Characterizing the Endoribonuclease Activity of APE1 Wan Cheol Kim BSc, Simon Fraser University, 2007 Thesis Submitted in Partial Fulfillment of The Requirements for the Degree of Master of Science In Mathematical, Computer, and Physical Sciences (Chemistry) The University of Northern British Columbia June 2009 © Wan Cheol Kim, 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-60814-2 Our file Notre reference ISBN: 978-0-494-60814-2 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada Abstract Recent evidence shows that mRNA degradation is a major control point in the regulation of gene expression. APE1, apurinic/apyrimidinic DNA endonuclease 1, has recently been discovered to possess an endoribonuclease activity against c-myc messenger RNA (mRNA) in vitro. We showed that APE1 shares catalytic residues to cleave both RNA and AP-DNA. Our results also suggested that the roles of active site residues in each reaction are not entirely identical. A consensus RNA secondary structures and sequences that are preferentially cleaved by APE1 were determined. Our results revealed that APE1 has a preference for cleaving the single stranded regions or weakly base paired regions of the RNA. Also, preferred sequences of cleavage were determined to be UA, UG, and CA. When APE1 was knocked down in HeLa cells, an increased level of c-myc mRNA and half-life was detected. This demonstrated that APE1 is involved in the regulation of c-myc mRNA turnover. TABLE OF CONTENTS Abstract i Table of Contents iii List of Tables vi List of Figures viii Acknowledgements xi Candidate's Publications Relevant to this Thesis xii Reference List xiii CHAPTER 1 - Introduction 1.1 Mechanism of messenger RNA (mRNA) degradation 1 1.2 Role of trans-acting factors in mRNA stability and turnover 4 1.2.1 Non-coding RNA (miRNA) 4 1.2.2 RNA-binding proteins (RBPs) 5 1.2.3 Ribonucleases (RNases) 7 1.3 Ribonucleases (RNases) associated with cancer 7 1.3.1 RNases from conventional mRNA decay pathway: CCR4b, PARN, XRN1 9 1.3.2 RNases from specific contexts: RNase L, IRE1, and PMR1 11 1.3.3 RNases from miRNA pathway: Drosha, DICER, Argonaute 2 18 1.3.4 RNases of the nucleus/cytoplasm: Angiogenin, G3BP, and FEN1 22 1.4 Apurinic/apyrimidinic endonuclease 1 (APE1) as an endoribonuclease 27 1.4.1 Possible role of APE1 in cancer 27 1.4.2 AbasicDNA incision activity of APE1 30 1.4.3 Variants of APE1 identified in human populations 34 1.4.4 3'-5'Exonuclease activity of APE 1 35 1.5 Research objectives 37 iii CHAPTER 2 - Identifying the critical amino acid residues for endoribonuclease activity of APE1 and characterizing its biochemical properties 2.1 Methodology 40 2.1.1 Reagents and buffer preparation 40 2.1.2 PCR generation of linear templates 40 2.1.3 Standard phenol/chloroform extraction and ethanol precipitation 42 2.1.4 Generation of unlabeled RNA substrates 42 2.1.5 Generation of 5'-radiolabeled RNA substrates 44 2.1.6 Generation of 5'-radiolabeled abasic DNA substrate 45 2.1.7 SDS-PAGE analysis on recombinant human APE1 47 2.1.8 Endoribonuclease assays using 5'-radiolabeled RNA 49 2.1.9 Abasic DNA endonuclease assays using 5'-radiolabeled abasic DNA 50 2.1.10 Dialysis of APE1 50 2.1.11 Determination of the nature of 3'-end of the RNA cleavage product 51 2.1.12 Electrophoretic mobility shit assay (EMSA) 52 2.2 Results and discussion 55 2.2.1 Generation of unlabeled and 5'-radiolabeled RNA 55 2.2.2 Identification of essential residues for RNA incision activity of APE1 56 2.2.3 Assessing the RNA-binding abilities of APE1 structural mutants 65 2.2.4 RNA incision activity of the population variants of APE1 66 2.2.5 Assessing the RNA-binding abilities of APE1 population variants 72 2.2.6 Possible mechanism of RNA incision by APE1 74 2.2.7 The effect of divalent metal ions in APE1 RNA catalysis 77 2.2.8 The effect of RNase inhibitors on APE1 activity 79 CHAPTER 3 - Establishing the RNA structure and sequence cleaved by APE1 3.1 Methodology 82 3.1.1 Plasmid linearization and PCR generation of linear templates 82 3.1.2 Utilization of enzyme probes for RNA secondary structure determination 84 3.1.3 In vitro effects of APE1 RNase activity on DICER product formation 86 3.2 Results and discussion 88 3.2.1 Generation of unlabeled and 5'-radiolabeled RNA 88 3.2.2 APE1 cleaves RNA components of SARS-corona virus at specific sites.. .89 3.2.3 APE1 cleaves pri-miR-21 and pri-miR-lOb at specific sites 94 3.2.4 Pre-treatment with APE1 interferes with processing of pre-miR-lOb and pre-miR-21 by DICER 98 IV CHAPTER 4 - Assessing the endoribonuclease activity of APE1 in biological systems 4.1 Methodology 103 4.1.1 Assessing the steady-state c-myc mRNA level in APE1 knocked down cells 103 4.1.2 Assessing c-myc mRNA half-life in APE1 knocked down cells 110 4.1.3 Preparation of LB-antibiotic plates for Origami cells 110 4.1.4 Preparation of Origami competent cells 110 4.1.5 Transformation and induction of protein expression by IPTG Ill 4.2 Results and discussion 112 4.2.1 APE1 knock down in HeLa cells up-regulates c-myc mRNA expression 112 4.2.2 Optimization of monitoring c-myc mRNA stability in HeLa cells 116 4.2.3 Knock down of APE1 in HeLa cells stabilizes c-myc mRNA 118 4.2.4 Origami cell transformation with pGEX4T3-Thioredoxin A andRNaseA 119 4.2.5 Induction of pET15b-WT-APEl in Origami cells is toxic 121 CHAPTER 5 - General discussion 5.1 Role of post-transcriptional control in cancer 125 5.2 Significance of identifying and studying RNases 127 5.3 Benefits of studying RNases in health and therapeutics 128 5.4 Identification of active site residues critical for RNA-cleaving activity of APE 1 129 5.5 Role of metal ions in APE1 RNA-cleaving activity 135 5.6 Role of N-terminus in APE1 136 5.7 RNA secondary structure and sequences preferentially cleaved by APE 1 139 5.8 Assessment of biological significance of RNA-cleaving activity of APE1 141 5.9 Concluding remarks 144 v List of Tables Table 1: Summary of abasic DNA incision activities of active site mutants (or structural mutants) 32 Table 2: Summary of dissociation constants and abasic DNA incision activities of APE1 population variants (Hadi et al. 2000) 35 Table 3: Composition of Reagents used in this chapter 40 Table 4: List of RNA substrates used in assessing the RNA-cleaving activity of APE1 and its mutants 41 Table 5: PCR primers and their sequences used in the generation of linear DNA template for c-myc (1705-1792) CRD RNA by in-vitro RNA transcription 41 Table 6: Composition of reagents for SDS-PAGE 48 Table 7: SDS-PAGE gel staining & destaining reagents 49 Table 8: The composition of reagents used in EMSA experiments 54 Table 9: Summary of fold reduction in WT APE 1 and structural mutants 59 Table 10: Summary of RNA incision activity of human population variants of APE 1 on c-myc-1705 -1792 CRD RNA and Oligo IB 68 Table 11: List of RNA substrates generated and their cDNA sequences 83 Table 12: List of primers and their sequences used in the generation of cDNA templates for in-vitro RNA transcription 84 Table 13: Sequences of Scrambled-Negative DICER substrate double stranded RNAi (dsRNAi) and dsRNAi against APE 1 mRNA 104 Table 14: Composition of the reagents used in Western blot 107 Table 15: Primer sequences used in qRT-PCR 109 Table 16: Sample table of CT values generated from qRT-PCR 116 vi Table 17: Summary of the results of Origami cell transformations using pET15b based plasmids coding for APE1 mutants and their RNA cleaving activities in vitro 123 vn List of Figures Figure 1: A schematic view of mRNA decay 3 Figure 2: A schematic representation on the modes of action for trans-acting factors..