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

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

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.

Dedicated to my loving family and friends

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

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. brucei ……………………………………………………………….…...58 3.3.3 Over-expression of recombinant proteins……………... …...59 3.3.4 TbADAT2 and TbADAT3 antibody generation and purification………………………………………………………………61 3.3.5 Tandem affinity purification of the A34 → I34 tRNA editing enzyme……...... …...61 3.3.6 Immunoprecipitation assays of TbADAT2………………. …...65 3.4 Discussion……………………………………………………………. …...66

4. The C-terminus of ADAT2 plays a critical role in tRNA binding and editing activity in Trypanosoma brucei

4.1 Abstract……………………………………………………………….. …...88

xi 4.2 Introduction…………………………………………………………… …...89 4.3 Materials and methods 4.3.1 Cloning and expression of the tRNA adenosine 34 deaminase from T. brucei ...... …...92 4.3.2 One and two dimensional TLC in vitro assays……………………………………………………………... …...93 4.3.3 Electrophoretic mobility shift Assay……………………… …...93 4.3.4 Footprint Analysis…………………………………………. …...94 4.4 Results 4.4.1 TbADAT2 and TbADAT3 is a Functional Heterodimer... …...95 4.4.2 Kinetic Characterization of the Recombinant T. brucei A34 Deaminase Enzyme…………………………….. …...96 4.4.3. The Pseudo-active site is important in catalysis………. …...96 4.4.4 The C-terminal Region of TbADAT2 is Essential for Binding tRNA Substrates………………………………………... …...98 4.4.5 TbADAT2 Binds the D-arm and T ΨC Arm of tRNA Val …. ….100 4.5 Discussion……………………………………………………………. ….100

5. Conclusion……………………………………………………………………... ….114

References………………………………………………………………………... ….119

xii

LIST OF TABLES

1.1 Table 1.1 RNA-editing reactions involving base deamination………………..20

4.1 Kinetic properties of the wild type TbADAT2/ADAT3 and mutant enzymes ...... 112

xiii

LIST OF FIGURES

Figure Page

1.1 Alignment of deaminases……………………………………………….. …...21

2.1 Anticodon stem loop sequences of A34 containing tRNAs from

T.brucei ……………………………………………………………………. …...43

2.2 A to I editing of Figure 1. tRNA Thr (AGU) allows decoding of the

C-ending threonine codon……………………………..………………... …...44

2.3. tRNA Thr (AGU) undergoes two different editing events in the

same anticodon loop………………….…………………………………. …...45

2.4. Effects of salt, Mg2+, and temperature on the A34 to I34 deaminase from

T. brucei …………………………………………………………………… …...46

2.5. tRNA Thr (AGU) is efficiently edited in vitro ……………………………...…...47

2.6. Two-dimensional-TLC analysis of an in vitro A to I editing reaction... …...48

2.7. An interdependence model for the double editing of tRNA Thr ……….. …...49

2.8. The presence of U32 stimulates A to I conversion at the wobble base….50

2.9. tRNA Thr (AGU) is efficiently aminoacylated in vitro regardless of its editing

state…………………………………………………………………………...... 51

2.10 The edited and pre-edited substrates are functional substrates for

aminoacylation in vivo ………………………………………………………...52

xiv 3.1 Ammonium sulfate precipitation of L. tarentolae S100…………………....71

3.2 Q Sepharose chromatography fractionation of ammonium sulfate

precipitated proteins from L. tarentolae ………………………………... …...72

3.3 Heparin chromatography fractionation of Q sepharose pool from L.

tarentolae …………………………………………………………………..…...73

3.4 Mono Q chromatography fractionation of Heparin pool from L.

tarentolae ……………………………………………………………………….74

3.5 Partial purification of the A34 → I34 tRNA deaminase from L.

tarentolae ……………………………………………………………………….75

3.6 sulfate precipitation of T. brucei S100…………………………………. …...76

3.7 Q Sepharose chromatography fractionation of ammonium sulfate

precipitated proteins from T. brucei ……………………………………..…...77

3.8 Superdex 200 gel filtration fractionation of T. brucei extract………… …...78

3.9 Expression and A34 → I34 tRNA deaminase assessment of recombinant

Tb ADAT2 and Tb ADAT3………………………………………………... …...79

3.10 Expression of Tb ADAT2 in L. tarentolae and purification of the

recombinant enzyme…………………………………………………………..80

3.11 Anti-ADAT2 purification and detection of endogenous Tb ADAT2 from T.

brucei extracts………………………………………………………………….81

3.12 Expression of Tb ADAT2 with Protein A and streptavidin tags for tandem

affinity purification……………………………………………………………...82

xv 3.13 Tandem affinity purification of the A34 → I34 editing complex from T.

brucei ……………………………………………………………………………83

3.14 Activity assessment of the Tandem affinity purified A34 → I34 tRNA

deaminase……………………………………………………………………...84

3.15 Anti-ADAT2 immunoprecipitation of T. brucei extracts………………...... 86

4.1 The native tRNA A34 to I34 L. tarentolae and T. brucei enzymes have

molecular masses of 218 kDa………………………………………………106

4.2 Multiple sequence alignment of tRNA-specific adenosine deaminases..107

4.3 Recombinant tRNAA34 →I34 L. tarentolae and T. brucei enzymes have

molecular masses of 72 kDa……………………………………………. ….108

4.4 Kinetic and binding analysis of recombinant ADAT2/ADAT3……….. ….109

4.5 Tb ADAT2 Binds the D-arm and T ΨC Arm of tRNA Val ………………... ….111

xvi

LIST OF ABBREVIATIONS

°C Degrees Celsuis

2-D Two-Dimensional

2xYT Two times yeast tryptone media

A Adenosine

A280 Absorbance at 280 nm

A600 Absorbance at 600 nm

ADAT Adenosine deaminase acting on tRNA

ADAR Adenosine deaminase acting on mRNA

AID Activation induced deamination

APE Apurinic/apyrimidinic endonuclease

ApoB Apolioprotein B

APOBEC-1 Apolioprotein B editing enzyme catalytic subunit 1

ASL Anticodon Stem Loop

BLAST Basic Local Alignment Search Tool

C Cytidine

CDAR Cytidine deaminase acting on RNA

CNS Central nervous system

CSR Class switch recombination

xvii C-terminus Carboxy terminal

DNA Deoxyribonucleic Acid

DTT Dithiothreitol

FT Flow through

G Guanosine

G Grams

Gln Glutamine

GluR Glutamate-gated ion channel receptor

GluR-B Glutamate receptor subunit gRNA Guide RNA

H Hours

Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HIV Human immunodeficiency virus

I Inosine

IP6 Inositol hexakisphosphate

IPTG Isopropyl-B-D-thiogalactopyranoside

KCl Potassium Chloride kDa Kilodalton

L Liter

M Molar

M1A 1-methyladenosine

M1I 1-methylinosine

xviii Mg Milligrams

Min Minutes

Ml Milliliter mM Millimolar mM Nanomolar mRNA Messenger RNA

Ng Nanogram

OD Optical Density

PCR Polymerase Chain Reaction

PMSF Phenylmethanesulphonylfluoride

Q Glutamine

R Arginine

RNA Ribonucleic Acid

RT Reverse Transcriptase

SBP Streptavidin Binding Protein

SDS PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SMH Somatic hypermutation

TAP Tandem Affinity Purification

TEV Tobacco Etch Virus

TLC Thin Layer Chromatography

Tris Tris-(hydroxymethyl) aminomethane tRNA Transfer RNA

xix U Uridine

µg Microgram

µM Micromolar

UNG uracil-DNA glycosylase

UV Ultraviolet

Vif Viral infectivity factor

VLDL Very low density lipoprotein

VSG Variant surface glycoprotein

WT Wild Type

CHAPTER 1

INTRODUCTION

1.1 Parasites and their significance to human health

Parasitism is a symbiotic relationship between two organisms of different species where one organism benefits from the interaction while the other is harmed. Parasitic infections claim the lives of millions of infected individuals each year. These parasitic infections are most common in tropical, subtropical, and temperate regions of the world (1;2). Among the most common human parasites are protozoans (3), single-celled eukaryotes that are either free living in marine environments or parasitize a human host. The human parasitic protozoans include members of the genera Trypanosoma, Leishmania, Giardia , and

Plasmodium . In humans, medical conditions associated with these protozoan parasites include sleeping sickness and Chagas disease ( trypanosomes) (4), mucocutaneous, cutaneous, and visceral Leishmaniasis ( Leishmania) , diarrhea

(Giardia) (5), and malaria ( Plasmodium ) in human hosts. Altogether, these diseases involve minor infections in the case of cutaneous leishmaniasis to life threatening conditions in the case of Chagas disease, sleeping sickness and

1 Malaria. With the high incidence of parasitic infections among human populations around the world, there is great need for basic research to understand the cellular processes of these organisms with the goal of designing therapies to combat infection.

The major focus of the Alfonzo laboratory is to investigate unique cellular processes of the protozoan parasite Trypanosoma brucei , the causative agent of sleeping sickness in Africa. T. brucei is a member of the order Kinetoplastida (6).

Members of this order have a single tubular mitochondrion that contains a kinetoplast, a catenated mass of circular mitochondrial deoxyribonucleic acid

(DNA) located near the basal body of the flagellum (7;8). The kinetoplast DNA

(kDNA) is made up of thousands of minicircles and a few dozen maxicircles (9).

In T. brucei, each minicircle and maxicircle contain one origin of replication (10).

Maxicircles encode ribosomal ribonucleic acid (rRNA) and mitochondrial proteins involved in respiration (11;12). Interestingly, there are no tRNA genes in either maxicircle or minicircle DNA; therefore all mitochondrial tRNAs are nucleus- encoded and imported into the mitochondrion (13;14). Studies of the genomically encoded mRNAs of the maxicircle DNA revealed the presence of "cryptogenes" encoding RNAs with premature stop codons that would render them untranslatable (11).

2 1.2 Uridine insertion/deletion RNA editing in kinetoplastids.

The existence of "cryptogenes" led to the discovery of a unique cellular process called RNA editing, first observed in transcripts of the mitochondrial cytochrome oxidase (COX) subunit II gene. The mature messenger RNA

(mRNA) transcript contained four additional nucleotides (uridines, U) not encoded in its DNA template (11). Remarkably, the mRNA could yield a predicted full-length protein only when the reading frame was restored by insertion of uridines (15). This process by which the mRNA was posttranscriptionally changed from what is encoded in the genome was termed RNA editing. It is now well accepted that U insertion/deletion editing of mitochondrial mRNAs is directed by short guide RNAs (gRNAs), which are primarily encoded in the minicircles (few are maxicircle-encoded). While all gRNAs play similar functions in providing the genetic information for editing, the organization of these gRNAs differs between species. gRNAs are mostly complementary to the pre- mRNA (completely complementary to the mature edited mRNA) but contain a few mismatches at each insertion or deletion site (16). An endonuclease then cleaves the mRNA at a mismatch in a gRNA-dependent manner, followed by U insertion (or deletion) and ligation of the mRNA (17-19). While U insertion/deletion editing of kinetoplastid mRNA requires a complementary gRNA, cytidine (C) or U insertions have also been observed in transfer RNAs

(tRNAs) of Physarum polycephalum and Didymium nigripes without the need for gRNAs (20). These insertions in tRNAs restore proper base pairing and may play

3 a role in proper tRNA processing (21). While RNA editing originally referred to mRNA editing, it is now an all-encompassing term to describe a variety of post- transcriptional changes in all types of RNA. Some examples of RNA editing involving base deamination are listed in Table 1.1.

1.3 Role of two forms of apolipoprotein B in mammals.

In addition to U insertion/deletion editing of mRNA, cytidine to uridine editing of mRNA has been described in mammals (22;23). The intestinal apolipoprotein B (apoB) exists in two forms, a full-length protein and a shorter version. Analysis of transcripts for the intestinal apolioprotein B (apoB) protein revealed a tissue-specific post-transcriptional cytidine deamination event that resulted in conversion of a genomically encoded CAA glutamine (Gln) to a UAA stop codon allowing expression of both forms of the protein from a single mRNA

(22;23). Mammals use the two forms of the protein to transport cholesterol and triglycerides in the blood. Full length apoB100 is synthesized in the liver and is the major component of very low density lipoproteins (VLDL) used to transport cholesterol and triglycerides into the circulatory system (24). The truncated version (apoB48) is synthesized in intestinal cells, integrated into chylomicrons

(after tissue-specific editing of the mRNA) and is responsible for dietary lipid absorption (22;25). An important balance must be maintained between these two proteins, as high levels of apoB100-associated VLDL increase the susceptibility of atherosclerosis (26). In contrast, chylomicron-associated apoB48 leads to

4 much lower susceptibility to atherosclerosis. Therefore, RNA editing regulates expression and maintains a proper balance of the long and short versions of the apoB proteins.

1.4 C to U editing of mRNA by APOBEC-1.

The enzyme responsible for C → U editing of apoB mRNA was originally purified from rat intestines (27). This 27 kDa enzyme called APOBEC-1

(Apo lipoprotein B mRNA editing enzyme catalytic subunit 1) is a member of the cytidine deaminase family (28). Since its original purification APOBEC-1 has been cloned and expressed from human and rabbit intestines (29;30). While

APOBEC-1 is the catalytic component of the C → U mRNA deaminase, additional factors are necessary for its activity (27). The minimal and sufficient components of the C → U mRNA deaminase are a dimer of the APOBEC-1 enzyme and ACF (APOBEC-1 complementation factor) (31). The establishment of an in vitro assay proved invaluable in determining the specific substrate requirements of the enzyme (32;33). First, cell extracts and eventually purified recombinant enzymes were incubated with ApoB substrates, which were determined to be tripartite in nature. They consist of a 22 nucleotides motif with a regulatory element, a spacer element, and an eleven nucleotide mooring sequence located four nucleotides downstream of the target C (34). Once proper substrates were established, C → U deamination of mRNA was detected by a sensitive primer extension assay and confirmed by DNA sequencing (32). Once

5 the mechanism for C → U deamination of mRNA was understood, studies were performed with mice to determine levels of APOBEC-1 editing under differing dietary conditions. Mice given diets high in carbohydrates showed increased editing of ApoB whereas in fasting mice, ApoB editing decreased by 50% (35).

Since high levels of VLDL often lead to atherosclerosis, there is appeal to the idea that over-expression of APOBEC-1 may lead to a possible therapy for atherosclerosis. As predicted, Transgenic mice expressing 15-fold more

APOBEC-1 than wild type mice had reduced levels of VLDL, but they also developed tumors due to APOBEC-1 hyperactivity (36).

1.5 C to U editing of DNA.

Genome searches revealed three homologs of APOBEC-1. They were termed APOBEC-2, activation induced deaminase (AID), and APOBEC-3G.

APOBEC-2 is found in cardiac and skeletal muscle but has no ApoB deaminase activity and to date no additional substrates for this deaminase are known (37).

Recent crystal structures of this enzyme give potential insights into the structural composition of other cytidine deaminase family members such as AID. The crystal structure identified regions involved in tetrameric oligomerization in

APOBEC-2. When the homologous residues were mutated in AID, the enzyme showed decreased deaminase activity (38). In contrast to APOBEC-1, both AID

(39) and APOBEC-3G (40) are able to deaminate C → U in DNA substrates.

6 1.6 AID is required for somatic hypermutation and class switch recombination.

In response to foreign pathogens, the immune system of mammals generates in excess of 10 9 different antibody varieties. This diversity of antibodies greatly exceeds the coding capacity of the inherited genome (41). To generate this number of antibodies, “requires at some stage of early embryonic development a genetic process for which there is no available precedent (42).”

The first step in the somatic hypermutation (SHM) mechanism is the deamination of deoxycytidine to deoxyuridine by AID (39). The resulting U:G mismatch is recognized by a uracil-DNA glycosylase (UNG) which excises uracil from the

DNA (43;44). The resulting abasic site stalls the progression of the replicative polymerase and a specialized error-prone polymerase is recruited to fill in the missing base with any of the four dNTPs generating errors that account for some of the diversity found in antibodies (45-47). Deamination by AID is also seen as an initiating factor of class switch recombination (CSR) by a poorly understood mechanism involving UNG. Interestingly, knockout mice where the UNG gene has been eliminated cannot generate antibody diversity by CSR (48). AID must first generate a U:G mismatch in the G-rich tandem repeated switch region (S region) located upstream of the heavy chain constant region. The resulting U:G mismatch is excised by UNG creating an abasic site. The abasic site is nicked by an apurinic/apyrimidinic endonuclease (APE) and mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-

7 strand breaks with complementary DNA ends appropriate for end-joining recombination (49). By these specific deamination events, AID plays critical roles in generating diversity of antibodies by SHM and CSR (41;42).

1.7 APOBEC-3G is an intracellular guardian against HIV.

APOBEC-3G was first identified as an interacting protein with the human immunodeficiency virus 1 (HIV-1)-encoded viral infectivity factor (Vif) (50). The

Vif protein is required for HIV infection by suppression of human innate antiviral activity (51). APOBEC-3G is packaged into the retrovirus virions and is the protein responsible for the human innate antiviral activity (52;53). This innate antiviral activity results because APOBEC-3G hypermutates deoxycytidines to deoxyuridines in the newly synthesized viral minus strand DNA (54). The Vif protein counteracts this innate immunity by binding to APOBEC-3G and tagging it with ubiquitin for degradation by the proteasome (55). Recent studies show that a

Vif/APOBEC-3G interaction can target APOBEC-3G’s proteasomal degradation without ubiquitination and Vif itself may transport APOBEC-3G to the proteasome

(56). These studies have renewed the hope that a long lasting treatment against

HIV can be generated by the inhibition of the Vif protein or a disruption of the

Vif/APOBEC-3G interaction (57). This is significant because current treatments of

HIV are relatively short lived because of the high mutagenic rate of the virus and the subsequent resistance to drug therapies (58).

8 1.8 A to I editing of mRNA by ADAR1 and ADAR2

Perhaps the most puzzling example of RNA editing which occurs in the mRNA for the glutamate-gated ion channel receptor (GluR) and is catalyzed by

Adenosine Deaminase that Act on mRNA (ADAR2). L-Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and plays a vital role in dendrite stimulation across synapses (59). Each GluR is assembled from protein subunits and form ion permeable channels in the post-synaptic membrane (60). The GluR-B, a subunit of the post-synaptic membrane of the glutamate receptor is of great importance in the mediation of excitatory stimulation from the pre-synaptic membrane (61). These heteromeric ion channels are involved in fast synaptic transmission and synaptic plasticity resulting in memory acquisition (62). A physiological adenosine (A) to inosine (I) editing event occurs in excess of 99% of the mRNA transcripts of the GluR-B subunit where a genomically encoded glutamine (Q) codon (CAG) is changed to a CIG codon specifying arginine (R) in the Q/R site by ADAR2 (63-65). This editing event alters the amino acid specificity because inosine is recognized as guanosine by the translational machinery (66). The Q → R change at the Q/R site drastically decreases the permeability of the ion channel to Ca 2+ ions (63).

The physiological role of editing at the Q/R site was established when mutant mice were generated to express an unedited Q at the Q/R site. These mice developed severe epileptic seizures and died within three weeks of birth as a result of decreased Ca 2+ permeability in their GluRs (67). Mice lacking the

9 ADAR2 gene displayed similar behavior indicating that the resulting phenotype might be the result of a lack of A → I editing at the Q/R site (68). Interestingly, in mice where the genomically encoded glutamine CAG codon was changed to an arginine CCG codon in the GluR-B allele, no phenotypic consequences were observed (69). In addition, point mutations where the glutamine CAG codon was changed to an arginine CCG codon in the GluR-B allele in ADAR2-/- mice rescued lethality (68). Taken together these data emphasize the importance of R at the Q/R site of the GluR mRNA generated by ADAR2-driven RNA editing. mRNA editing by ADAR1 or ADAR2 at another site (R/G) on the GluR-B transcript results in an arginine to glycine (G) change in the GluR-B subunit protein (70). In this case the change in amino acid composition modulates the channel gating kinetics where edited channels recover faster from desensitization. The level of editing of the R/G site is directly coordinated to the development of the brain (71). The fact that the Q/R site is edited 99% of the time raises the still unanswered question as to why R is not genomically encoded at the Q/R site. The answer must be similar to the ApoB example where different versions of the protein have different roles in fat metabolism. The unedited GluR-

B must confer and undetermined advantage in the CNS.

1.9 A to I editing of viral RNA by ADAR1

A → I RNA editing has also been observed outside of neuronal substrate in the Hepatitis Delta Virus (HDV). Hepatitis D is caused by the HDV and can

10 propagate only in the presence of another virus, Hepatitis B (72). The HDV encodes one protein in its RNA genome for replication known as the delta antigen (73). A deamination event by ADAR1 in the viral RNA coverts an amber stop codon (UAG) into a tryptophan codon (UIG) (74;75). This editing event extends the open reading frame generating a larger protein that suppresses replication and is involved in viral packaging (76). Without this editing event, the viral lifecycle cannot be completed.

1.10 C to U editing of mitochondrial tRNAs

The discovery of C or U insertions in tRNA was one of the first examples of RNA editing outside of an mRNA substrate. However, soon after, the first example of C → U editing at a single position in the anticodon of a tRNA was reported in marsupial mitochondria, where the second position in the anticodon of glycyl-tRNA (GCC) undergoes editing. Sequence analysis of the entire mitochondrial genome of the marsupial Didelphis virginiana revealed the absence of a GUC containing tRNA. Without this tRNA, aspartic acid (Asp) could not be incorporated into newly synthesized peptides because the GAC codon could not be decoded in the mitochondrion. When the GCC anticodon undergoes

RNA editing to generate a GUC anticodon the resulting tRNA can recognize the

GAC codon and Asp can be incorporated into proteins (77). This editing event is not complete and only about 50% of the GCC containing tRNA is converted to

GUC. This allows for the unedited tRNA to retain its ability to decode the glycine

11 codons. The RNA editing event also affects aminoacylation of the tRNAs. When the tRNA is not edited, it is charged with Gly while the edited version is charged with Asp (78). In this way, RNA editing allows one gene to encode two tRNAs with different specificities.

For many years the genetic code was suggested to result from a “frozen accident” universal in all organisms (79). However, in 1979, an interesting observation was made that in yeast and human mitochondrial genomes UGA codons encoded the aminoacid tryptophan rather than the canonical stop codon used in cytoplasmic translation (80;81). L. tarentolae is no exception and 88% percent of the tryptophan codons are UGA, but since no tRNA genes exist in the

Leishmania mitochondria, UGA codon usage raised the possibility of the existence of a tRNA containing a UCA anticodon encoded in the nuclear genome and later imported into the mitochondria for UGA decoding (82;83). However, only one tryptophanyl tRNA with a CCA anticodon is genetically encoded by L. tarentolae and while it can decode UGG codons as tryptophan both in the cytoplasm and mitochondria, it can not decode the mitochondrial UGA codons

Trp (84). To remedy this problem, in Leishmania, tryptophanyl-tRNA (tRNA CCA)) undergoes C to U editing at the wobble position (position 34) (83). The resulting tRNA then contains a UCA anticodon, which recognizes the UGA codon and permits the incorporation of tryptophan into proteins during translation.

12 1.11 Possible evolution of RNA editing enzymes.

A great deal of effort has been put forth to determine possible evolutionary relationships of RNA editing deaminases. All of the known RNA editing enzymes that deaminate their substrates contain a conserved catalytic core that resembles cytidine deaminases that act on mononucleotides or on RNA (70). This observation led to the suggestion that the ancestor of all RNA editing enzymes most likely was a cytidine deaminase that acted on single nucleotides (CDA).

The discovery of Adenosine Deaminases that Act on tRNA (ADAT) with similar catalytic cores to the CDAs supported this idea. In bacteria the enzyme responsible for A to I deamination at the wobble position of tRNA Arg is composed of homodimeric ADATA proteins (85;86). In yeast and other eukaryotes, a heterodimeric enzyme composed of ADAT2 and ADAT3 proteins facilitates A to I deamination at the wobble position (87). ADAT2 and ADATA both show a great sequence similarity to CDAs (34). Since ADATA is present in bacteria and

ADAT2 is found in eukaryotic organisms, it is believed that ADAT2 and ADATA evolved from a common CDA ancestor while ADAT3 likely arose after the divergence of bacteria and eukaryotes by a gene duplication of the ADAT2 or

ADATA (70). Over time the eukaryotic enzymes acquired the ability to deaminate more substrates compared to ADATA that can act only on tRNA Arg . Genetic drift may have allowed ADAT1 to evolve from ADAT2 by the acquisition of a C- terminal domain (Figure 1.1) changing the site-specificity from position 34 to 37 of tRNA Ala (70). ADAT1 is not found in bacteria but is found in archaea and

13 eukaryotes (88). Further evolution may have resulted in the rise of the ADARs with the acquisition of a double stranded RNA binding domain. This hypothesis is supported by the fact that the catalytic cores of ADAT1 and ADARs are much more similar to each other than to other deaminases (Figure 1.1) (34). In addition a recently published crystal structure of ADAR2 shows a unique inositol hexakisphosphate (IP6) molecule in the catalytic core, which is essential for activity (89). Molecular modeling of ADAT1 revealed that IP6 is likely to be present as well. The CDARs are somewhat harder to place into an evolutionary scheme, by sequence similarity, they fall somewhere in between CDA and

ADATA/ADAT2s. It was previously thought that CDARs evolved from CDA independent of the ADATs (34). Since CDARs have only been found in eukaryotic organisms it is somewhat hard to imagine that they could have given rise to ADATs. One possible explanation is that the ADATs could have given rise to the CDARs after the divergence of bacteria and eukaryotes. Some CDARs such as APOBEC can edit both DNA and mRNA substrates (90). We have shown that the ADAT2/ADAT3 heterodimer from T. brucei can also deaminate tRNA as well as DNA substrates and provides a potential missing link to the evolutionary scheme (91). Our data challenges the idea that CDARs evolved independently of ADATs and supports the idea that ADATA/2/3s may have given rise to CDARs.

14 1.12 A to I editing of tRNAs outside of the anticodon

As described above, A → I tRNA editing at the wobble position is an essential process found in two of the three domains of life. A → I editing events found outside the anticodon of tRNAs occur at only two known positions, 57 and

37 (92). Several species of Archaea deaminate adenosine to inosine at position

57 of their tRNAs (93). The formation of inosine at position 57 is a two step process that first requires adenosine to be methylated to 1-methyladenosine by

(m 1A) MTase (93;94). 1-Methyladenosine is then deaminated to 1-methylinosine

(m 1I) by an unknown deaminase. The only other known example of A → I editing in tRNAs occurs at position 37 of the anticodon loop of tRNAs and is catalyzed by ADAT1 (88). This enzyme catalyzes the first step in the site-specific

(Ala) 1 conversion of A → I at position 37 of tRNA which then is methylated to m I37 .

ADAT1 is restricted to the eukaryotic domain of life and has been recombinantly expressed from yeast, mice, human, and fruit fly (88;88;95;96). It is therefore

1 unclear what the role of m I37 is in vivo since an ADAT1 gene disruption in yeast yields viable cells that are not able to deaminate A37 → I37. Interestingly,

(Ala) 1 tRNA containing m I37 is the target of specific auto-antibodies present in patients with the inflammatory muscle disease myositis (92;97). Perhaps more interesting is the similarity of Sc ADAT1 to the known ADARs. ScADAT1 is 22% identical to the C-terminal portion of ADAR1 and ADAR2 and appears more related to these ADARs than to other tRNA deaminases from a phylogenic standpoint (88;98) In addition, these enzymes share a structural IP6 cofactor in

15 the catalytic core. While ADAT1 deaminates a tRNA substrate, it most likely represents an early ancestor to all ADARs.

1.13 An enzyme that can edit DNA and RNA in T. brucei .

Genomic searches of T. brucei database revealed members of the cytidine deaminase super-family named for their ability to deaminate tRNA,

ADAT2 and ADAT3 respectively. Interestingly, these enzymes can perform C →

U editing of ssDNA (91). While the relevance of cytidine deamination of ssDNA in

T. brucei is unclear at the moment, it is hypothesized that C → U editing of ssDNA may play a role in antigenic variation (91). Similar to the role of AID in

CSR and SHM, C → U deamination by ADAT2/3 may be the root of the diversity of the variant surface glycoprotein (VSG).

In addition to deamination of ssDNA, Tb ADAT2/3 can deaminate A to I in at the wobble position in 8 tRNAs (Arg, Ala, Pro, Ser, Thr, Ile, Val, and Leu) (99).

RNA editing of tRNA at the wobble position is an essential process that expands the decoding capabilities of tRNAs. Of the possible 61 codons that specify amino acids, only a portion of the tRNA genes necessary to decode them are encoded in the T. brucei genome (100). This apparent paradox is resolved by the ability of

I to base pair with C, U, or A as proposed by Crick and the wobble rules over 40 years ago (ref). Consequently a tRNA containing I at the first position of the anticodon (the wobble position) can easily be likened to a skeleton key. Just as a

16 skeleton key has the ability to open many different locks with one key, a tRNA containing inosine at the wobble position has the ability to decode three codons containing a C, U, or A at the last position of the codon, effectively increasing the decoding capacity of the organism. In all known cases I formation is the result of hydrolytic deamination of adenosine by post-transcriptional RNA editing (34).

This essential editing event in bacteria is orchestrated by a homodimeric enzyme composed of two ADATA (85;86;101;102) proteins or an ADAT2/ADAT3 heterodimer in eukarya (87;103). While the subunit composition varies between domains of life, the catalytic cores are highly conserved. Interestingly, while these enzymes catalyze A → I deamination, phylogenetic analysis groups them into a clade with the cytidine deaminase superfamily (34). The catalytic core consists of an HAE domain as well as a PCXXC (where X denotes any amino acid) domain where the histidine and two cysteines residues coordinate a Zn 2+ ion. Three independent crystal structures revealed that a fourth ligand in the tetrahedrally coordinated Zn 2+ ion is a water molecule (86;101;102).The glutamate residue stabilizes the transition-state and actively shuttles a proton from an activated water molecule to the N1 position of the newly formed I molecule (104). ADAT3 contains an HPV domain in place of the HAE domain and was thought to be solely a structural component because it lacked a proton shuttling glutamate. Recently the Staphylococcus aureus ADATa homodimer was crystallized in complex with the anticodon stem loop (ASL) of tRNA arg . This co- crystal was made possible by the use of a nebularine-containing ASL at the

17 wobble position. Nebularine, a non-hydrolyzable adenosine analog, served to stabilize the structure, which has given great insight into enzyme-substrate interactions. This crystal shows the Sa ADATA homodimer binds the ASL with its active site residues (86). In addition, kinetic analysis of Ec ADATA has shown that it can utilize an ASL as a substrate in vitro , implying that a full-length tRNA substrate is not required for deaminase activity with the bacterial enzymes (101).

While a great deal of effort has been put forth to characterize bacterial ADATs, little is known about the eukaryotic enzymes and their interactions with tRNA substrates. A recently report shows that I at the wobble position is essential for cell cycle progression in the G(1)S and G(2)M transitions in fission yeast (103).

This finding reaffirms the delicate and complex importance of these enzymes in biology.

Since the original discovery of RNA editing in kinetoplastid mitochondrion, many other types of RNA editing have been discovered and characterized both inside and outside of the mitochondria in all domains of life. While a great deal of effort has been put forth to characterize eukaryotic enzymes that edit mRNA little is known about the eukaryotic enzymes responsible for tRNA editing. We have shown that the enzymes responsible for A to I editing of the wobble position in T. brucei also edits DNA. The ability of this enzyme to recognize and deaminate two different substrates suggests a possible evolutionary connection between DNA

18 and RNA editing enzymes. My thesis work has focused on identifying the A to I tRNA editing enzyme of trypanosomes as well as determining the bases for tRNA binding and recognition.

Table 1.1 RNA-editing reactions involving base deamination. This figure was reproduced from Trends in Biochemical Sciences (34).

Figure 1.1. Sequence alignment of deaminase domains and phylogenic tree of their relationship. A. Highly conserved residues are framed in black, similar ones in gray. The deaminase motifs I, II, and III are overlined. Zn 2+ -chelating residues are shown in yellow and proton shuttling amino acid is in red. Conserved Phe residues in CDARs boxed in green. The DDBJ/EMBL/GenBank accession numbers are indicated in a column next to the names of the enzymes. B. Phylogenic tree of the deaminase domains encompassing the 36 amino acids that are underlined in A.

Figure 1.1. Sequence alignment of deaminase domains and phylogenic tree of their relationship. This figure was reproduced from Trends in Biochemical Sciences (34).

CHAPTER 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

Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNA Thr (AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo , every inosine-containing tRNA Thr is also C to U edited at position 32. In vitro , C to U editing stimulates conversion of A to I at the wobble base. Although the in vivo and in vitro requirements differ, in both cases, the C to

U change plays a key role in A to I editing. Due to an unusual abundance of A34- containing tRNAs, our results also suggest that the unedited and edited tRNAs are functional, each dedicated to decoding a specific threonine codon. C to U editing of cytoplasmic tRNA expands the editing repertoire in eukaryotic cells, and when coupled to A to I changes, leads to an interrelation between editing sites.

23 2.2 Introduction

The degeneracy of the genetic code is implied in the need for 61 sense codons to specify 20 different amino acids and, with the exception of methionine and tryptophan, each amino acid is encoded by more than one codon (105). This discrepancy between codon and amino acid numbers was first explained by

Crick's wobble hypothesis, which invoked flexibility between the first anticodon and third codon positions during decoding (106). Since the inception of the wobble rules, over 100 post-transcriptional modifications have been described with the largest number affecting the anticodon of tRNA (107;108). As anticodon modifications accrue, new findings lead to a constant reinterpretation of the wobble rules to include novel effects on tRNA function. Although some anticodon modifications play key roles in translational fidelity and efficiency (105;109), anticodon-sequence alterations that permit decoding of multiple codons are part of a growing number of post-transcriptional changes collectively known as tRNA editing. Thus decoding changes imparted by tRNA editing provide a mechanism to effectively accommodate genetic code degeneracy. To date, well characterized anticodon editing events include editing of C34 to lysidine of methionyl tRNAs in bacteria, which permits decoding of AUA codons as isoleucine (110;111), cytidine

(C) to uridine (U) editing in eukarya, which reassigns tRNA Gly and tRNA Trp to new codons in mitochondria (78;83), and adenosine (A) to inosine (I) editing, which expands tRNA decoding capacity and is found in organisms from each of the three domains of life (34;108).

24 Although inosine was first discovered over 40 years ago in tRNA (112), its involvement in codon alterations in eukaryotes was first demonstrated by the discovery of A to I editing in mRNAs (113;114). Inosine in mRNA expands the number of proteins that can be encoded from a single gene and is a significant source of genetic diversity (90). In tRNA, adenosines at the first position of the anticodon (A34, wobble position) are almost universally changed to inosine by hydrolytic deamination of the 6-amino group of the base (104;108). This editing reaction is so efficient that under steady-state conditions, A34-containing tRNAs are difficult to detect, tRNA Thr from Mycoplasma, thus far, being the only naturally occurring exception (115).

C to U editing of tRNA is less prevalent and, until now, restricted to eukaryotic organelles. In marsupial mitochondria, a single C to U editing event at the second position of the anticodon (C35) changes a tRNA such that it recognizes aspartate in place of glycine codons (77;78;116;117). Some evidence supports the requirement for methylation and pseudouridylation reactions prior to editing (117). C to U editing in this system is also required for creating the proper substrate for further modification of the first anticodon position (G34) (117). This

C to U editing also generates structural features important for aminoacyl-tRNA synthetase recognition (78). The only other example of C to U editing of tRNA occurs in the mitochondria of trypanosomatids (83), where the nucleus-encoded tryptophanyl tRNA (tRNA Trp ) is transcribed with a CCA anticodon. A

25 subpopulation of this tRNA is imported into the mitochondrion, where RNA editing

of C 34 creates the U 34 CA anticodon required to translate the UGA tryptophan codons found in mitochondrial mRNAs. Following mitochondrial import, tRNA Trp undergoes an unprecedented number of posttranscriptional modifications, which, as in the marsupial system, may play a role in editing specificity (83;118).

In the current study, we have shown that the cytoplasmic tRNA Thr -(AGU) of

Trypanosoma brucei undergoes two distinct editing events in the anticodon loop, where A34 is changed to inosine and C32 is changed to uridine. We demonstrated that C to U editing at position 32 affects the efficiency of A to I editing of the anticodon. These findings represented the first example of C to U editing of tRNAs outside organelles and demonstrated an interrelation between two different editing sites in a single anticodon loop. Unlike most organisms, we also reported an abundance of unedited tRNAs, which are substrates for aminoacylation in vivo . Together, our findings have raised new and important questions about the prevalence of tRNA editing in eukaryotes and demonstrated a functional role for double editing of tRNAs in trypanosomatids.

2.3 Materials and Methods:

2.3.1 Cell Culture and Preparation of Cell-free Extracts

T. brucei cells were grown in SDM-79 medium supplemented with 10% fetal bovine serum (Fisher) and 10 g/ml hemin (Calbiochem). Exponentially

26 growing cultures (2 x 10 6 cells/ml) were harvested by centrifugation at 4,000 x g and washed with phosphate-buffered saline. The resulting pellets were suspended in buffer containing 50 mM Hepes, pH 8.0, 50 mM KCl, 2.5 mM

EDTA, 1 mM DTT. The suspension was sonicated with a Sonifier 450 sonicator

(Branson) using a microprobe at 50% output for a total of five intervals with 1-min rest between sonication. The resulting lysate was initially spun at 10,000 rpm in a

Beckman Coulter Avanti J-25 centrifuge JA25.50 rotor for 15 min at 4 °C followed by a 30-min centrifugation at 100,000 x g in a Beckman Coulter Optima L-90K ultracentrifuge Type 60TI rotor at 4 °C. To the clarified lysate, glycerol was added to a final concentration of 20% and stored frozen in 4 mg/ml aliquots at –80 °C.

2.3.2 cDNA Synthesis and Amplification by PCR

RNA was isolated from cells (total RNA) and/or nuclear fractions by the guanidinium thiocyanate/phenol/chloroform extraction method (119) and as described previously by us (120). RNA was further treated with RQ1 (RNA qualified) RNase-free DNase I (Promega). Two picomoles of reverse oligonucleotide primer (57R: 5'-AGGCCACTGGGGGGATCGAACCC-3') complementary to the 3'-end of tRNA Thr (AGU) was added to 5 µg of total or nuclear RNA with 10 µmol of all four deoxynucleotide triphosphates and heated at 65 °C for 5 min and then quick-cooled at 4 °C for 1 min followed by the addition of 1 µl of SuperScript TM II reverse transcriptase-(RT) in 1x first strand buffer and incubation at 50 °C, as described (Invitrogen). Following the RT

27 reaction, the cDNA was amplified from 2 µl of the 20-µl RT reaction as a template in a 100-µl (PCR) with 40 pmol of forward (56F: 5'-

GGCCGCTTAGCTCAATGGCAGAG-3') and 40 pmol of reverse (57R) oligonucleotide primers. PCR reactions were performed using Taq DNA polymerase and incubated in a thermal cycler using a program consisting of a 94

°C denaturation step, a 50 °C annealing step for 40 s, and an elongation step of

72 °C repeated for a total of 20 cycles, following manufacturer's instructions

(PerkinElmer Life Sciences). Controls included a mock reaction in which the RT was left out of the reaction and used as a negative control to test for DNA contamination in the RNA samples and a reaction in which total genomic DNA was used as a template serving as a positive control for amplification. RT-PCR products were cloned into pCR2.1-TOPO (Invitrogen). Independent clones were isolated after transformation of DH5 Escherichia coli and sequenced using

Sequenase TM Version 2.0 DNA polymerase (USB), per the manufacturer's instructions. The dideoxynucleotide terminated sequencing reactions were separated in a 6% acrylanmide/7 M urea denaturing gel, and the resulting sequences were used to ascertain the state of editing for each clone.

2.3.3 In vitro Editing Assays

In vitro transcribed tRNAs with internally incorporated [ -32 P]ATP were heated in water at 70 °C for 3 min and allowed to cool to room temperature. After

1 min, reaction buffer was added to a final concentration of 50 mM Tris-HCl (pH

8.0), 25 mM KCl, 2.5 mM MgSO 4, 0.1 mM EDTA (pH 8.0), 2 mM dithiothreitol), and the mixture was allowed to cool for an additional 5 min. The reaction was started by the addition of cell extract and incubated at 27 °C. For the time course experiments, a large reaction (446 µl) containing 40 pmol of RNA (200,000 cpm) in reaction buffer was assembled. As a negative control, an aliquot of 49 µl was transferred into a separate tube and incubated at 27 °C for 480 min. To the remaining 397-µl reaction mix, 8.1 µl of cell extract was added and incubated at

27 °C. Eight individual aliquots of 50 µl were removed after 1, 30, 50, 120, 180,

240, 360, and 480 min, respectively). Each sample was extracted using an equal volume of phenol (previously saturated with 10 mM Tris-HCl, pH 8). The RNA in the aqueous phase was recovered after precipitation with a 0.1 volume equivalent of 3 M sodium acetate (pH 5.2), 2.5 volumes of ethanol and incubated at –20 °C. After centrifugation, the resulting pellet was dissolved in 30 mM ammonium acetate and 10 mM zinc acetate containing 0.4 units nuclease P1 in a

20-µl reaction (MPBiomedicals). The digestion reaction was incubated at 37 °C for at least 12 h. The reaction was dried down in a SpeedVac DNA 110 concentrator system (Savant) for 10 min under high heat. The dried sample was

resuspended in 3 µl of double distilled H 2O, where 1 µl (13.33 pmol) was spotted and dried individually onto a cellulose Thin Layer Chromatography (TLC) sheet

(EMD Chemicals). On the same sheet, 2.5 pmol of a cold mix containing adenosine 5'-monophosphate and inosine 5'-monophosphate was spotted in a separate lane and used as cold markers. The TLC was allowed to develop using

29 liquid chromatography in Solvent C (0.1 M sodium phosphate (pH

6.8):ammonium sulfate:n-propyl alcohol (100:60:2, v/w/v). The TLC plate was allowed to dry and was then exposed to a PhosphorImagerTM screen. The resulting images were visualized and quantified using an Amersham Biosciences

Storm® imaging system with an ImageQuant® program (Amersham

Biosciences). Cold markers were visualized by a hand-held ultraviolet lamp at

260 nm and used to assess the relative migration of the 32 P-labeled individual nucleoside 5'-monophosphates from the radiolabeled samples. Two-dimensional

TLC was used to further confirm the relative positions of nucleoside 5'- monophosphates assignments. The first dimension of the TLC plate was

developed in Solvent A (isobutyric acid:25% ammonium hydroxide:H 2O;

50:1.1:28.9, v/v/v). The TLC plate was removed and allowed to dry before separation in the second dimension by developing in Solvent B (isopropyl alcohol:concentrated HCl: water, 68:18:14, v/v/v) or solvent C (0.1 M sodium phosphate (pH 6.8): ammonium sulfate:n-propyl alcohol, 100:60:2, v/w/v).

Nucleotide assignments were made using published maps (121).

2.2.4 In Vitro Aminoacylation and Oxidation Assays

To corroborate the editing state of aminoacylated species, total aminoacyl- tRNAs were extracted under acidic conditions (using phenol equilibrated with 0.3

M sodium acetate, pH 4.5, and 10 mM EDTA), ethanol-precipitated, and

30 resuspended in 10 mM sodium acetate, pH 4.5, and 1 mM EDTA. The RNA was then split into two fractions. One fraction was deacylated by incubation at 37 °C for 1 h in a basic buffer (10 mM Tris, pH 9.0) followed by oxidation of the 3'-ribose

by treatment with 40 mM NaIPO 4 in ice for 90 min. The second fraction was

directly oxidated by NaIPO 4 followed by deacylation as above. Both fractions were individually polyadenylated by incubation of the RNA at 37 °C for 45 min in

buffer containing 20 mM Tris, pH 7.0, 50 mM KCl, 0.7 mM MgCl 2, 0.2 mM EDTA,

1 mM DTT, 0.1 mg/ml bovine serum albumin, 10% glycerol, 500 µM ATP and

1,700 units of yeast poly-A polymerase in 100 µl of reaction buffer. The reaction was then supplemented with 30 µl of 5x E. coli poly-A buffer (200 mM Tris, 7.0, 1

M NaCl, and 25 mM MgCl 2), 15 µl of 5 mM ATP, 1 µlof 0.1 M DTT, 3.5 µl of Mn

Cl 2, and 3 units of E. coli poly-A polymerase and incubated further for 45 min at

37 °C. The reactions were phenol-extracted and ethanol-precipitated. Both reactions were then used in RT-PCR reactions. First, a 3'-primer specific for the poly-A tail was used to RT-PCR the poly-adenylated RNA followed by PCR with the RT primer and a 5'-specific primer specific for tRNA Thr (AGU) in a 100-µl PCR reaction as above. One µl of this reaction was used as a template for a second

PCR reaction in which both primers were specific for tRNA Thr -(AGU). The resulting product was purified, cloned into pCR2.1-TOPO (Invitrogen), and transformed into E. coli , and individual clones were sequenced to establish editing levels.

31 For in vitro aminoacylation, all assays were performed at 37 °C as follows.

A 35-µl pre-reaction mixture was first prepared containing 100 mM Hepes (pH

7.5), 25 mM KCl, 10 mM MgCl 2, 10 mM ATP, 5 mM DTT, 15 µM in vitro transcribed tRNA Thr variants (see "Results" for details), and 28 µM L-[3-

3H]threonine. The reaction was started by the addition of 10 µl (1 µg/µl total protein) of T. brucei extract. Eight-µl aliquots were removed periodically and spotted onto 3MM filter disks presoaked in 5% trichloroacetic acid (w/v), washed three times in 5% trichloroacetic acid (w/v), rinsed in ethanol, and dried, and the remaining radioactivity was quantified by scintillation counting.

2.4 Results

2.3.1 A to I editing exists in T. brucei

In the search for examples of interdependence between different editing sites, we have focused on the formation of inosine at the wobble base (position

34) of tRNAs in T. brucei . In these organisms, eight different tRNA species contain an encoded A at the first position of the anticodon (A34). These tRNAs are proposed to undergo A to I editing to allow the decoding of the C-ending codons for the amino acids Ile, Ala, Leu, Pro, Val, Ser, Arg, and Thr (Figure 2.1).

We decided to determine the A to I editing levels of threonyl tRNA. The genome of T. brucei encodes three different tRNA Thr genes with anticodons UGU, CGU, and AGU responsible for decoding ACA, ACG, and ACU codons, respectively. As in most organisms, trypanosomatids use a fourth threonine codon (ACC), which

32 presumably cannot be decoded by tRNA Thr (AGU), because A34 cannot efficiently wobble with C at the third codon position (Fig. 2.2 A). To permit wobbling, this tRNA must undergo an A to I editing at position 34, where inosine (a guanosine analog) can then pair with the third position C (Fig. 2.2). To determine the levels of A to I editing in vivo , we designed oligonucleotide primers specific for tRNA Thr (AGU) (Fig. 2.2B), where a 3'-specific oligomer was used to reverse- transcribe tRNA Thr from total T. brucei RNA. The resulting cDNA was then used as a template for PCR amplification with the same 3'-primer and a 5'-specific oligomer. A specific amplification product was obtained with this set of primers when the reaction was performed in the presence of reverse transcriptase (Fig.

2.3A) but was absent in a mock control in which the enzyme was omitted from the reaction. A product of identical size was obtained when both primers were used to amplify tRNA Thr from total genomic DNA used as a positive control for amplification (Fig. 2.3A). Both the cDNA-derived and the genomic DNA-derived products were then cloned into a plasmid vector and transformed into E. coli , and

30 independent clones were sequenced to assess the levels of A to I editing. As expected, we found that A34 is posttranscriptionally changed to I34 (G34 in the

DNA sequence), where 18 out of 30 clones (60%) contained a G at position 34

(corresponding to inosine in the RNA sequence) (Fig. 2.3, B and C).

33 2.3.2 C to U and A to I editing are found in the same anticodon stem loop

These sequences also showed a second editing event at position 32 of the same tRNA. Position 32 is a genomically encoded cytidine, which as shown here is posttranscriptionally changed to uridine (Fig. 2.3B). In fact, under the conditions described, all of the inosine-containing tRNAs also have the C to U change (Fig. 2.3C). These results demonstrate the first example of two different editing events in a single tRNA in any organism. A similar type of editing, but at much lower levels, was also observed with L. tarentolae , a close relative of T. brucei .

The findings above raised questions as to a possible connection between the two processes. First, we tested for A to I editing in vitro . A 32 P-labeled tRNA Thr (AGU) was generated by in vitro transcription whereby every adenosine is radioactively labeled. This substrate was then incubated for various times with total cell-free extracts from T. brucei .

2.3.3 Effect of MgSO 4, KCl, NaCl on the deamination reaction

To optimize the in vitro reaction conditions, components of the reaction mix were titrated to determine which concentrations yielded optimal A34 → I34 deaminase activity. Interestingly, both KCl and NaCl inhibited the A34 → I34 deaminase (figure 2.4A). A concentration of 0.5 mM MgSO 4/MgCl 2 also was necessary to achieve optimal A34 → I34 deaminase activity. Concentrations of

34 MgSO 4/MgCl 2 higher or lower than 0.5 mM resulted in decreased levels of inosine formation (Figure 2.4B). The ability of the enzyme to deaminate A34 →

I34 was also decreased when the enzyme was stored at 23 °C while storage at 4

°C had minimal effects compared to the enzyme stored at -20 ° C (Figure 2.4B).

For all remaining experiments, 0.5 mM MgSO4 and 0 mM KCl/NaCl were used in the deaminase reaction mixture. Following this incubation, the labeled tRNA was gel-purified followed by digestion with nuclease P1. The nucleotide mixture generated by the nuclease treatment was then separated by TLC as described previously (78). Unlabeled adenosine and inosine were used as cold markers during the TLC separation. These markers, when visualized by UV shadowing, serve to corroborate the position of labeled adenosine and inosine generated during the assay. We found that >50% of adenosine 34 was efficiently converted to inosine by the T. brucei extract under the assay conditions described (Figs.

2.5A and 2.8C, and data not shown). No detectable inosine was observed in a control reaction using a substrate in which A34 was changed to G34, indicating that the observed A to I conversion is specific for position 34 (Fig. 2.5B). A two- dimensional-TLC was also performed to confirm the identity of the reaction products (Fig. 2.6).

2.3.4 The interdependence model

We recently proposed an interdependence model to explain the connection between editing and modification of tRNA Trp in trypanosomatid

35 mitochondria. We have now expanded this model to include cytoplasmic tRNAs in these organisms. We propose that double editing of tRNA Thr occurs in a sequential manner, where editing at one position affects subsequent editing at a second position (Fig. 2.7). To test a possible connection between the two sites, we created in vitro transcribed tRNA substrates representing the unedited tRNA and a possible intermediate in the editing reaction (Fig. 2.7, I and II) so that every adenosine in the various tRNAs is radioactively labeled. Upon incubation of the different substrates with cell-free extracts, we found that the C32-containing substrate could support editing; however, a similar substrate in which C32 was replaced by U32 (Fig. 2.7, II) supported editing with reproducibly higher efficiency and significantly higher initial rate (compare Fig. 2.5A and Fig. 2.8, A and C). We also found that tRNA substrates, in which C32 was replaced by A or G, showed no stimulation (data not shown). Taken together, the in vivo observation that every inosine-containing tRNA Thr is also edited at position 32 and the observed in vitro stimulation of inosine formation led us to conclude that editing at one site affects editing at a second site and that indeed, the two editing events are interrelated.

In our model, editing at position 32 occurs first, and it promotes efficient A to I editing at position 34 of the anticodon. If C to U editing at position 32 occurs first, this may impart subtle changes in the loop structure, providing the proper substrate for further editing at position 34 (Fig. 2.7, I). In this scheme, C to U

36 editing may also affect tRNA aminoacylation in addition to modulating the ability of the tRNA to undergo further A to I editing. Alternatively, C to U editing may affect translational efficiency by affecting A to I formation, thus regulating wobbling.

2.3.5 Effect of A to I editing on aminoacylation efficiency

Recent evidence supports a role for I34 as a key determinant for synthetase recognition of tRNA Ile in yeast (122). To test the possibility that A to I editing affects charging of tRNA Thr , substrates were generated corresponding to the two partially edited intermediates (Fig. 2.7, I and II) and incubated with partially purified synthetase fractions from T. brucei in the presence of 3H-labeled threonine. No significant difference in aminoacylation efficiency was observed when the in vitro transcripts were compared with native tRNA (99). However, in vitro , the presence of C32, in the A34-containing tRNA (the unedited tRNA), supported a reproducible 2-fold difference in aminoacylation when compared with a similar substrate with a U at position 32. Interestingly, similar experiments performed with either substrate but with a G at position 34 supported similar charging efficiencies as the unedited tRNA (Fig. 2.9). The observed in vitro aminoacylation efficiency rules out the possibility that the differences in editing levels in vitro between the various substrates could be due to problems in the global folding of the in vitro transcribed tRNA substrates when compared with native substrates.

To further assess the editing state of the aminoacylated tRNAs in vivo a coupled oxidation/polyadenylation assay was employed(99). In this assay, RNA fractions were isolated under acidic conditions and then subjected to a combination of in vitro oxidation, polyadenylation, and RT-PCR (see "Materials and Methods"). In these reactions, oxidation by sodium periodate lead to formation of a dialdehyde at the 3'-end of uncharged tRNAs, whereas the 3'-end of aminoacylated tRNAs is protected from oxidation by the covalently attached amino acid. The oxidized tRNA is not a substrate for polyadenylation, whereas following deacylation, only the charged tRNA will have an intact 3'-end and will thus serve as a substrate for poly-A polymerase (Fig. 2.10A). Under these conditions, we observed an RT-PCR product when total RNA was oxidized as described, whereas no product was detected in a similar reaction, where the total

RNA was deacylated prior to oxidation to de-protect every tRNA present in the mixture (Fig. 2.10B). The product from the reaction above was then purified, cloned, and sequenced. We found that, as in the in vitro situation, both the edited and the unedited tRNA were substrates for aminoacylation, where the majority of the charged species (23 out of 30 clones) corresponded to that of the double- edited tRNA (Fig. 2.10C).

38 2.4 Discussion

We previously proposed an interdependence model for editing and modification in tRNA. This model suggests that editing and modifications at multiple sites act in concert to help achieve the degree of substrate specificity that different systems demand. To further expand this model, we have focused on the process of inosine formation in the tRNAs of trypanosomatids. Here we have described the first example of two different editing events in a single tRNA anticodon loop, whereby positions 32 and 34 of tRNA Thr undergo C to U and A to

I editing, respectively. The finding that every inosine-containing tRNA also undergoes C to U editing at position 32 (5' of the wobble position) raised important questions as to what role the two editing events play in the function of this tRNA. In vivo , every I34-containing tRNA Thr (AGU) also has the C to U change at position 32. By establishing an A to I editing assay, we have demonstrated that

C to U stimulates A to I editing in vitro , indicating interdependence between the two editing sites. The fact that the C32-containing in vitro transcribed tRNA

(devoid of naturally occurring posttranscriptional modifications) can still be edited in vitro raises the possibility that in addition to C to U editing, other factors in vivo

(e.g. posttranscriptional modifications) make the requirements for double editing stricter. The observed interdependence also suggested that these cells might be able to regulate A to I editing of tRNA Thr through changes in C to U editing activity.

39 A to I editing is conserved in many organisms and occurs by hydrolytic deamination of adenosine (92). In yeast, the enzyme involved has two subunits, resembling cytidine deaminases, that upon association specifically deaminates

A34 to generate I34 and requires an intact tRNA structure for activity (87;92). E. coli contains a homolog of the smaller, but not the larger, subunit of the yeast enzyme (85). The E. coli enzyme catalyzes the same A to I editing but, unlike the yeast enzyme, deaminates much smaller substrates, including molecules that are essentially short versions of the anticodon stem-loop (85;87;123). The E. coli enzyme, however, is very specific in that it is able to deaminate the cognate bacterial tRNA Arg but is unable to edit any of the eukaryotic A34-containing tRNAs

(85;92). Although the enzyme that performs A to I editing in trypanosomatids has not been identified, it is likely that this enzyme also utilizes a similar mechanism to that described for yeast and E. coli . The mechanism of C to U editing at position 32 is less clear. To date, a C to U tRNA editing enzyme has not been identified in any organism. However, C to U editing via a deamination mechanism plays a key role in the processing of the apoB mRNA in mammalian cells

(27;32;124). Given this precedent, it is possible that the tRNA C to U editing enzyme also utilizes a deamination mechanism. Furthermore, in the case of ribose methylation, one enzyme, TRM7, is responsible for the methylation of both positions 32 and 34 in yeast tRNAs (125). A similar situation may have arisen in

40 the double editing of tRNA Thr in trypanosomatids, where a single tRNA could be edited twice in the anticodon loop by a single deaminase with multisite and multinucleotide specificity.

Although the analysis of tRNA Thr (AGU) in T. brucei confirmed the presence of the two editing events, it also revealed that under steady-state conditions, one could detect A34-containing tRNAs. This is unlike other organisms, where the A to I reaction occurs so efficiently in vivo that the levels of the A34 intermediate are difficult to detect. The fact that both the in vivo and the in vitro data (Figs. 2.9 and

2.10) demonstrate the ability of the A34-containing tRNA to support amino acylation also argues for it to be functional in cytoplasmic translation.

The wobble rules establish that inosine at position 34 may decode codons ending in A, C, or U. Inosine in tRNA Thr may thus be sufficient to decode both the

ACU and the ACC codons by wobbling. Why then do these cells keep a lower but significant number of A34-containing tRNAs? We suggest that in the trypanosomatid system, the I34-containing tRNA cannot readily wobble with a U- ending codon. Therefore both tRNAs are dedicated to the decoding of one specific codon, where the ACC codon is decoded by the double-edited and ACU codon by the unedited tRNA, respectively. In addition, recent evidence supports the view that posttranscriptional modifications play an essential role in achieving tRNA functional uniformity helping offset differences among various aminoacyl-

41 tRNAs regarding their binding to the ribosome (126). We also envisage a situation in which C to U32 editing is not only required for inosine formation in vivo but also enhances translational efficiency by providing the necessary changes for structural tRNA uniformity during translation. However, an in vitro translation system is not currently available for trypanosomatids, and answering these important questions will thus await further experimentation.

42 Figure 2.1. Anticodon stem loop sequences of A34 containing tRNAs from T. brucei . A to I editing of these anticodons is essential to decode the respective C-ending codons for their cognate amino acid. The anticodon sequence is boxed.

Figure 2.2. A to I editing of tRNA Thr (AGU) allows decoding of the C-ending threonine codon. A, the four threonine codons used in trypanosomatid translation and their respective tRNAs. A possible decoding of the GCA codon by UGU wobbling is shown in brackets . The arrows indicate the sequence polarity. Isoaccepting tRNAs, which may decode the ACU, ACA, and ACG codons, are genomically encoded. No tRNA that may decode the remaining codon (ACC) is encoded in the genome and must be formed by editing. B, tRNA Thr (AGU) is proposed to undergo A to I editing, where the wobble position inosine can then decode the remaining threonine codon by wobbling. Arrows indicate the position of the primers (56F and 57R) used in the RT and PCR reactions. The short arrow denotes the edited position.

Figure 2.3. tRNA Thr (AGU) undergoes two different editing events in the same anticodon loop. A, RT-PCR analysis of tRNA Thr (AGU) and PCR analysis of genomic DNA. RT + refers to reverse transcription reactions using a tRNA Thr (AGU)-specific primer and total T. brucei RNA. RT – is a negative control in which the reverse transcriptase was left out of the reaction. gen DNA refers to a PCR reaction (positive control) using the same oligonucleotides above, and marker refers to a 50-bp DNA size marker. The 72-bp band is of the size expected for a product from PCR or RT-PCR reactions using the primers specific for tRNA Thr (AGU) as per Fig. 1. B, representative sequencing gels from independent isolates generated by cloning the products from A into a plasmid. The arrows denote the two-nucleotide changes observed in the cDNA sequences derived from total tRNA but absent from amplified genomic DNA. C, a summary table of the results of sequencing 30 independent clones derived from cDNA copies (total RNA) or genomic DNA copies. Edited refers to the presence of both editing events. Conditions for the different reactions above are as described under "Materials and Methods." Tb , T. brucei . LT , Leishmania tarentolae .

A Salt Effects on Deamination + [Salt]

100

80 NaCl 60 KCl 40

DeaminaeActivity (%) 0 0 200 400 600 800 1000 [Salt] (mM)

B C

-20 °C Mg 2+ Effe ct on Deamination Enzyme Stability 4 °C 23 °C 100 100 MgSO4 80 80 MgCl 2 60 60 40 40 20 20 Mol I/ Mol tRNA Mol I/ Mol

0 (%) Activity Deaminase 0 0 1 2 3 4 5 0 50 100 150 200 250 Mg 2+ (mM) Time (Hrs)

Figure 2.4. Effects of salt, Mg 2+ , and temperature on the A34 to I34 deaminase from T. brucei . A, NaCl and KCl titrations were conducted to determine the optimal concentration of salt in the deaminase reaction. The A to I deaminase functions optimally without salt present. B, The effect of Mg 2+ concentrations on A to I deaminase activity. The activity of the A to I enzyme is plotted vs increasing magnesium concentrations. C. The effect of temperature on the enzyme’s ability to deaminate A. The enzyme was incubated at room temperature (23 °C), 4 °C, and -20 °C for 200 hours. Time points were taken and the enzymes were assayed for A to I activity.

Figure 2.5. tRNA Thr (AGU) is efficiently edited in vitro . tRNA Thr (AGU) was labeled by in vitro transcription in the presence of [ -32 P]ATP. The labeled tRNA was incubated with total T. brucei extracts for increasing lengths of time followed by nuclease P1 digestion and separation by TLC. A, TLC analysis of the reaction in the presence or absence of extract, where pA and pI denote the positions of unlabeled 5'-AMP and 5'-IMP used as markers and visualized by UV shadowing (not shown). B, a reaction where a similar tRNA as in A but containing a G34 was used as a control for specificity. This reaction also served as a background control. The relative fraction of pA converted to pI was calculated by dividing the amount of radioactivity in the pI spot by the sum of the radioactivity in the pA+pI spots (pI/pI+pA). The specific percent conversion at a single site ( i.e. A34) was then calculated by normalizing the amount of radioactivity at A34 to the total number of label adenosines ( n = 14), where conversion at one site will yield a maximum theoretical value of 7.7% (or possible adenosines). The specific yield of pI was then calculated by dividing the percent total by the relative percentage at one site or %pI 34 /%pA 34 x 100, where the theoretical maximum of 7.1% equals 100% conversion A to I conversion at position 34.

Figure 2.6. Two-dimensional-TLC analysis of an in vitro A to I editing reaction. Radioactive tRNA Thr (AGU), where every adenosine is labeled, was used as a substrate in an in vitro editing assay by incubation with total T. brucei extract as described under "Materials and Methods." Following incubation, the tRNA was digested to nucleotides, and the products were separated on two- dimensional thin-layer chromatography. –E and +E refer to reactions performed in the absence or presence of extract, respectively. pA, pG, pC, pU, and pI refer to the migration of nucleotides as corroborated by comparison with published maps and/or by separation of individual nucleotides used as cold markers and visualized by UV shadowing (not shown) also depicted by dashed circles. Arrows denote the direction of migration during TLC. A–C refer to the different solvents used during chromatography as described under "Materials and Methods."

Figure 2.7. An interdependence model for the double editing of tRNA Thr . In the event of interdependence, tRNA maturation may take one of two paths, indicated by 1 and 2. 1) either U32 is first converted into C32, and that affects A34 to I 34 conversion, or 2) A34 is converted to I34 first followed by the C32 to U32 conversion. If no interdependence occurs between the sites then 3 will prevail.

Figure 2.8. The presence of U32 stimulates A to I conversion at the wobble base. An [ -32 P]ATP-labeled pre-edited tRNA Thr (AGU), where C32 was replaced by U32, was incubated with T. brucei extracts for various times. A, TLC analysis of an [ -32 P]ATP labeled U32-containing tRNA in the presence or absence of enzyme and increasing incubation times. B, TLC analysis of an [ -32 P]ATP labeled U32-containing tRNA in which A34 was replaced by G34 used as a control for specificity. C, a plot of the percentage of conversion of A to I at 34 versus time. The products of the reaction and calculations of percent conversion are as described in the legend for Fig. 3. The values shown are the result of five independent experiments, where values were averaged to obtain a measure of error between experiments and data points (error bars). pI and pA, refer to unlabeled inosine-5-monophosphate and adenosine-5-monophosphate used as markers for TLC.

Figure 2.9. tRNA Thr (AGU) is efficiently aminoacylated in vitro regardless of its editing state. Different versions of tRNA Thr (AGU) were generated by in vitro transcription and then used in aminoacylation reactions in the presence of L-[3- 3H]threonine and partially purified synthetase fractions from T. brucei . A, a tRNA containing a G34 and either the pre-edited C32 or the edited U32 was used during the reaction. B, a similar experiment as in A, but the reactions were performed with tRNA substrates that are pre-edited at position 34 (A34) and either a pre-edited or an edited position 32 (C32 or U32). Control refers to a reaction where the in vitro transcript was left out of the reaction. This control serves as background during quantitation; pmol refers to the amounts of Thr- tRNA Thr generated at various times expressed in picomoles.

Figure 2.10. The edited and pre-edited substrates are functional substrates for aminoacylation in vivo . Total RNA was isolated from T. brucei under acidic conditions, oxidized, and polyadenylated, and the resulting template was used for RT-PCR to determine the editing state of the aminoacylated species. A, a – scheme of the oxidation/tailing reaction. IO 4 refers to a reaction where total acidic RNA was treated with sodium periodate. OH – refers to the incubation of the acidic RNA at pH 9.0 for 30 min, leading to deacylation. RT refers to a cDNA synthesis reaction using the tailed tRNA or a negative control where the RNA was deacylated prior to oxidation. PCR1 and PCR2 are reactions in which the cDNA from the previous step was first used as a template for PCR using primers specific for the poly(A) tail and tRNA Thr (PCR1) followed by a PCR reaction with tRNA Thr -specific primers (PCR2). These reactions were performed in succession. aa, aminoacyl. B, the reaction products from A were separated on a 4% agarose gel and visualized by staining with ethidium bromide. RT+ and RT–refer to reactions performed in the presence or absence or reverse transcriptase as described. Marker refers to the 10-bp size marker used during electrophoresis. C, results of sequencing 30 independent clones, where Edited refers to double- edited tRNAs.

CHAPTER 3

PURIFICATION AND IDENTIFICATION OF THE A TO I EDITING ENZYME.

3.1 Introduction

Inosine formation at the wobble position is an essential process that is known to occur in two of the three domains of life. In bacteria, a homodimeric enzyme composed of two ADATa proteins is responsible for this essential RNA editing event. In yeast a heterodimeric complex composed of ADAT2 and ADAT3 proteins facilitates this editing process. In the yeast system, the two proteins were recombinantly expressed separately, mixed together, and full editing activity was observed. In contrast when we recombinantly expressed ADAT2 and ADAT3 from T. brucei in E. coli or in L. tarentolae and mixed them together, no editing activity was observed. From these data, we hypothesized that the editing complex from T. brucei may contain additional factors required for tRNA editing.

To determine the identity of these additional factors a native purification procedure was undertaken where the active complex was partially purified.

53 3.2 Materials and Methods

3.2.1 Cloning of Trypanosome tRNA ADAT2 and ADAT3 from genomic DNA

The ADAT2 open reading frame was amplified from 10 ng of genomic

DNA by PCR with primers JAG 12 (GTGCAAGATCTAGACAAAGACAC) and

JAG 13 (AACAACTGACAAATCTAGACGTTTTC) while the ADAT3 open reading frame was amplified with primers JAG 9 (TTCCTGAGTCTAGACAACAAATTAC) and JAG 14 (AGGCACCGTAAGGTCTAGACAGTACG). This PCR product was gel purified and ligated into the pTrcHis2-TOPO TA vector then transformed into

E. coli . To ensure proper ligation of PCR fragments into the pTrcHis2-TOPO TA vector, independent colonies were grown and plasmid DNA was purified. The purified DNA was incubated with 10 units of EcoRI overnight at 37 °C and subjected to gel electrophoresis. Once positive results were obtained, the inserts were sequenced to ensure the proper sequence was obtained.

3.2.2 Preparation of the native enzyme complex from L. tarentolae/T. brucei.

Cells were grown to a final concentration of 10 7 cells/ml. Cells were harvested by centrifugation, suspended in lysis buffer (50 mM Tris-HCl (pH 8.0),

25 mM KCl, 2.5 mM MgSO 4, 0.1mM EDTA, and 1 mM DTT) and sonicated 7 times with a Sonifier 450 sonicator using a microprobe at 50% output using 20 second intervals with 20 second rest on ice between sonication bursts. An S100 was produced by centrifugation at 100,000 g for 30 min. The S100 was stored at

-80 ºC in 20% (v/v) glycerol.

3.2.3 Immunoprecipitation of T. brucei extract with anti-ADAT2 antibody.

Six hundred µl of T. brucei S100 extract was incubated with Protein A beads for 15 minutes at 4 ºC. The mix was centrifuged for 10 min at max speed and the supernatant was collected. 45 µl of the supernatant was incubated with anti-ADAT2 antibodies for 1 hour at 4 ºC, 10 µl of Protein A beads were then added and incubated for 1.5 hours at 4 ºC. The mixture was centrifuged at 16.1 g for 10 min and the supernatant was assayed for A34 → I34 deaminase activity.

3.2.4 One and two dimensional Thin Layer Chromatography (TLC) in vitro assays.

tRNA substrates were prepared as previously described by us (99). Radio- labeled substrate was first heated to 70 ºC for 3 min to denature the RNA and allowed to refold at room temp for 5 min in reaction buffer (50 mM Tris-HCl (pH

8.0), 25 mM KCl, 2.5 mM MgSO 4, 0.1mM EDTA, and 1 mM DTT). Reactions were incubated for 45 min in reaction buffer at 27 ºC then phenol extracted and ethanol-precipitated. Pellets were washed with 70% ethanol and resuspended in

8 µl of P1 buffer (30 mM ammonium acetate and 10 mM zinc acetate containing),

1µl cold total yeast tRNA and 1 µl of P1 nuclease (0.4 units) (MPBiomedicals) overnight at 37 ºC. The reaction was dried in a SpeedVac DNA 110 concentrator system (Savant) under high heat. Dried samples were supended in 3 µl ddH 20 and 1 µl was spotted on a TLC plate. Reaction products were separated by one

55 or two dimensional TLC in solvent C (0.1 M sodium phosphate (pH

6.8):ammonium sulfate:n-propyl alcohol (100:60:2, v/w/v)) and visualized using an Amersham Biosciences Storm imaging system and quantified using the

ImageQuant program. Nucleotide assignments were made using published TLC maps and cold nucleotide markers (121).

3.3 Results

3.3.1 Purification of the A → I tRNA deaminase from L. tarentolae.

Proteins were precipitated with 0%-35%, and 36%-55%, and 56-99% ammonium sulfate respectively from a L. tarentolae S100 extract. To determine into which ammonium sulfate fraction the A34 → I34 deaminase partitioned, fractions were incubated with radio-labeled substrate, phenol extracted, digested with nuclease PI, and subjected to TLC (Figure 3.1). The fraction containing proteins that precipitated with 35% ammonium sulfate were able to fully deaminate A34 containing tRNA. These proteins were dialyzed against buffer A

(50 mM Hepes pH 8.0) overnight then loaded onto a Q Sepharose column.

Proteins that did not bind to the Q Sepharose column were washed away with 20 column volumes of buffer A. The column was then washed with buffer B (50 mM potassium chloride (KCl) and 50 mM Hepes pH 8.0) to elute any loosely proteins.

A linear gradient of an increasing concentration of KCl starting with buffer B and ending with buffer C (1 M KCl and 50 mM Hepes pH 8.0) was used to elute all remaining proteins from the column. The absorbance at 280 nm indicated where

56 proteins eluted from the column and determined a starting point to search for the

A34 → I34 deaminase (Figure 3.2A). Every third fraction beginning with fraction

25 was incubated with radioactive substrate and assayed for A34 → I34 deaminase activity by TLC (Figure 3.2B). Fractions 25-40 (proteins eluting at 200 mM-400 mM KCl) corresponding to peak A34 → I34 deaminase activity were pooled and dialyzed against 50 mM Hepes pH 8.0. While fractions 41-46 contained the A34 → I34 deaminase enzyme, these were not pooled as the specific activity was lower in these fractions. Fractions 25-40 eluted from the Q

Sepharose column were bound to a heparin column. The column was washed with 20 column volumes of Buffer A. A linear gradient beginning with buffer A and ending with Buffer C was used to elute proteins from the heparin column.

Spectrophotometric analysis (A280) revealed peaks corresponding to fractions

16, 29, and 32 (Figure 3.3A). Proteins corresponding to these peaks were assayed for A34 → I34 deaminase activity by incubation with radio-labeled substrate and were subjected to TLC (Figure 3.3B). Fractions 12-16

(corresponding to proteins that eluted at 150 mM KCl to 275 mM KCl) were capable of A34 → I34 deaminase activity with fraction 12 being the most active fraction. Fractions 10-16 were pooled and dialyzed against 50 mM Hepes pH 8.0.

These proteins were subjected to Mono Q column chromatography using identical buffers and conditions as described above for Q Sepharose column chromatography. A280 spectrophotometric analysis revealed a single symmetrical peak corresponding to fractions 15-20 (DNS). Proteins

57 corresponding to this peak were assayed for A34 → I34 deaminase activity by incubation with radio-labeled substrate, phenol extraction, digestion with nuclease PI, and were subjected to TLC (Figure 3.4). Fractions 15-17 were capable of A34 → I34 deaminase activity with fraction 16 being the most active fraction. The individual protein components of each fractionation were subjected to SDS-PAGE and stained with Coomassie brilliant blue (Figure 3.5). Nine prominent bands resulted after elution from the Mono Q column. Two of these bands correspond in size to that expected for ADAT2 and ADAT3, components of the A34 → I34 tRNA deaminase, which have predicted molecular masses of

25 kDa and 37 kDa respectively.

3.3.2 Purification of the A → I tRNA deaminase from T. brucei.

An S100 was produced from T. brucei cells and proteins were precipitated with 0%-35% and 36%-80% ammonium sulfate respectively. Fractions were incubated with radio-labeled substrate and assayed for A34 → I34 activity. The

36%-80% ammonium sulfate fractionation was the most active for A34 → I34 deaminase activity (Figure 3.6). These fractions were dialyzed against 50 mM

Hepes, pH 8.0 and subjected to Q-Sepharose column chromatography (as above). Every third fraction was assayed for A34 → I34 deaminase activity

(Figure 3.7A). Fractions 24-36 were pooled, concentrated, and dialyzed against

50 mM Hepes pH 8.0. Western analysis was used to confirm the pressence of

Tb ADAT2 and Tb ADAT3 in the Q-sepharose fractions (Figure 3.7B). To

58 determine the size of the T. brucei A34 → I34 tRNA deaminase, active fractions from the Q-Sepharose pool were subjected to Superdex 200 gel filtration chromatography using a buffer containing 50 mM Hepes pH 8.0 and 200 mM

KCl. Fractions corresponding to proteins with molecular masses ranging from

5kDa to 600 kDa were assayed for A34 → I34 tRNA deaminase activity (Figure

3.8). Fractions 27-34 appeared to contain proteins capable of a low level of A34

→ I34 tRNA deaminase activity corresponding to molecular masses of 135-600 kDa (fractions 27-34) respectively. Attempts to reconstitute activity by mixing fractions corresponding to molecular masses of 25kDa and 37kDa failed to restore A34 → I34 tRNA deaminase activity (DNS).

3.3.3 Over-expression of recombinant proteins

Since the proteins responsible for A34 → I34 tRNA deaminase activity are known in yeast (87), Sc ADAT2 and Sc ADAT3 were subjected to Basic Local

Alignment Search Tool ( BLAST) analysis and searched against the T. brucei genome to find potential homologs. By sequence comparison, Tb ADAT2 is

22.7% identical and 31.4% similar to Sc ADAT2, but in the catalytic region it is

42.3% identical and 56.7% similar. Tb ADAT3 is 18% identical and 27.5% similar to Sc ADAT3, but in the catalytic region it is 39.6% identical and 56.3% similar.

Given this degree of similarity, we hypothesized that Tb ADAT2 and Tb ADAT3 would bear a similar function in their respective systems. To test this hypothesis, the Tb ADAT2 gene was amplified from T. brucei genomic DNA and introduced

59 into the pTrcHis2-topo vector for expression in bacterial cells. This vector is unique in that there are C-terminal Myc and 6xHis tags. A commercially available anti-Myc antibody allows for detection of the recombinant protein while the 6xHis tag aids in purification. The recombinant proteins were expressed and subjected to sequential purification by Ni-NTA (nickel-nitrilotriacetic acid), Mono-Q, and

Superose 12 chromatography to ensure a high level of purity. The full-length

Tb ADAT2 protein is 225 amino acids in length and has a molecular mass of 23.7 kDa while the full-length Tb ADAT3 protein is 365 amino acids in length and has a molecular mass of 40 kDa (Figure 3.9A). The recombinant proteins were unable to catalyze A34 → I34 tRNA deaminase activity (Figure 3.9B). This observation differs from the yeast system where recombinant Sc ADAT2 and Sc ADAT3 proteins were over-expressed separately in yeast, mixed, and the two recombinant proteins then yielded an active enzyme (87). Our goal was then to express Tb ADAT2 and Tb ADAT3 in trypanosomes with the idea that once expressed they would be active. We cloned Tb ADAT2 and Tb ADAT3 with C- terminal Myc and 6xHis tags into a pX vector which allows for expression in L. tarentolae cells (127;128). Once electroporated, the cells were harvested and recombinant Tb ADAT2 was purified by Ni-NTA chromatography and probed with the anti-Myc antibody by western analysis (Figure 3.10). Tb ADAT3 was unable to be expressed and purified using the pX system.

3.3.4 TbADAT2 and TbADAT3 antibody generation and purification

Full length Tb ADAT2 and Tb ADAT3 containing C-terminal Myc and 6xHis tags were sent to BioPharm for polyclonal antibody generation. The resulting antibody recognized recombinant Tb ADAT2 efficiently (Figure 3.11B). To determine how efficiently the antibody detects endogenous Tb ADAT2, an S100 was prepared and probed by western analysis. A prominent signal corresponding to a protein with a molecular mass of 75 kDa was observed as well as faint bands at 28 kDa and 50 kDa (Figure 3.11A). To determine if endogenous

Tb ADAT2 migrates differently than recombinant Tb ADAT2 expressed in E. coli, the protein was expressed in T. brucei with a TY tag by F. Nina Papavasiliou and coworkers. When the S100 of wild type T. brucei and the S100 of T. brucei expressing Tb ADAT2 with a TY tag were probed by western analysis with an anti-TY antibody, a lone band at 75 kDa was observed in only T. brucei cells expressing Tb ADAT2 with a TY tag (Figure 3.11C).

3.3.5 Tandem affinity purification of the A34 → I34 tRNA editing enzyme

Tb ADAT2 was cloned into the pLew82 vector 5’ of the Protein A and streptavidin binding protein (SBP) tags (129). This vector allows for controlled expression of proteins by a tetracycline inducible T7 promoter when grown in a strain of T. brucei with T7 polymerase incorporated into its genome. To determine if the Protein A and SBP tagged Tb ADAT2 was being expressed T.

61 brucei cells electroporated with the vector were grown in the presence or and absence of tetracycline. S100s were prepared and probed by western analysis with an anti-PAP reagent which is specific to Protein A (130) (Figure 3.12A). T. brucei cells grown in the presence of tetracycline produced a strong signal corresponding to 50 kDa (the size of Tb ADAT2 with the Protein A and SBP) while cells grown in the absence of tetracycline produced a very weak signal. T. brucei extract not containing the pLew82 vector did not produce a signal when probed with anti-PAP reagent by western analysis. Coomassie-stained gels confirmed that relatively the same amount of total protein (30 ug) was present in each sample (Figure 3.12B). The samples were probed with anti-ADAT2 and a signal at 50 kDa was observed in addition to the 75 kDa signal in the induced and uninduced samples but not in the T. brucei sample (Figure 3.12C). Once it was determined that tagged Tb ADAT2 was expressed, a time course was performed to determine when the protein was expressed optimally. Cells were collected at

24, 48, and 72 hours. S100s were prepared and probed with anti-PAP reagent by western analysis (figure 3.12D). Cells harvested after 24 h yielded the strongest signal and decreased with time. We harvested cells after 24 h for all subsequent experiments. Once the expression of the tagged protein was optimized, two liters of T. brucei were harvested. An S100 was generated and incubated with IgG sepharose beads for 3 hours at 4º C. The beads were then washed and incubated with Tobacco Etch Virus (TEV) protease overnight at 4º C. The TEV protease is a highly site-specific protease that is found in the Tobacco Etch Virus.

62 It is a cysteine protease and recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln-

Gly and orchestrates cleavage between the Gln and Gly residues (131). The supernatant was then incubated with Streptavidin sepharose and the complex was eluted from the column with biotin. To ensure the TEV protease had cleaved efficiently, the IgG sepharose beads were incubated with glycine pH 2.5 and the supernatant was quickly neutralized. The low pH should have release any non-

TEV protease cleaved Protein A tagged protein from the IgG sepharose beads.

The fractions were subjected to gel electrophoresis and stained with coomassie blue as well as probed with PAP reagent and anti-ADAT2 by western analysis.

Seven noticeable bands are present after TEV protease was added to release the complex from the immobilized IgG sepharose (Figure 3.13A). No bands were seen in on the stained gel corresponding to the fractions that were eluted from the streptavidin sepharose. Six prominent bands were observed in the fraction where glycine pH 2.5 had been used to elute the bound proteins from the IgG sepharose beads. The same fractions were probed with the PAP reagent (figure

3.13B). A band corresponding to 50 kDa was observed in the S100, flow through, and the washes but was not observed in any other fractions. These results were expected as the antibody is specific to the Protein A tag and after TEV protease treatment the Protein A tag was immobilized on the IgG sepharose beads. Since the protein A tag would have been cleaved after the TEV elution, anti-Tb ADAT2 was used to determine if Tb ADAT2 was present in any other fractions. The same membrane was reprobed with anti-Tb ADAT2 without stripping (Figure 3.13C).

63 The appearance of a 24 kDa protein in the TEV protease and biotin elution fractions confirms the presence of Tb ADAT2. The fractions taken from the various columns were assayed for the ability to catalyze A34 → I34 tRNA deaminase activity (Figure 3.14A). None of the fractions were active except the

S100. Since the fraction containing the proteins that did not bind to the column

(FT) was unable to edit the tRNA, we hypothesized that either the IgG column led to inactivation of the enzyme or the enzyme was bound to the column in a manner that could not be removed by the elution procedure. To test the latter, the bound enzyme was released from the column with biotin, but the eluted enzyme was still unable to edit the tRNA substrate (Figure 3.14A).

In an attempt to reconstitute activity, the proteins that were released with

TEV protease were combined with the proteins in the different washes of the column. In addition the proteins in the FT were combined with the proteins in the different washes. When the FT fraction was mixed with the proteins in the third wash, some editing activity was restored (Figure 3.14B). Wash two and wash three differ only in the amount of DTT present where wash three contains 100 mM DTT and wash two has none. To investigate whether the concentration of

DTT or the proteins present in the fraction containing DTT were the reason activity was restored, DTT was added to the FT fraction (Figure 3.14 C). When

DTT was added to the fraction containing the proteins in wash two, editing activity was observed. The proteins released from the column with the TEV

64 protease were incubated with DTT and no editing activity was observed. It is possible that the washes disrupted the tRNA editing complex. Triton X-100 has been shown to keep complexes together (132). To determine if Triton X-100 has detrimental effects on deamination, the S100 was incubated with Triton X-100 and assayed for A34 → I34 tRNA activity by TLC and found to have no negative effect on editing (Figure 3.14B). In the future, Triton X 100 will be used in the

TAP of the A34 → I34 editing complex. To determine if the activity could be re- gained with the independently expressed recombinant Tb ADAT2 and Tb ADAT3 proteins in the presence of DTT, the two proteins were incubated with DTT and assayed for editing activity. No inosine formation was detected by TLC analysis

(Figure 3.14C).

3.3.6 Immunoprecipitation assays of TbADAT2

To determine if Tb ADAT2 was a component of the A34 → I34 tRNA editing complex in T. brucei , T. brucei extract was incubated with anti-ADAT2 antibodies. The antibody-antigen complex was incubated with Protein A sepharose beads and centrifuged. The supernatant was collected and assayed for A34 → I34 tRNA deaminase activity by TLC. As the concentration of anti-

ADAT2 increased, the level of deamination decreased (Figure 3.15A). To determine if the decrease in A34 → I34 tRNA deaminase activity was a result of the immunoprecipitation of anti-ADAT2 and the endogenous protein or the inhibition of the reaction by other factors present in the serum, we did a titration

65 with the serum and assayed the fractions (Figures 3.15B and 3.15C). The immunoprecipitation with anti-ADAT2 resulted in an 80% decrease in A34 → I34 tRNA deaminase activity while the control immunoprecipitation with serum resulted in a 19% decrease. When recombinant Tb ADAT2 was added to the supernatant of the immunoprecipitated fractions no activity was recovered. In addition, when the pelleted beads with anti-ADAT2 and the antigen complex were assayed for A34 → I34 tRNA deaminase activity no activity was recovered.

3.4 Discussion:

The A34 → I34 tRNA deaminase from L. tarentolae was partially purified and subjected to SDS PAGE. Nine clear bands were present on Coomassie stained gel. Three of the bands correspond to molecular masses of proteins thought to be present in the hypothetical A34 → I34 tRNA deaminase. Tb ADAT2 and TbADAT3 are predicted to have molecular masses of 24 kDa and 40 kDa respectively and correspond to bands present in the Coomassie stained gel. We believe that the band present on the Coomassie stained gel migrating at approximately 75 kDa is Tb ADAT2 but migrates differently than we expect. We have supplied three independent lines of evidence to support the hypothesis that

Tb ADAT2 can localize with proteins corresponding to molecular masses of 75 kDa when subjected to SDS-PAGE. We have generated an antibody that recognizes recombinant Tb ADAT2. When an S100 from both T. brucei and L. tarentolae are probed with this antibody by western analysis, a clear band

66 corresponding to a protein with a molecular mass of 75 kDa is observed. When

Tb ADAT2 is expressed with a C-terminal TY tag in T. brucei a band corresponding to a protein with a molecular mass of 75 kDa is observed when probed with an anti-TY antibody by western analysis (Figure 3.11D). In addition we expressed Tb ADAT2 with C-terminal 6xHis and Myc tags in L. tarentolae using a pX vector. An S100 was generated and probed with an anti-myc antibody by western analysis. Clear bands were observed corresponding to proteins with molecular masses of 27 kDa (TbADAT2 with Myc and 6xHis tags) at 75 kDa respectively (Figure 3.10B). To ensure the anti-Myc antibody was not cross- reacting to endogenous proteins present in T. brucei , a wild-type (WT) S100 was generated and probed with anti-Myc. No signal was observed. The identities of the other proteins corresponding to the remaining six bands are currently unknown. It is possible that some of these proteins may be merely contaminants that co-purified with the A34 → I34 tRNA deaminase; however, the 2 known catalytic components together do not represent the size of the endogenous enzyme recovered from gel filtration chromatography. When a T. brucei S100 was subjected to gel filtration chromatography (and L. tarentolae in the next chapter), the peak of A34 → I34 tRNA deaminase activity corresponds to a complex of proteins with a molecular mass of >200 kDa. A similar observation was made with the mammalian enzyme when an S100 prepared from a nucleated rabbit reticulocytes was subjected to gel filtration chromatography. The fractions collected were assayed and a molecular mass of 200 kDa was

67 determined for the enzyme responsible for the A34 → I34 tRNA deaminase activity (104). Taken together, we suggest that the endogenous enzymes from T. brucei and L. tarentolae may contain additional subunits, implying that the T. brucei A34 → I34 tRNA deaminase is a multimeric enzyme. This is not unprecedented in that other deaminases such as the deoxycytidylate deaminase

(133) and the E. coli cytosine deaminase (134) are both hexameric enzymes.

To reaffirm our hypothesis that Tb ADAT2 is one component of the T. brucei A34 → I34 tRNA deaminase enzyme, the T. brucei extract was immunoprecipitated with anti-ADAT2 antibody. The results show a decrease in

A34 → I34 tRNA deaminase activity in samples incubated with anti-ADAT2 antibody. Concurrently, our collaborators used RNAi to knock-down the level of

Tb ADAT2 in T. brucei cells. A decrease in endogenous Tb ADAT2 was shown by western analysis and resulted in a corresponding decrease in ability to deaminate tRNA (135). Taken together, these data show Tb ADAT2 is an essential component of the A34 → I34 tRNA deaminase in T. brucei . To determine the other protein factors that might comprise the T. brucei A34 → I34 tRNA deaminase, Tb ADAT2 was used as “bait” in tandem affinity purification.

After incubation with IgG sepharose no A34 → I34 tRNA deaminase was observed in the flow-through. Our original hypothesis was that all of the endogenous enzyme bound the bait and no deaminase activity was found because the entire complex was bound to the IgG sepharose. Western analysis

68 supports this idea as the band present at 27 kDa is not seen in the flow though, and the band at 75 kDa is significantly decreased when probed with anti-ADAT2 by western analysis. Once TEV protease was used to release the complex from the column, clear bands at 27 kDa corresponding to endogenous Tb ADAT2 as well as a slightly larger band corresponding to recombinant Tb ADAT2 with a SBP tag were seen when probed with anti-ADAT2 by western analysis. While it is clear Tb ADAT2 was present in this fraction the proteins were unable to facilitate

A34 → I34 tRNA deaminase activity. We believe the washes designed to rid the complex of non-specific binding proteins may have been too harsh and proteins important for A34 → I34 tRNA deaminase activity were inadvertently eliminated.

The addition of DTT restored some A34 → I34 tRNA deaminase activity in the flow through fraction and suggests a reduced environment is important for the deamination reaction. The question as to how the environment was changed from one conducive to deamination to one so oxidized only a small amount of deamination was observed arises. The addition of the IgG sepharose beads which must be in an oxidized state to keep the antibodies intact may have been the cause of the shift in redox state. The addition of DTT was not sufficient to restore deaminase activity in the proteins eluted from the column with TEV protease suggesting the A34 → I34 tRNA deaminase is not a tightly bound complex because essential proteins were eliminated in the washes. To stabilize the complex formation Triton X-100 will be used in subsequent TAPs (132).

Expression of C-terminally tagged recombinant Tb ADAT2 and Tb ADAT3 in

69 bacteria and in trypanosomes failed to yield an active enzyme. Our original hypothesis that the proteins had to be expressed in trypanosomes to be active was incorrect. The reason for the inactivity was rooted in the necessity of the enzymes to be co-expressed. Co-expression of protein complexes increase solubility and biological activity (136) as well as allow for coupled protein folding

(137). Once expressed together in E. coli full A34 → I34 tRNA deaminase activity was observed. While these two proteins supply the minimal and sufficient components of the A34 → I34 tRNA deaminase, given the native size of the A to

I enzyme in partially purified fractions from T. brucei , it may still be possible that other proteins contribute to functioning in vivo , but this will remain an open question.

N E S100 35% 55% 80%

1ug 5ug 1ug 5ug 1ug 5ug 1ug 5ug

Figure 3.1. Ammonium sulfate precipitation of L. tarentolae S100. An S100 was produced from L. tarentolae cells and ammonium sulfate was added until concentrations of 35%, 55%, and 80% were achieved. tRNA Val (AAC) was labeled by in vitro transcription in the presence of [ α-32P] ATP. The labeled transcript was incubated with either 1 ug or 5 ug of the ammonium sulfate precipitated proteins followed by nuclease P1 digestion and separated by TLC. Cold 5’AMP (pA) and 5’IMP (pI) were used as markers to show the locations of the nucleotides respectively. When tRNA Val (AAC) was incubated without extract (NE) no inosine formation was observed. Fractions corresponding to proteins precipitated with 0 to 35% ammonium sulfate had the highest specific activities.

A Q-Sepharose Chromatography 1.2 1 Total Protein A to I activity 0.8 0.6 OD280 0.4 0.2 0 15 20 25 30 35 40 45 50 55 60 65 70 Fraction (#)

B NE 2p W FT 25 28 31 34 37 40 43 46 49 52 55

Figure 3.2. Q Sepharose chromatography fractionation of ammonium sulfate precipitated proteins from L. tarentolae . A. Graphical representation of the fractions collected from the Q sepharose column vs the OD280 observed. The OD280 is shown as solid circles and the deaminase activity is shown as solid squares. B. Labeled tRNA Val (AAC) was incubated with every third fraction following purification by Q sepharose chromatography, digested with nuclease P1, and separated by TLC. When tRNA Val (AAC) was incubated without extract (NE), the flow through from the column (FT), and with a 50 mM KCl wash (W) no inosine formation was observed. Incubation of the substate with recombinantly purified Tb ADAT2 (2p) resulted in two spots not corresponding to inosine.

Heparin Chromatography 1

0.8 Total Protein A to I activity 0.6 0.4 OD280 0.2

0 0 5 10 15 20 25 30 35 40 -0.2 Fraction (#)

B 1 2 3 4 5 6 7 8 9 10 11 12

Figure 3.3. Heparin chromatography fractionation of Q sepharose pool from L. tarentolae . A. Graphical representation of the fractions collected from the Heparin column vs the OD280 observed. The OD280 is shown as solid circles and the deaminase activity is shown as solid squares. B. Labeled tRNA Val (AAC) was incubated with various fractions corresponding to major peaks in OD280, digested with nuclease P1, and separated by TLC. When tRNA Val (AAC) was incubated without extract (Lane 1) no inosine was observed. Lane 2: Q Sepharose pool 1ug Lane 3 Q Sepharose pool 2 ug Lane 4: flow through Lane 5: 50 mM KCl Wash Lane 6: Fraction 12 Lane 7: Fraction 14 Lane 8: Fraction 16 lane 9 Fraction 18 Lane 10: Fraction 20 Lane 11: Fraction 29 Lane 12 Fraction 32.

73 1 2 3 4 5 6 7 8 9 10

Figure 3.4. Mono Q chromatography fractionation of Heparin pool from L. tarentolae . Labeled tRNA Val (AAC) was incubated with various fractions corresponding to the major peak in OD280, digested with nuclease P1, and separated by TLC. When tRNA Val (AAC) was incubated without extract (Lane 1) no inosine was observed. Lane 2: heparin pooled fractions, Lane 3: flow through, Lane 4: Fraction 14, Lane 5: Fraction 15, Lane 6: Fraction 16, Lane 7: Fraction 17, Lane 8: Fraction 18, Lane 9: Fraction 19, lane 10: Fraction 20.

M Am Sul Q Seph Heparin Mono Q kDa 125

100 75

25 20 15

Figure 3.5. Partial purification of the A34 → I34 tRNA deaminase from L. tarentolae. Proteins recovered from each purification step ((0-35% ammonium sulfate precipitation (Am Sul), Q sepharose chromatography (Q seph), heparin chromatography (Heparin), and mono Q chromatography (Mono Q)) were subjected to SDS-PAGE and the gel was stained with Coomassie brilliant blue. Nine prominent bands corresponding to proteins of varying molecular masses were seen in the final purification step (Mono Q) and are denoted by black arrows.

NE AmLt Tb35 Tb80

Figure 3.6. Ammonium sulfate precipitation of T. brucei S100. An S100 was produced from T. brucei cells and ammonium sulfate was added until concentrations of 35%, and 80% were achieved. Labeled tRNA Val (AAC) was incubated without extract (NE), with proteins that precipitated at 0-35% (Tb35) and 36-80% (Tb80), and the proteins present in the ammonium sulfate precipitation of L. tarentolae S100 as a positive control digested with nuclease P1, and separated by TLC.

NE IN FT W 18 21 24 27 30 33 36 39 A

42 45 48 51 54 57 66 69 72 75 78 81

Anti-ADAT2 Anti-ADAT3

Ext Ext kDa

75 50 37 25

Figure 3.7. Q Sepharose chromatography fractionation of ammonium sulfate precipitated proteins from T. brucei . A. Labeled tRNA Val (AAC) was incubated with without extract (NE), digested with nuclease P1, and separated by TLC. The ammonium sulfate pool (IN), flow through from the column (FT), the a 50 mM KCl wash (W) and every third fraction following purification by Q sepharose chromatography (number denotes fraction number) are denoted on the TLC plate. B. Pooled active fractions were probed with anti-ADAT2 and anti- ADAT3 by western analysis.

NE IN 27 31 34 37 40 43 46 49 52

Figure 3.8. Superdex 200 gel filtration fractionation of T. brucei extract. Labeled tRNA Val (AAC) was incubated with without extract (NE), pooled fractions from the Q sepharose pool (IN), and fractions isolated from the column (fraction number denoted), digested with nuclease P1, and separated by TLC.

78 A TbTad2p TbTad3p

kDa M S F W E WS kDa M S F W E WS 100 100 75 75 50 50 37 37

25 25 15 15 10 10 B NE S 2p 2/3p

Figure 3.9. Expression and A34 → I34 tRNA deaminase assessment of recombinant Tb ADAT2 and Tb ADAT3. A. Proteins from the crude S100 (S), nickel column flow through (F), 50 mM imidizole wash (W), and elution (E) were subjected to SDS-PAGE and the gel was stained with Coomassie brilliant blue. The recombinant proteins were probed with an anti-Myc antibody and visualized by western analysis (WS). B. Labeled tRNA Val (AAC) was incubated with without extract (NE), with T. brucei S100 (S), Tb ADAT2 alone (2p), Tb ADAT2 and Tb ADAT3 together (2p/3p), then digested with nuclease P1, and separated by TLC.

A S FT W 27 29 30 31 32 33 34 36 40 44

S FT W 27 29 30 31 32 33 34 36 40 44

Figure 3.10. Expression of Tb ADAT2 in L. tarentolae and purification of the recombinant enzyme. A. Proteins from the crude S100 (S), nickel column flow through (F), 50 mM imidizole wash (W), and elution fractions (fraction number denoted) were subjected to SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. B. The recombinant proteins were probed with an anti-Myc antibody and visualized by western analysis.

g AB u kDa g kDa 2p u 0

1 5 75 100 50 75 37 50 37 25 25

C Tetracycline kDa - +

100 75 50 37 25

Figure 3.11. Anti-ADAT2 purification and detection of endogenous Tb ADAT2 from T. brucei extracts. A. T. brucei S100 extract (both 5 ug and 10 ug total protein) was probed with anti-ADAT2 by western analysis. B. Tb ADAT2 (2p) was probed with anti-ADAT2 by western analysis. C. T. brucei S100 extracts containing a vector that allows for expression of Tb ADAT2 with a C- terminal TY tag under control of a tetracyline inducible promoter were probed with an anti-Ty antibody by western analysis. Cells were either grown in the presence (Tet +) or absence (Tet -) of tetracycline.

A B

kDa S100 Tet - Tet + kDa Tet - Tet + S100 100 75 100 75 50 50

37 37 25 20 25 20

C D

kDa S100 Tet - Tet + Time kDa 100 75 100

75 50 50 37 37 25 25 20 20

Figure 3.12. Expression of Tb ADAT2 with Protein A and streptavidin tags for tandem affinity purification. A. T. brucei S100 extracts containing a vector that allows for expression of Tb ADAT2 with C-terminal Protein A and streptavidin tags under control of a tetracyline inducible promoter were probed with an PAP reagent by western analysis. Cells were either grown in the pressence (Tet +) or absence (Tet -) of tetracycline. Wild type T. brucei S100 extract (S100) was also probed ensure no cross-reactivity with the PAP reagent was observed. B. The same samples were stained with Coomassie Brilliant Blue and C. probed with the PAP reagent and D. anti-ADAT2 by western analysis.

kDa SFT M W1W2W3TSFSWSE G 150 85 63 47.5 33 25 20 15

B M SFT kDa W1W2W3TSFSWSE G 85 63 47 33 25

C M SFT kDa W1W2W3TSFSWSE G

85 63 47 33 25

Figure 3.13. Tandem affinity purification of the A34 → I34 editing complex from T. brucei. A. Proteins from the crude S100 (S100), Protein A flow through (FT), wash 1 (W1), wash 2 (W2), wash 3 (W3), elution with TEV protease (T), flow through from the streptavidin column (SF), wash from streptavidin column (SW), elution from streptavidin column (SE), and elution from Protein A with glycine (G) were subjected to SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. The molecule mass marker (M) is displayed in kDa. B. The same fractions were probed with the PAP reagent and C. anti-ADAT2 by western analysis.

Figure 3.14. Activity assessment of the Tandem affinity purified A34 → I34 tRNA deaminase. A. Proteins from the crude S100 (S100), Protein A flow through (FT), wash 1 (Wash1), wash 2 (Wash 2), wash 3 (Wash 3), elution with TEV protease (TEV elution), flow through from the streptavidin column (Streptavidin FT), wash from streptavidin column (Streptavidin Wash), elution from streptavidin column (Streptavidin Elution), and elution from Protein A with glycine (Glycine Elution) were assayed for A34 → I34 tRNA deaminase activity and subjected to TLC analysis. B. Different combinations of each fraction were done in an attempt to restore deaminase activity. C. DTT was added to each fraction to determine if a reduced environment is necessary for A34 → I34 tRNA deaminase activity.

A 1 2 3 4 5 6 7 8 9 10 11 1. No Enzyme 2. S100 3. FT 4. Wash 1 pI 5. Wash 2 6. Wash 3 7. TEV Fraction 8. Streptavidin FT pA 9. Streptavidin Wash 10. Streptavidin Elution 11. Glycine Elution

B 1 2 3 4 5 6 7 8 9 1. S100

2. TEV + 1 ul FT pI 3. TEV + 2 ul FT 4. TEV + 5 ul FT 5. TEV + 10 u l FT 6. FT + Wash 1 pA 7. FT + Wash 2

8. FT + Wash 3 9. S100 + Triton X 1 00

C 1 2 3 4 5 6 7 8 9 10 1 water + 0.1M DTT 2. S100 3. FT without DTT 4. FT + 0.1 M DTT pI 5. FT + Wash 2 6. FT + Wash 2 + 0.1M DTT 7. FT + Wash 3 pA 8. TEV eluted Frac tion + DTT 9. Recom binant ADAT2 + DTT 10. Rec ombinant ADAT2 no DTT + Tad3p + DTT

Figure 3.14. Activity assessment of the Tandem affinity purified A34 → I34 tRNA deaminase

Figure 3.15. Anti-ADAT2 immunoprecipitation of T. brucei extracts. A. Fractions collected from the immunoprecipitation reactions were incubated with Labeled tRNA Val (AAC) and subjected to TLC analysis. When tRNA Val (AAC) was incubated without extract (NE) no inosine formation was observed. Incubation with T. brucei S100 ectract (E) yeilded full activity. The volume/volume ratio of antibody to reaction mix is denoted above each appropriate lane. B. The experiment was repeated as well as a control experiment with serum containing no antibody. C. Graphical representation of the fold decrease in activity vs. amount of antibody or serum added to the reaction mix.

86 A a-ADAT2 NE 1:5K 1:1K 1:100 1:50 1:10 E

B a-ADAT2 serum NE E 1:50 NE E 1:50

C Antibody Dilution vs Fold Decrease in Acitivity 6 5 Anti -ADAT2 4 S erum 3 2 1

Fold Decrease Activity Decrease Fold 0 0 0.005 0.01 0.015 0.02 0.025

Antibody Dilution

Figure 3.15. Anti-ADAT2 immunoprecipitation of T. brucei extracts.

CHAPTER 4

THE C-TERMINAL END OF ADAT2 PLAYS A CRITICAL ROLE IN TRNA BINDING AND EDITING ACTIVITY IN TRYPANOSOMA BRUCEI

4.1 Abstract

Inosine at the first position of the anticodon (“wobble” position) in tRNAs has been observed in two of the three domains of life and is essential for cell viability (85;87). Inosine formation at the wobble position expands the decoding capabilities of a tRNA, as inosine can base pair with A, C, or U, at the third position of codons allowing the decoding of multiple codons by a single tRNA

(138). The Adenosine Deaminases Acting on tRNA (ADATs) are a group of enzymes responsible for inosine formation in tRNA. The anticodon-specific bacterial enzyme is a homodimer that recognizes only one tRNA as a substrate

Arg (tRNA ACG ) (85). The eukaryal A to I enzyme is composed of two non-identical subunits, ADAT2 and ADAT3, which upon heterodimerization can recognize seven to eight different tRNAs as substrates depending on the organism (87).

Previous studies in yeast demonstrated that ADAT2 is the catalytic subunit while

ADAT3 plays a structural role. Although a deamination mechanism is common to all ADATs, the precise residues that provide substrate binding and thus tRNA

88 recognition with the eukaryotic enzymes are not known. Here we explore the contribution of individual residues in ADAT2 and ADAT3 from Trypanosoma brucei in both, the ability to catalyze the deamination reaction and the ability to recognize the tRNA substrate. We show that mutations to conserved residues in the active site of the enzyme lead to catalytic defects but unexpectedly many of these mutations have little effect on substrate binding. In addition, deletion of a stretch of charged amino acids (lysines and arginines) at the C-terminus of

ADAT2 abolishes both the binding and the deamination activity. We have termed this domain the KR-motif, which provides a first glance at key residues involved in tRNA binding.

4.2 Introduction

RNA editing was first observed in trypanosomes where mitochondrial messenger RNA (mRNA) contained post-transcriptional insertion or deletion of uridines (U) not directly encoded by the genome(11). While this type of editing occurs exclusively in kinetoplastids, RNA editing by base deamination is found in all domains of life (139). Cytidine to uridine (C to U) and adenosine to inosine (A to I) deamination of mRNA by apolipoprotein B (APOBEC-1) (140) and adenosine deaminases that act on m RNA (ADARs) respectively result in a change of information from the genomic message giving rise to protein isoforms that have different roles in cells (141). RNA editing is thus an effective way to generate protein diversity besides alternative RNA splicing and post-

89 transcriptional frameshifting mechanisms (34). In addition to deamination of mRNA, RNA editing of tRNA at the wobble position is an essential process that expands the decoding capabilities of tRNAs. Of the possible 61 codons that specify amino acids, only a portion of the tRNA genes necessary to decode them are encoded in the genome (100). This apparent paradox is rationalized by the ability of I to base pair with C, U, or A in a so-called “wobble” base pair.

Consequently a tRNA containing Inosine (I) at the 34 th or wobble position can easily be likened to a skeleton key. Just as a skeleton key has the ability to open many different locks with one key, a tRNA containing inosine at the wobble position has the ability to decode three codons containing a C, U, or A at the last position of the codon. Without this tRNA degeneracy, proper translation would not be possible, and thus the generation of inosine containing tRNAs is essential

(85;87). In all known cases I formation is the result of hydrolytic deamination of adenosine by post-transcriptional RNA editing. This essential editing event is orchestrated by a homodimeric enzyme that act on tRNA (ADATa) in bacteria or an ADAT2/ADAT3 heterodimer in Eukarya. While the subunit composition varies between domains of life, the catalytic cores are highly conserved. Interestingly, these enzymes that catalyze A to I deamination phylogenically form a clade with the cytidine deaminase superfamily (34). Like all cytidine deaminases, the catalytic core of ADATs consists of an HAE domain as well as a PCXXC domain where the histidine and the two cysteines coordinate a Zn 2+ ion (Figure 1.1). The glutamate residue stabilizes the transition-state and actively shuttles a proton

90 from an activated water molecule to the N1 position of the newly formed I molecule (104). Through evolution the HAE domain of ADAT3 has been replaced by the HPV motif and is now thought to be solely a structural component of the enzyme, as it lacks a proton shuttling glutamate. The recently crystalized

Staphylococcus aureus ADATa homodimer complexed with the anticodon stem loop (ASL) of tRNA Arg has given great insights into enzyme-substrate interactions by the bacterial enzyme. This crystal structure clearly shows that the Sa ADATa homodimer binds the ASL via active site residues (86), whereas the tRNA backbone contributes minimally to RNA binding. In turn, kinetic analysis of

Ec ADATa has shown that an ASL derived from tRNA Arg is a substrate for the enzyme and a full-length tRNA is not required for deaminase activity (101). While a great deal of effort has been put forth to characterize bacterial ADATs, little is known about the eukaryotic enzymes and their interactions with tRNA substrates.

Here we report the importance of critical residues from the T. brucei tRNA adenosine deaminase enzyme on substrate binding and A to I activity. These studies reveal the importance of the C-terminal region of ADAT2 protein, give insights into the contribution of the HPV domain of Tb ADAT3 in catalysis, and provide the first steady-state kinetic analysis of a member of the eukaryotic

ADAT family.

91 4.3 Materials and Methods:

4.3.1 Cloning and expression of the tRNA adenosine 34 deaminase from T. brucei.

The ADAT2/3 open reading frames were cloned into E. coli recombinant expression vectors as previously described by us (91). Twenty-five ml of a starter culture was added to 1.0 L of media and grown at 37 ºC until an OD 600 =0.8 was reached. The culture was induced with 0.5 mM IPTG and grown at 25 ºC for overnight until an OD 600 of 2.5 was reached. All protein purification procedures were conducted at 4ºC and in buffers containing a protease inhibitor cocktail and phenylmethanesulphonylfluoride (PMSF) (Sigma-Aldrich). Cells were washed with lysis buffer (50 mM hepes pH 8.0, 200mM KCl, and 50 mM imidazole) and sonicated 7 times with a Sonifier 450 sonicator using a microprobe at 50% output for 20 s intervals with 20 s rest on ice between sonication bursts. The S100 was collected and run through a Ni 2+ -nitrilotriacetic acid (NTA) agarose column

(volume 1ml) and eluted with a linear gradient of imidazole (50 mM to 1M). Peak fractions were dialyzed overnight in storage buffer (50 mM hepes pH 8.0 and

1mM 1,4-Dithiothreitol (DTT)) and loaded onto a Mono Q column and eluted with a linear gradient of KCl (0 mM to 1 M). Peak fractions were loaded onto a

Superdex 200 gel filtration column. The active fraction eluted at 75 kDa instead of 212 kDa as previously stated (91). Fractions were pooled and dialyzed overnight against storage buffer and stored at -80 ºC with 20% glycerol.

4.3.2 One and two dimensional TLC in vitro assays

tRNA substrates were prepared as previously stated by us (99). Radio- labeled substrate was first heated to 70 ºC for 3 minutes to denature the RNA and allowed to refold at room temp for 5 min in reaction buffer (50 mM Tris-HCl

(pH 8.0), 25 mM KCl, 2.5 mM MgSO 4, 0.1mM EDTA, and 1 mM DTT). Reactions were incubated for 45min then phenol extracted and ethanol-precipitated. Pellets were washed with 70% ethanol and resuspended in 8 µl of P1 buffer (The components), 1ul cold total yeast tRNA and 1 µl of P1 nuclease (MPBiomedicals) overnight at 37 ºC. The reaction was dried in a SpeedVac DNA 110 concentrator system (Savant) under high heat. Dried samples were resusupended in 3 ul ddH 20 and 1 ul was spotted on TLC plates. Reaction products were separated by one or two dimensional TLC in solvent C (0.1 M sodium phosphate (pH

6.8):ammonium sulfate:n-propyl alcohol (100:60:2, v/w/v)) and visualized using an Amersham Biosciences Storm imaging system and quantified using an

ImageQuant program. Nucleotide assignments were made using published TLC maps and cold nucleotide markers (121). Steady-state kinetic constant were calculated by linear regression using the Sigma Plot kinetic software.

4.3.3 Electrophoretic mobility shift Assay

Radio-labeled tRNA (GAC) was incubated with reaction buffer (50 mM hepes pH 8.0, 0.1 mM DTT 1 mM MgCl 2 and 5 mM KCl) and various concentrations of

93 protein for 30 min at 4°C. Glycerol was added to samples (10% total) and separated on 6.5% non-denaturing poly-acrylamide gel at 100 volts for 3 h at 25

ºC. The gel was dried and exposed to a PhosphorImager screen. The shift was visualized using an Amersham Biosciences Storm imaging system and quantified using an ImageQuant program and fit to a single ligand binding curve using

Sigma Plot kinetic software.

4.3.4 Footprint Analysis

Transcript tRNA was dephosphorylated with calf intestinal alkaline phosphatase (GibcoBRL); 1ug of the tRNA was then 5’-end labeled using [ γ-

32 P]ATP and T4 polynucleotide kinase using standard protocols (New England

Biolabs). The labeled transcripts were purified on a 7% denaturing polyacrylamide gel. Before use tRNA was denatured at 70 ° in 40mM Tris (pH

7.5) and 0.1mM EDTA for 5 minutes followed directly by refolding at 37 ° for 10 min and placed at room temperature. Folded tRNA (approximately 1pmole per reaction) was placed on ice for binding with or without protein (3ug-18ug) in

50mM KCl, 10mM MgCl 2, and 0.1mM DTT for 30 minutes. Nuclease was then added (0.005-0.1U T1, Ambion) and reactions placed at room temperature for 20 min. The reaction was stopped with phenol/chloroform, and RNA precipitated with ethanol. Reactions were run on a 15% denaturing polyacrylamide gel with

8M urea and results visualized with a PhosphorImager.

4.4Results

4.4.1 TbADAT2 and TbADAT3 is a Functional Heterodimer

Gel filtration chromatography demonstrated that the Leishmania tarentolae native tRNA A34 deaminase enzyme partitioned in fractions that eluted at a molecular weight of 200 kDa (Figure 4.1). Fractions corresponding to various molecular weights were assayed for A to I activity and run on Thin Layer

Chromatography (TLC) plates. Sequence alignments of known bacterial ADATA proteins as well as yeast ADAT2/3 proteins revealed potential ADAT2 and

ADAT3 proteins from T. brucei (Figure 4.2). Tb ADAT2 and Tb ADAT3 were co- expressed recombinantly with Tb ADAT2 containing an N-terminal 6xHis tag and

Tb ADAT3 containing a C-terminal Flag tag (91). The recombinant Tb ADAT2 and

Tb ADAT3 heterodimer was capable of 1 mole of inosine formation per 1 mole of tRNA after 40 min of incubation (figure 4.3A,B time course). Gel filtration chromatography of the recombinant enzymes revealed that two peaks eluted corresponding to the void volume of the column and to the proteins that eluted at

70 kDa respectively (Figure 4.3C,D). While the fraction isolated from the void volume had a small amount of activity, the bulk of the activity is found in the 70 kDa peak (figure 4.3C). SDS PAGE and western analysis probed with anti-6xHis antibody showed localization of the proteins in both fractions with the prominent signal at 70 kDa in agreement with the size expected for a heterodimeric enzyme

(Figure 4.3D).

4.4.2 Kinetic Characterization of the Recombinant T. brucei A34 Deaminase

Enzyme

32 P-labelled tRNA was used to determine the kinetic parameters of the reaction under steady-state conditions where the concentration of substrate ranged from 0.1 µM to 1.6 µM. A K m value of 0.72 ± 0.06 µM was determined for the native enzyme. When subjected to the same TLC-based assay the recombinant enzyme yielded a K m value of 0.78 ± 0.11 µM and k cat = 0.118 ±

0.006 min -1. Based on this data we determined that the recombinant enzyme had a comparable K m to that of the native enzyme (Figure 4.4A). Electrophoretic mobility shift assays (EMSA) were performed to assess the ability of the recombinant enzyme to bind tRNA. This experiment yielded an apparent K d of

0.15 ± 0.02 µM (Figure 4.4B).

4.4.3 The Pseudo-Active Site is Important in Catalysis

The homodimeric bacterial A34 deaminase active site is composed of three zinc-chelating residues and a proton shuttling glutamate residue (85). The heterodimeric eukaryotic A34 deaminase contains a homologous ADAT2 protein that shares these residues and an ADAT3 protein containing a valine in place of the catalytic glutamate residue in a region we named the pseudo-active site (87).

While the active site found in bacterial ADATA and ADAT2 contains two distinct domains HAE and PCXXC respectively, the pseudo-active site present in ADAT3

96 contains HPV and PCXXC domains. The glutamate in the ADATA/ADAT2 active site is believed to shuttle a proton in the deaminase reaction. This observation has lead researchers to believe that ADAT2 is the catalytic component of the heterodimeric enzyme in contrast to ADAT3 which lacks this glutamate.

Mutations attempting to swap a true active site into Sc ADAT3 and a pseudo- active site into Sc ADAT2 were unable to restore A34 deamination (87). A similar experiment done in the T. brucei A34 deaminase also resulted in an inactive enzyme (Table 4.1B Mutant 2). When wild type Sc ATAD2 and Sc ADAT3 containing a glutamate in the pseudo-active site were incubated with substrate, full deaminase activity was observed (87). Interestingly, a similar experiment done in the T. brucei enzyme resulted in an enzyme unable to catalyze A34 deamination (Table 4.1B-Mutant 6). Site-directed mutagenesis was used to introduce aspartate, alanine, and threonine into the Tb ADAT3 pseudo-active site in place of valine. These mutant enzymes, with the exception of the threonine substitution, were unable deaminate A34 (Table 4.1B-compare Mutant 9, V243D;

Mutant 11, V243A and Mutant 12, V243T). The Val to Thr substitution, although active, showed a 50-fold decrease in activity (Table 4.1B-Mutant 12). While these mutants showed catalytic deficiencies, they were all able to bind their tRNA substrates (Table 4.1B).

97 4.4.4 The C-terminal Region of TbADAT2 is Essential for Binding tRNA

Substrates

The crystal structure of Sa ATADA bound with tRNA Arg furnished insights on tRNA-Tb ADAT2 interactions given the 23% sequence identity of Sa ADATA and Tb ADAT2. A unique C-A base-pair between positions 32 and 38 in tRNA Arg was identified as a key determinant for binding by K106 in the Sa ADATA dimer

(86). Of the T. brucei tRNAs that are A → I edited at position 34, 4 (Arg, Ile, Ser,

Thr) of the 7 substrates contain a potential C32-A38 base-pair. The residue corresponding to K106 in Tb ADAT2 is R159 while ADAT3 has no similar residue.

An R159A mutation in Tb ADAT2 abolished editing activity but no decrease in binding was observed suggesting that R159 in Tb ADAT2 is not required for substrate binding (Table 4.1B Mutant 3). Since eukaryotic ADATs recognize and deaminate seven to eight different substrates while the bacterial enzymes specifically recognize only one tRNA substrate, we hypothesized that there must be specific features lacking in the bacterial enzymes but present in the eukaryotic enzymes that permits recognition of multiple substrates. Primary sequence comparison between bacterial ADATA proteins and the Tb ADAT2 protein revealed a stretch of 10 amino acids (KRKRKDLSVV) at the C-terminus, which we have named the R/K domain. This R/K domain is present in all eukaryotic

ADAT2 proteins but does not exist in bacterial ADATA enzymes (Figure 4.2).

RNA binding domains are commonly found to be rich in positively charged amino acids (142) and the R/K domain may represent a general tRNA binding domain.

98 Tb ADAT2 was analyzed for potential RNA binding residues with the RNABindR program (http://bindr.gdcb.iastate.edu/RNABindR/ ) and the R/K motif showed the highest probability of being a potential RNA binding domain. C-terminal mutants were constructed to determine the role the R/K domain may play in substrate binding and/or deaminase activity. Two mutants were generated, mutant 7 retained all positively charged residues but the five C-terminal residues (DLSVV) were deleted, while mutant 8 had one additional charged residue removed and terminated in KRKR (Table 4.1A). In addition, in mutant 5, all of the last 10 C- terminal residues (KRKRKDLSVV) were deleted (Table 4.1A). When five amino acids were deleted from the C-terminus (mutant 7) the recombinant enzyme showed wild type levels of substrate binding and deaminase activity (Table 4.1B).

The deletion of one additional lysine residue (mutant 8) resulted in a 40-fold decrease in Kcat for adenosine to inosine conversion at position 34 (Table 4.1B).

When all ten of the C-terminal residues were deleted (mutant 5), both substrate binding and deamination activity were abolished (Table 4.1B). Since the T. brucei deaminase is a heterodimer instead of a homodimer like in ADATA, it was thought that residues critical to substrate binding in ADATA not found in ADAT2 might be present in ADAT3s. Furthermore, the substitution through evolution of an ADAT3 subunit for one of the ADATA subunits may be what provides the eukaryotic enzymes the ability to recognize several substrates. The S. aureus crystal structure showed a clear interaction between ADATA R125 and the 5’ phosphate of C40 of the tRNA. While this residue was missing in ADAT2,

99 sequence alignments showed that ADAT3 contained an R335 that might substitute for the role of the missing residue in eukaryotic deaminases (Figure

4.2). An R335A mutation resulted in similar levels of tRNA binding as the wild type but led to a 10-fold decrease in deaminase activity (Table 4.1B Mutant 4).

4.4.5 TbADAT2 Binds the D-arm and T ΨC Arm of tRNA Val

While the Sa ADATa co-crystalized with its tRNA Arg ASL gave insights into where bacterial deaminases interact with their substrates, nothing is known about the eukaryotic ADATs substrate interaction. tRNA Val(GAC) incubated with recombinant Tb ADAT2/ Tb ADAT3 was subjected to T1 and RNAse A digestion

(Figure 4.5). Nucleotides 25 to 31 and 52-54 corresponding to the D and T ΨC arms respectively on the tRNA were protected by nuclease digestion and represented regions bound by the A34 deaminase (Figure 4.5B). When tRNA Val(GAC) was incubated with the recombinant enzyme lacking the R/K domain

(mutant 5) no protection was observed when exposed to T1 or RNAse A nucleases suggesting the importance of the charged C-terminal region of

Tb ADAT2 for substrate binding (Figure 4.5).

4.5 Discussion

Bacterial ADATs recognize and deaminate one solitary tRNA molecule while eukaryotic ADATs recognize either 7 or 8 different tRNA molecules. This level of specificity suggests very stringent recognition between the bacterial

100 enzymes and the substrate. The question to how eukaryotic A34 deaminases recognize 8 substrates while bacterial A34 deaminases recognize only one substrate is yet unanswered. The Sa ADATA crystal structures indicate that the majority of tRNA Arg binding takes place in the active site. Since eukaryotic wobble deaminases must recognize more substrates we hypothesized that there must be a general tRNA binding domain able to accommodate multiple substrates somewhere within the eukaryotic enzymes. Our observation that the C-terminal

R/K domain is present only in eukaryotic enzymes may represent this binding domain. We have proposed a model in which the tRNA binding domain moved away from the active site in eukaryal deaminases to the C-terminal R/K domain.

Thus, this general RNA binding domain may allow for binding and catalysis of additional tRNA substrates than would be tolerated by the bacterial enzymes. Of the C-terminal R/K domain residues, five carry a positive charge and represent a potential RNA binding domain (142). Unlike bacterial ADATs that bind the ASL at the active site, the T. brucei A34 deaminase recognizes an intact tRNA with the

R/K domain at the C-terminus of the ADAT2 protein. Deletion of the R/K domain resulted in an enzyme unable to bind tRNA or catalyze A to I deamination demonstrating the importance of the R/K domain as a major determinant for substrate binding.

In addition to the C-terminal region of ADAT2, we identified potential RNA binding and catalytic residues based on corresponding residues in the Sa ADATA

101 crystal structure. Since the crystal structure of the bacterial enzyme indicates the active site contacts the substrate, we wondered if the active site and pseudo- active site of the T. brucei enzyme may play a role in tRNA binding and catalysis.

We hypothesized that the pseudo-active site of ADAT3 which was believed to be non-catalytic, probably played a role in accommodating the additional substrates, and the ADAT2 protein facilitated the deamination reaction. As expected, the proton shuttling glutamate as well as other residues in the active site were essential for deaminase activity. Interestingly, when mutations were made to these residues, no binding deficiency was observed. Since mutations to the pseudo-active site in the yeast enzyme did not result in decreased deaminase activity, our prediction was that these mutations in the T. brucei enzyme would yield similar results. However our data show that mutations to the pseudo-active site completely abolish deaminase activity and suggest the role of Tb ADAT3 is catalytic as well. We are able to restore deaminase activity when we make more conservative mutations but the k cat is decreased by 40-fold. Interestingly, all of these pseudo-active site mutants bind tRNA substrates with similar efficiencies as the wild type enzyme. Taken together, it appears that the relaxed specificity of the T. brucei enzyme is rooted in the charged C-terminal residues and not the pseudo-active site residues.

Comparison of the footprinting pattern of the wild type enzyme and the C- terminal mutants revealed nucleotide positions in the tRNA backbone important

102 for ADAT2/3 interaction. Interestingly, these included not only regions of contact between the T ΨC and D-arms but also included portions of the acceptor stem.

Although, this type of analysis does not pinpoint residues directly contacted by the enzyme, it does highlight regions of the tRNA where key determinants may lie. It also may explain why a full-length tRNA is required for activity, in that the protected area includes key interaction points in the 3D-tRNA structure. Future mutagenesis analysis will be needed to provide, a more precise view of how these regions of the tRNA are recognized and how they may or may not differ between different tRNA substrates.

We have shown that A to I deamination occurs at the wobble position of tRNAs in T. brucei . Our sensitive assay allowed us to detect A to I deamination in crude extract and follow this deaminase activity as we attempted to purify the native enzyme. In our search for the A to I wobble deaminase found in T. brucei we uncovered many interesting findings. One interesting disparity is the size of the native enzyme versus the size of the recombinantly expressed enzyme. The native enzyme elutes at a larger molecular weight than the recombinant enzyme suggesting that the native enzyme either contains additional protein components or post-translational modifications which may affect its solution structure. There is currently a debate ongoing with the solution structure of the ADARs as well. It is still not conclusively established if the enzyme is a dimer or a monomer. Some studies suggest that the enzyme is a monomer at low concentrations and then

103 binds the substrate mRNA at higher concentrations and dimerizes upon interaction with its substrate (143). Other studies where the RNA binding domains are mutated in ADARs still dimerize suggesting that dimerization is independent of substrate interactions but still may depend on protein concentration (144). Futher investigation is required before any conclusive statements can be made about the in vivo solution structure of the wobble deaminase from T. brucei . It is clear that these accessory components or modifications are not required for A34 deaminase activity because Tb ADAT2 and

Tb ADAT3 are the minimal components required for in vitro deaminase activity.

Interestingly, when Tb ADAT2 and Tb ADAT3 are expressed separately no deaminase activity is observed (chapter 3) and deaminase activity can only be reconstituted when the proteins are co-expressed.

To investigate if the recombinant enzyme represented a fully active deaminase steady-state kinetic techniques were employed. Steady-state kinetic analysis yielded similar values for the recombinant and partially purified native deaminases supporting the claim that we have identified the minimal but fully active components of the T. brucei A34 deaminase. The partially purified T. brucei native enzyme exhibited a K m value of 0.72 ± 0.06 uM while the recombinant enzyme has a K m value of 0.78 ± 0.11 uM and Kcat = 0.118 ± 0.006

-1 -1 min . The bacterial ADATA enzyme yielded kcat = 13 ± 1 min , K m = 0.83 ± 0.22 uM (101). A partially purified yeast ADAT2/3 exhibited a much lower K m value

104 (2.3 ± 0.4 nM) when assayed with tRNA Ser (104). Therefore, while the subunit composition of the T. brucei deaminase resembles the heterodimeric eukaryotic deaminase, the K m is more similar to the bacterial enzyme. This is especially interesting because the substrate used for the bacterial enzyme is an ASL instead of a full tRNA. It was thought that the T. brucei deaminase would resemble other eukaryotic deaminases because it recognizes multiple tRNA substrates, like other eukaryotic ADATs and cannot recognize an ASL like bacterial ADATs

In summary we have provided the first steady-state kinetic characterization of a recombinant eukaryotic A34 tRNA deaminase. Our results demonstrate the importance of the pseudo-active site, previously thought to merely play a structural role, in catalysis of A to I deamination at the wobble position of tRNAs. In addition, we have shown the critical importance of the C- terminal region of Tb ADAT2 in substrate binding and A to I deamination.

Futhermore, we believe descriptions of the activity and unique substrate recognition of this ADAT provide crucial information of fundamental importance for all eukaryotic deaminases.

105

Peak = 218K

- P 24 25 26 27 28 29 30 31 32 33 34 35

Figure 4.1. The native tRNAA34 →I34 L. tarentolae and T. brucei enzymes have molecular masses of 218 kDa. A. tRNAVal (AAC) was labeled by in vitro transcription in the presence of [ α-32P] ATP. The labeled transcript was incubated with Superdex 200 precolumn (P) partially purified L. tarentolae fractions (#) followed by nuclease P1 digestion and separated by TLC. Cold 5’AMP (pA) and 5’IMP (pI) were used as markers to show the locations of the nucleotides respectively. A peak of activity was observed at fractions corresponding to 218 kDa.

106

(158) 158 170 180 190 200 210 220 230 SaTadA (1) ------MTNDIYFMTLAIEEAKKAAQLGEVPIGAIITK------D Tb Tad2p (2) VQDTGKDTNLKGTAEANESVVYCDVFMQAALKEATCALEEGEVPVGCVLVKADSSTAAQAQAGDDLAL----Q Ec TadA (1) ----MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVH------N AaTadA (1) ------MGKEYFLKVALREAKRAFEKGEVPVGAIIVK------E Sc Tad2p (1) ------MQHIKHMRTAVRLARYALDHDETPVACIFVHTP------T Rr Tad2p (1) ------MLDKEANFNNFFMEQALKQAKIAFDKNEVPVGAVVVDRL------N Mm Tad2p (1) -MEEKVESTTTPDGPCVVSVQETEKWMEEAMRMAKEALENIEVPVGCLMVY------N Gg Tad2p (1) ------MEDEAAWMERAFSMAQEALEAGEVPVGCLLVY------D TbTad3p(158) WPFATPKPRAPTQLSECEMGSIQRIFRTVVMPLAKRLRTDETLGIAAVLVDPSDGYRVLVSSGEEHALKRGNS Consensus(158) FM ALRLAK A E EVPVGAVLV * (231) 231 240 250 260 270 280 290 303 SaTadA (34) DEVIARAHNLRETLQQ--PTAHAEHIAIERAAKVLGSWR------LEGCTLYVTLEPCVMCAG Tb Tad2p (71) KLIVARGRNATNRKGH--ALAHAEFVAVEELLRQATAGTSENIGGGGNCGAVSQDLADYVLYVVVEPCIMCAA Ec TadA (49) NRVIGEGWNRPIGRHD--PTAHAEIMALRQGGLVMQNYR------LIDATLYVTLEPCVMCAG AaTadA (33) GEIISKAHNSVEELKD--PTAHAEMLAIKEACRRLNTKY------LEGCELYVTLEPCIMCSY Sc Tad2p (35) GQVMAYGMNDTNKSLT--GVAHAEFMGIDQIKAMLGSRG------VVDVFKDITLYVTVEPCIMCAS Rr Tad2p (41) QKIIASTHNNTEEKNN--ALYHAEIIAINEACNLISSKN------LNDYDIYVTLEPCAMCAA Mm Tad2p (52) NEVVGKGRNEVNQTKN--ATRHAEMVAIDQVLDWCHQHGQSPS------TVFEHTVLYVTVEPCIMCAA Gg Tad2p (34) GAAIGKGRNEVNETKN--ATRHAEMVAIDQVLEWCQQHKKDHE------EVFSHSVLYVTVEPCIMCAA TbTad3p(231) AACLGYVSNSGCRKSNRVVLDHPVTFVLKEVTRKQCKDREVEG------DASYLANGMDMFVSHEPCVMCSM Consensus(231) VIAKG N N ATAHAEMVAID V L L LYVTLEPCIMCAA

(304) 304 310 320 330 340 350 360 370 387 SaTadA (89) TIVMSRIPRVVYGADDPKGGCSGSLMNLLQQ------SNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN------Tb Tad2p(142) MLLYNRVRKVYFGCTNPRFGGNGTVLSVHNSYKGCSGEDAALIGYESCGGYKAEEAVVLLQQFYRRENTNAPLGKRKRKDLSVV Ec TadA(104) AMIHSRIGRVVFGARDAKTGAAGSLMDVLHH------PGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD-- AaTadA (88) ALVLSRIEKVIFSALDKKHGGVVSVFNILDE------PTLNHRVKWEYYPLEEAS-ELLSEFFKKLRNNII------Sc Tad2p (94) ALKQLDIGKVVFGCGNERFGGNGTVLSVNHDTCTLVPKNNSAAGYESIPGILRKEAIMLLRYFYVRQNERAPKPRSKSDRVLDK Rr Tad2p (96) AIAHSRLKRLFYGASDSKHGAIESNLRYFNS------SACFHRPEIYSGILAEDSGLLMKEFFKRIRTVISSHRMK------Mm Tad2p(113) ALRLMKIPLVVYGCQNERFGGCGSVLNIASADL-----PNTGRPFQCIPGYRAEEAVELLKTFYKQENPNAPKSKVRKKDCQKS Gg Tad2p (95) ALRLMKIPRVVYGCRNERFGGCGSVLSISSDDI-----VDTGEPFECIAGYRAKDAVEMLKAFYRQENPNAPKSKVRKKDNRK- TbTad3p(297) ALVHSRVRRVFYCFPNPVHGGLGSTVSIHAIQ-----ELNHHFRVFRCDSRWLSDPEGVSSDHDNPYWEDLTVP------Consensus(304) ALV SRI RVVYG N K GG GSVL I R E G LAEEA LL FFK N K K

Figure 4.2. Multiple sequence alignment of tRNA-specific adenosine deaminases. The enzymes are denoted as follows: T. brucei ( Tb ), S. aureus (Sa ), E. coli ( Ec ), S. cerevisiae ( Sc ), Rickettsia rickettsii (Rr) , Mus musculus (Mm ), Gallus gallus ( Gg ). The first 157 amino acids of TbADAT3 were deleted for clarity of presentation as they do not align with the other deaminases. The highly conserved residues are boxed in yellow. Zinc chelating residues are denoted by red arrows, the proton shuttling glutamate residue or residue of the pseudo active site in Tb ADAT3 is denoted by a green asterix, the residue shown to be important in recognition of the unique C-A base pair is marked by a purple arrow, the residue found in common with bacterial ADATAs and with Tb ADAT3 is marked with a blue arrow, and the RK domain is denoted by a brown bar.

107 A. Time (min) 0 10 20 30 40 50 60 B.

A to I Timecourse pI 1.2 1 0.8 0.6

tRNA) 0.4 0.2 pA I/mol Ito(mol A 0 0 10 20 30 40 50 60 Time (min) mol I/ mol tRNA 0 0.36 0.6 0.75 0.94 0.97 1.03 C. D. (Fra ction #) NE 31 33 35 36 37 38 39 41 43 kDa 30 31 32 33 34 35 36 37 38 39 40 41 42 50 37 25

29 30 31 33 35 36 37 38 39 40 41 42

pI 50 37 pA 25

mol I/ mol tRNA 0 .03 .05 .16 .22 .26 .24 .2 .12 .06

Figure 4.3. Recombinant tRNAA34 →I34 L. tarentolae and T. brucei enzymes have molecular masses of 72 kDa. A. A time course was performed where recombinant ADAT2/ADAT3 was incubated with radio-labeled tRNA (AAC) followed by digestion with P1 nuclease and separated by TLC. pI and pA denote the location of cold 5’- IMP and 5’-AMP markers respectively as seen by UV shadowing. A 40 minute incubation resulted in maximal editing of radio-labeled tRNA (AAC) . B. Graphical representation of data from panel A. C. Recombinant ADAT2/ADAT3 were subjected to Superdex 200 gel filtration chromatography. Fractions were assayed for I formation with a peak activity found in fractions corresponding to molecular masses of 72 kDa (fraction 37). D. SDS PAGE (upper panel) and Western analysis (lower panel) of the fractions collected from the gel filtration purification. The fraction numbers are on the top of the gels. A protein marker was used to determine the molecular masses of the proteins on the gels.

108

Figure 4.4. Kinetic and binding analysis of recombinant ADAT2/ADAT3. A. Steady state kinetics were used to determine the kinetic parameters of ADAT2/ADAT3. The left panel is a representative example where radio-labeled substrate was increased until a saturating level was achieved. The right panel represents the average Michaelis-Menten curve of three independent experiments for both the partially purified native A34 deaminase (native) and the recombinant ADAT2/ADAT3 enzyme (recombinant). A K m value of 0.72 ± 0.06 uM were determined for the native enzyme and a K m value of 0.78 ± 0.11 uM was determined for the recombinant enzyme. B. An electrophoretic mobility shift assay was used to determine apparent K ds for the recombinant enzymes and subsequent mutants. The top left panel is a representative example of increasing concentrations of recombinant ADAT2/ADAT3 enzymes incubated with radio- labeled tRNA (GAC) . tRNA only (-) shows the location of the unshifted band. The bottom left panel is a competition assay where increasing concentrations of cold tRNA (GAC) were incubated with the radio-labeled tRNA (GAC) -ADAT2/ADAT3 complex. The numbers above represent the molar ratio of unlabled to labeled tRNA (GAC) ranging from an equal molar ratio (1) to 128 fold excess of unlabeled to labeled tRNA (GAC) . The right panel is a binding curve generated by Sigma Plot where an apparent K d of 0.15 uM was determined.

109

Figure 4.4. Kinetic and binding analysis of recombinant ADAT2/ADAT3

110

Figure 4.5 Tb ADAT2 Binds the D-arm and T ΨC Arm of tRNA Val . A. The first lane (far right) represents the tRNA molecule that has been digested so a ladder has been created to see each individual nucleotide. The next lane is the tRNA digested with T1 nuclease and has no protein added. The next three lanes are the same but with increasing amounts of protein added. A protection is seen in 2 distinct areas (24-31) and (52-54). The next three lanes show no protection when mutant 5 (binding deficient mutant) is incubated with tRNA and digested with T1 nuclease. B. tRNA val shown with arrows indicating where protection from T1 nuclease digestion was observed.

111

Table 4.1 Schematic and kinetic properties of pseudo-active site and deletion mutants. A. Blue boxes represent Tb ADAT2 while yellow boxes represent Tb ADAT3 proteins. Red text denotes amino acid changes from the wild type enzyme (top). The corresponding mutant numbers are listed at the right. Tb ADAT2 C-terminal deletion mutants are shown as truncated blue boxes. The residues that have been deleted are denoted by the absence of the amino acid letters in the blue boxes. B. Kinetic data for active or binding mutants. Mutants 2, 10, 11, and 13 are not shown because they were not able to catalyze A to I deamination and we were not able to determine apparent K ds for these mutants. 4 µg of purified mutants 8 and 12, 600 ng of mutant 4, 150 ng of mutant 7, and 125 ng of wild type were used in the steady state kinetic experiments.

112

A. HAER KRKRKDLSVV HPV R Wild type

Pseudo-active and active site mutants

HA A R KRKRKDLSVV HPV R Mutant 1

R HAE R Mutant 2 HA A KRKRKDLSVV HAEA KRKRKDLSVV HPV R Mutant 3 HAER KRKRKDLSVV HPV A Mutant 4 HAER KRKRKDLSVV HAE R Mutant 6 HAER KRKRKDLSVV HP D R Mutant 9 HAER KRKRKDLSVV HP L R Mutant 10 HAER KRKRKDLSVV HP A R Mutant 11 HAER KRKRKDLSVV HP T R Mutant 12

Deletion mutants

HAER KRKRK HPV R Mutant 7

HAE KRKR HPV R Mutant 8 R Mutant 5 HAE R HPV R

Construct Km kcat k cat /K m Apparent Kd

(uM) (min -1) (uM -1 min -1 ) (uM)

Wild ty pe 0.78 ± 0.11 0.118 ± 0.0061 0.151 0.150

Pseudo-active site and active site m utants

Mutant 1 N/ A N/ A N /A 0.147

Mutant 3 N/ A N/ A N /A 0.344

Mutant 4 0.19 ± 0.04 0.012 ± 0.0050 0.063 0.311

Mutant 6 N/ A N/ A N /A 0.193

Mutant 9 N/ A N/ A N /A 0.177

Mutant 12 0.91 ± 0.13 0.003 ±. 0002 0.003 0.380

Binding mutants

Mutant 5 N/ A N/ A N /A N/ A

Mutant 7 0.60 ± 0.21 0.178 ±. 0280 0.297 0.150 Mutant 8 0.66 ± 0.11 0.003 ±. 0004 0.005 N/ A

Table 4.1 Schematic and kinetic properties of pseudo-active site and deletion mutants.

113

CHAPTER 5

CONCLUSION

RNA editing has been observed in all domains of life. While the types of

RNA editing seem to vary between domains of life, all editing events result in an

RNA sequence different from that predicted by the original DNA message. Since the original discovery of RNA editing, a great deal of effort has been put forth to understand these editing events. In some cases, the role of RNA editing seems to be a regulatory one. Therefore these editing events complement other cellular processes that allow for regulatory expression of protein isoforms. One example of this occurs in mammals where the intestinal ApoB exists in two forms, a full- length protein and a shorter version. Intracellular carbohydrate levels dictate if the long or short version should be synthesized. In this way RNA editing regulates expression and maintains a proper balance of the long and short versions of the ApoB proteins. In addition to this regulatory role, RNA editing of tRNAs at the wobble position results in the degeneracy of the genetic code. A to I editing at the wobble position of some tRNAs results in tRNAs that can recognize multiple codons. In our search for A to I editing in trypanosomes we discovered a unique C to U editing event in tRNA Thr(AGU) . This is the first example of two editing

114 events inside the same anticodon stem loop. Furthermore our discovery that C to

U editing stimulates A to I editing indicated an interdependence between the two editing sites. C to U editing in tRNAs had been observed at positions 34 and 35 but never outside of the anticodon. This discovery indicates that C to U editing in tRNAs is more widespread that previously thought and is not restricted to the anticodon.

The subunit composition of the A to I editing enzymes differs between different domains of life and perhaps from species to species. In bacteria, a 50 kDa homodimeric enzyme is responsible for I formation at the wobble position in tRNA Arg . In yeast, a 75 kDa heterodimeric complex composed of ADAT2 and

ADAT3 proteins can edit the wobble position of seven tRNAs. Little is known about the subunit composition in other eukaryotes, but there has been a report of a larger complex of about 200k Da in rabbit reticulocytes. When we use gel filtration chromatography to determine the size of the native enzyme from T. brucei , A to I editing is observed in fractions corresponding to proteins with molecular masses of 210 kDa. Recombinant enzymes co-expressed in E. coli were subjected to gel filtration chromatography and eluted with proteins corresponding to 70 kDa as expected as well at proteins in the null volume of the column. The proteins eluting at 70 kDa are able to deaminate tRNA at the wobble position and represent the minimal components necessary for this deamination reaction. It is unclear why the native enzyme elutes differently than the

115 recombinantly expressed proteins from the gel filtration column and suggests that the enzyme forms a higher order complex in vivo . When the recombinant enzymes are concentrated to 2 mg/ml, a major peak is observed in the null volume of the gel filtration column and may suggest concentration-dependent oligomerization where at high concentrations a higher order complex is observed.

Interestingly, antibodies generated to Tb ADAT2 detect proteins in cell extracts that migrate at 75 kDa when subjected to SDS PAGE in addition to the 25 kDa proteins and we can not explain this observation. Native purification of the A to I wobble deaminase from T. brucei resulted in 9 prominent bands on a coomassie stained SDS PAGE gel. Two of the bands correspond to Tb ADAT2 and

Tb ADAT3 respectively. Another band corresponds to a 75 kDa protein which most likely represents Tb ADAT2 as well.

In an attempt to isolate the native complex, a TAP strategy was conducted using Tb ADAT2 as bait. Tb ADAT2 was chosen as the bait protein because immunoprecipitation and RNAi experiments had shown Tb ADAT2 to be an essential protein in the T. brucei A to I deaminase complex. While the TAP approach did not result in the isolation of an active complex, it did lead to the discovery of the importance of a reduced environment for the deamination reaction. Titrations of other compounds revealed that 0 mM KCl, 0.5 mM MgSO 4, and 1 mM DTT constitute the ideal concentrations of the reactants in the deaminase reaction mix.

116

Co-expression of Tb ADAT2 and Tb ADAT3 resulted in a fully active complex that behaved very similarly to the native enzyme. Site directed mutagenesis was employed to determine the role of the pseudo-active site of Tb ADAT3 in A34 →

I34 tRNA deaminase activity. Our data shows that the psuedo-active site of

TbADAT3 plays an essential role in catalysis in T. brucei . This observation was surprising because similar mutations in the yeast A34 → I34 tRNA deaminase had no noticable effect on catalysis, and it was assumed that ADAT3 from all eukarotic organisms would not play a role in catalysis.

The greatest differences between bacterial A34 → I34 tRNA deaminase enzymes and eukaryotic enzymes is how the enzymes interact with their substrates. The bacterial enzymes can recognize only the anticodon stem loop of the substrate while eukaryotic enzymes require a full tRNA for catalysis. In addition, the bacterial enzyme only deaminates one tRNA while the eukaryotic enzymes recognizes seven or eight substrates. We reasoned that the decreased specificity of the eukaryotic enzymes might be the result of a general RNA binding domain present in eukaryotic enzymes but not in bacterial enzymes. We identified and deleted a possible RNA binding region at the C-terminus of

Tb ADAT2 and found that when this region was deleted, RNA binding was abolished. We also found that the D-arm as well as the T ΨC arm of the tRNA is contacted by the A34 → I34 tRNA deaminase enzyme. Crystal structures show that the bacterial enzymes contact the tRNA at the anticodon stem loop almost

117 exclusively with the active site. These results have given many answers but more importantly they represent preliminary data for more interesting questions. The future direction of this project seems poised to answer what role Tb ADAT3 plays in substrate specificity. More specifically does the pseudo-active site play a role in substrate binding? The interesting observation that the native enzyme appears larger than the recombinant enzyme raises the question as to the identity of the other factors that are present in the native enzyme. Once the identity of these additional factors have been elucidated, will the addition of these other factors allow this enzyme to edit C32 → U32 in tRNA Thr ?

118

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