. 4 LIST OF TABLES……………………………………………………………………………… 6 LIST OF ABREVIATIONS…………………………………………………………………… 8 PROLOGUE………………………………………………………………………………….. 10 CHAPTER 1. INTRODUCTION…………………………………………………………… 12 1.1 Central dogma of molecular biology…………………………………………….... 12 1.2 Ribonucleic acids………………………………………………………………….. 12 1.3 Non-protein coding RNAs………………………………………………………… 14 1.4 Major ncRNAs in cells…………………………………………………………….. 15 1.5 Significant cellular roles of major ncRNAs……………………………………….. 19 1.6 Classical methods for isolating the major ncRNAs……………………………….. 23 1.7 Contemporary methods for the characterization of major ncRNAs………………. 26 1.8 MALDI mass spectrometry for biomolecules…………………………………….. 27 1.9 Research goal……………………………………………………………………… 40 CHAPTER 2. SIGNATURE DIGESTION PRODUCT…………………………………… 41 2.1 Introduction………………………………………………………………………... 41 2.2 Experimental………………………………………………………………………. 41 1 2.3 Results……………………………………………………………………………. 44 2.4 Discussion………………………………………………………………………… 57 2.5 Conclusion………………………………………………………………………… 60 CHAPTER 3. MULTIPLE RIBONUCLEASES………………………………………….. 62 3.1 Introduction……………………………………………………………………….. 62 3.2 Experimental………………………………………………………………………. 62 3.3 Results…………………………………………………………………………….. 64 3.4 Discussion………………………………………………………………………… 83 3.5 Conclusion………………………………………………………………………… 90 CHAPTER 4. RELATIVE QUANTIFICATION OF SMALL RNA…………………….. 91 4.1 Introduction……………………………………………………………………….. 91 4.2 Experimental………………………………………………………………………. 92 4.3 Results…………………………………………………………………………….. 94 4.4 Discussion………………………………………………………………………... 111 4.5 Conclusion………………………………………………………………………... 112 CHAPTER 5. EXTENDED APPLICATIONS OF SIGNATURE DIGESTION PRODUCTS…………………………………………………………………………………. 113 5.1 General introduction……………………………………………………………... 113 Part A. Relative quantification of large ribosomal rRNAs of E. coli by their signature digestion products………………………………………………………... 114 2 5.2 Introduction……………………………………………………………………… 114 5.3 Experimental……………………………………………………………………... 114 5.4 Results……………………………………………………………………………. 117 5.5 Discussion………………………………………………………………………... 118 5.6 Conclusion……………………………………………………………………….. 119 Part B. Detection of mitochondrial transfer RNAs import of Saccharomyces cerevisiae by their signature digestion products…………………………………... 119 5.7 Introduction………………………………………………………………………. 119 5.8 Experimental……………………………………………………………………... 119 5.9 Results…………………………………………………………………………… 123 5.10 Discussion……………………………………………………………………… 129 5.11 Conclusion……………………………………………………………………… 131 CHAPTER 6. SCOPE……………………………………………………………………… 132 BIBLIOGRAPHY………………………………………..………………………………… 135 3 LIST OF FIGURES Figure 1.1 Structure of four common ribonucleotides………………………………………… 13 Figure 1.2 Schematic of a typical E. coli transfer RNA……………………………………….. 17 Figure 2.1 Overview of small RNA isolation procedure……………………………………… 43 Figure 2.2 Determination of signature digestion products. Digestion products in bold are unique or signature digestion products, the rest are common…………………………………….…… 46 Figure 2.3 Some selected common and signature digestion products of tRNATyr I found experimentally when digested with ribonuclease T1. (peaks marked with * are common peaks, whereas marked with # are signature peaks)…………………………………………………... 47 Figure 2.4 Ribonuclease T1 cleavage mechanism………………………………………….…. 48 Figure 2.5 MALDI mass spectra obtained from the RNase T1 digestion of mixtures of E. coli tRNAs. (a) tRNAs of Tyr I, Val III, and Glu II. (b) tRNAs of Phe, Val III, and Glu II. (c) tRNAs of Tyr I, Phe, and Glu II. (d) tRNAs of Tyr I, Phe, and Val III……………………………….. 50 Figure 2.6 Heat map representation of the results from Figure 2.5, along with m/z and sequences of signature digestion products. In all analysis, reproducible detection of the signature digestion products of the expected tRNAs is demonstrated……………….……………………………... 51 Figure 2.7 MALDI mass spectra obtained from the RNase T1 digestion of total tRNA mixtures of E. coli from Sigma. (a) m/z 850-1750; (b) m/z 1850-2600………………………………..… 52 Figure 2.8 MALDI mass spectra obtained from the RNase T1 digestion of E. coli tRNAs. (a) m/z 900-2700; (b) 2700-6000…………………………………………………………….… 54 Figure 2.9 Reproducibility study of RNase T1 digest of E. coli total tRNA mixture………… 56 Figure 3.1 tRNA isoacceptors……………………………………………………………….… 67 Figure 3.2 Digestion of four tRNA mixtures with ribonuclease A. (a) tRNAs of Tyr II, Val III, and Glu II. (b) tRNAs of Phe, Val III, and Glu II. (c) tRNAs of Tyr II, Phe, and Glu II. (d) tRNAs of Tyr II, Phe, and Val III ……………………………………………..…………….… 78 Figure 3.3 RNase TA digestion