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Identification and Characterization of the a to I Wobble Deaminase from Trypanosoma Brucei

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IDENTIFICATION AND CHARACTERIZATION OF THE A TO I WOBBLE DEAMINASE FROM TRYPANOSOMA BRUCEI DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Frank L. Ragone, M.S. ***** The Ohio State University 2008 Dissertation Committee: Approved by Dr. Juan D. Alfonzo, Advisor Dr. Michael Ibba _________________________________ Dr. Venkat Gopalan Dr. Douglas R. Pfeiffer Advisor The Ohio State Biochemistry Program ABSTRACT This study focuses on the adenosine deaminase that acts on tRNA (ADAT) at the wobble position found in Trypanosoma brucei. This essential enzyme facilitates the deamination of adenosine (A) to inosine (I) resulting in an edited tRNA that has the ability to recognize three unique codons. In our search for inosine containing tRNAs in trypanosomes, we discovered a unique cytidine (C) to uridine (U) editing event in tRNA Thr(AGU) at position 32. This was the first example of two editing events inside the same anticodon stem loop. Furthermore our discovery that C to U editing at position 32 stimulates A to I editing at position 34 indicated an interplay between the two editing sites which supports our previously proposed interdependence model for RNA editing. The observation that C to U editing occurs in cytoplasmic tRNAs indicated that editing in tRNAs is more widespread that previously thought and is not restricted to organellar tRNAs. In our search for the native A to I wobble deaminase found in T. brucei ,we determined that the native enzyme has a molecular mass of 210 kDa while the catalytically active recombinant enzymes elute with protein markers ii corresponding to molecular masses of 70 kDa when subjected to gel filtration chromatography. Similar observations have been made in rabbit reticulocytes where the deaminase eluted with proteins corresponding to molecular masses of 200 kDa. While we cannot conclusively explain this discrepancy, we speculate that the enzyme forms a higher order complex in vivo, possibly involving other protein factors not directly involved in catalysis. Recombinant co-expression of Tb ADAT2 and Tb ADAT3 resulted in an enzyme that is catalytically similar to the native enzyme. The partially purified T. brucei native enzyme exhibited similar kinetic behaviors as the recombinant -1 enzyme has a K m value of 0.78 ± 0.11 uM and a Kcat = 0.118 ± 0.006 min . Once it was established that the recombinant enzyme behaved similarly to the native enzyme, site directed mutagenesis was used to determine the role of various domains of Tb ADAT2 and Tb ADAT3 in the deamination reaction. Our data show that both Tb ADAT2 and Tb ADAT3 play critical roles in catalysis. These findings were in contrast to similar experiments conducted in yeast showing that mutations to conserved residues of ADAT3 had little effect on catalysis. The results of our mutagenesis study also indicate that the C-terminus of Tb ADAT2 contains critical residues that are involved in catalysis and tRNA binding. A 10 amino acid deletion from the C-terminus results in an enzyme with a greatly reduced ability to bind tRNAs as well as catalyze the A to I reaction. Footprint analyses suggests that this portion of the enzyme contacts the D and TΨC arm iii of the tRNA. We speculate that by contacting the tRNA substrates with a string of charged residues at the C-terminus of the enzyme, the deaminase uses this general RNA binding domain to interact with additional substrates, different than the bacterial deaminases which only recognize one substrate, tRNA Arg . In this fashion, the T. brucei ADAT enzyme is able to accommodate the eight different tRNAs that must be deaminated in trypanosomes. Although work has gone into dissecting the bacterial ADATs but little emphasis has been directed toward eukaryotic ADATs. This study provides novel insight into the catalytic and substrate recognition differences between bacterial and eukaryotic ADATs. iv Dedicated to my loving family and friends v ACKNOWLEDGMENTS First I would like to express my gratitude to my advisor Dr. Juan D. Alfonzo for his supervision, guidance, and extreme passion for science. I feel that I have learned a lot from him during my tenure here at The Ohio State University. I would also like to thank him for his assistance in preparing this dissertation and the work that comprises it. I feel privileged to have the opportunity to have Drs. Michael Ibba, Venkat Gopalan, and Douglas Pfeiffer as part of my committee. When they innocently signed my advisory committee form I do not think they could have ever imagined what they were getting themselves into. I have relied on them for much more than scientific expertise, but for personal growth and I can honestly say without them I would not be completing this dissertation. For their constant support I will forever be indebted. I am grateful to members of the Alfonzo lab, past and present. Mary Anne Rubio has been invaluable to my career. From guidance with my first experiment to practicing my presentations, she has always provided outstanding input and kindness. To Jessica M. Wohlgamuth-Benedum and Angela M. Smith I would like to thank you for your outstanding friendship through some of the toughest years vi of my life. It is clear that without you I would not have completed this degree and I am glad we all leaned on each other in our times of need. I would like to thank Jessica Spears, Ashlie Tseng and the rest of the Alfonzo lab for outstanding scientific input as well as good times and laughs. In addition I would like to thank all of my friends in the department for their continued support. It was a real pleasure going through this with them and I could not have done it without them. Finally, I want to thank my family (Judy, Lenny, Joanna, Manny, and Francesco) and friends. My mom and family have been the most supportive people throughout this process and my life. I thank them for shaping me into the person that I am and the one that I aspire to be. vii VITA May 12, 1978………………………Born - Fairview, Ohio 2001………………………………...B.S., Biology + Chemistry Magna Cum Laude, Baldwin-Wallace College Berea, OH 2001-2002….................................Research and Development Chemist I, Oakwood Laboratory, Oakwood Ohio 2002-2008………………………….Graduate Teaching Assistant The Ohio State University, Columbus Ohio PUBLICATIONS Rubio MA*, Ragone FL*, Gaston KW, Ibba M, Alfonzo JD. 2006. C to U editing stimulates A to I editing in the anticodon loop of a cytoplasmic threonyl tRNA in Trypanosoma brucei . Journal of Biological Chemistry 281, 115-120. * Denotes both authors contributed equally to the work Yakovich AJ, Ragone FL, Alfonzo JD, and Werbovetz KA. 2006. Leishmania tarentolae : purification and characterization of tubulin and its suitability for antileishmanial drug screening. Exp. Parasitology 114, 289-296. Rubio MA, Pastar I., Gaston KW, Ragone FL, Janzen CJ, Cross GM, Papavasiliou N, and Alfonzo JD. 2007. An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA. Proc Natl Acad Sci U S A. May 8;104(19):7821-6. viii FIELD OF STUDY Major Field: Biochemistry ix TABLE OF CONTENTS Page Abstract……………………………………………………………………………. …….ii Dedication……………………………………………………………………………..…v Acknowledgments………………………………………………………………... ……vi Vita………………………………………………………………………………….…..viii Field of Study……………………………………………………………..............……ix List of Tables……………………………………………………………………… …..xiii List of Figures…………………………………………………………………….. …..xiv List of Abbreviations……………………………………………………………....….xvii Chapters: 1. Introduction……………………………………………………………………..…….1 1.1 Parasites and their significance to human health………………… …….1 1.2 Uridine insertion/deletion RNA editing in Kinetoplastids………… …….3 1.3 Role of two forms of apolipoprotein B in mammals……………………..4 1.4 C to U editing of mRNA by APOBEC-1…………………………… …….5 1.5 C to U editing of DNA……………………………………………….. …….6 1.6 AID is required for somatic hypermutation and class switch recombination…………………………………………………………….. …….7 1.7 APOBEC-3G is an intracellular guardian against HIV…………… …….8 1.8 A to I editing of mRNA by ADAR1 and ADAR2…………………... ….....9 1.9 A to I editing of viral RNA by ADAR1……………………………… …...10 1.10 C to U editing of mitochondrial tRNAs…………………………… …...11 1.11 Possible evolution of RNA editing enzymes…………………….. …...13 1.12 A to I editing of tRNAs outside of the anticodon………………… …...15 1.13 An enzyme that can edit DNA and RNA in T. brucei …………… …...16 2. C to U editing stimulates A to I editing in the anticodon loop of a cytoplasmic threonyl tRNA in Trypanosoma brucei . 2.1 Abstract……………………………………………………………….. …...23 2.2 Introduction…………………………………………………………… …...24 x 2.3 Materials and methods……………………………………………… …...26 2.3.1 Cell Culture and Preparation of Cell-free Extracts…….. …...26 2.3.2 cDNA Synthesis and Amplification by PCR…………….. …...27 2.3.3 In vitro Editing Assays…………………………………….. …...28 2.3.4 In vitro Aminoacylation and Oxidation Assays…………. …...30 2.4 Results 2.4.1 A to I editing exists in T. brucei …………………………... …...32 2.4.2 C to U editing is found in the same anticodon stem Loop……………………………………………………………….. …...34 2.4.3 Effect of MgSO 4, KCl, NaCl on the deamination reaction……………………………………………………………. …...34 2.4.4 The interdependence model …………………………….. …...35 2.4.5 Effect of A to I editing on aminoacylation efficiency…… …...37 2.5 Discussion……………………………………………………………. …...39 3. Purification and Identification of the A to I editing enzyme. 3.1Introduction……………………………………………………………. …...53 3.2 Materials and methods 3.2.1 Cloning of Trypanosome tRNA ADAT2 and ADAT3 from genomic DNA……………………………………………………...…...54 3.2.2 Preparation of the native enzyme complex from L. tarentolae /T. brucei ………………………………………………. …...54 3.2.3 Immunoprecipitation of T. brucei extract with anti-ADAT2 antibody…………………………………………………………… …...55 3.2.4 One and two dimensional Thin Layer Chromatography (TLC)………………………………………………………………. …...55 3.3 Results 3.3.1 Purification of the A → I tRNA deaminase from L. tarentolae ……………............................................................... …...56 3.3.2 Purification of the A → I tRNA deaminase from T.
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  • A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family

    A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family

    Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 A High-Throughput Approach to Uncover Novel Roles of APOBEC2, a Functional Orphan of the AID/APOBEC Family Linda Molla Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Linda Molla June 2018 © Copyright by Linda Molla 2018 A HIGH-THROUGHPUT APPROACH TO UNCOVER NOVEL ROLES OF APOBEC2, A FUNCTIONAL ORPHAN OF THE AID/APOBEC FAMILY Linda Molla, Ph.D. The Rockefeller University 2018 APOBEC2 is a member of the AID/APOBEC cytidine deaminase family of proteins. Unlike most of AID/APOBEC, however, APOBEC2’s function remains elusive. Previous research has implicated APOBEC2 in diverse organisms and cellular processes such as muscle biology (in Mus musculus), regeneration (in Danio rerio), and development (in Xenopus laevis). APOBEC2 has also been implicated in cancer. However the enzymatic activity, substrate or physiological target(s) of APOBEC2 are unknown. For this thesis, I have combined Next Generation Sequencing (NGS) techniques with state-of-the-art molecular biology to determine the physiological targets of APOBEC2. Using a cell culture muscle differentiation system, and RNA sequencing (RNA-Seq) by polyA capture, I demonstrated that unlike the AID/APOBEC family member APOBEC1, APOBEC2 is not an RNA editor. Using the same system combined with enhanced Reduced Representation Bisulfite Sequencing (eRRBS) analyses I showed that, unlike the AID/APOBEC family member AID, APOBEC2 does not act as a 5-methyl-C deaminase.