Towards identifying New Human Ribonucleases that Cleave microRNA Using a High-Throughput Method by Suhua Ye B.Sc., University of Ottawa, 2007 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MATHEMATICAL, COMPUTER AND PHYSICAL SCIENCE (CHEMISTRY) THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA July 2011 © Suhua Ye, 2011 1+1 Library and Archives Canada Bibliotheque et Archives Canada Published Heritage Branch Direction du Patrimoine de I'edition 395 Wellington Street Ottawa ON K1A0N4 Canada 395, rue Wellington Ottawa ON K1A 0N4 Canada Your file Votre reference ISBN: 978-0-494-87574-2 Our file Notre reference ISBN: 978-0-494-87574-2 NOTICE: AVIS: The author has granted a non­ exclusive license allowing Library and Archives Canada to reproduce, publish, archive, preserve, conserve, communicate to the public by telecommunication or on the Internet, loan, distrbute and sell theses worldwide, for commercial or non­ commercial purposes, in microform, paper, electronic and/or any other formats. L'auteur a accorde une licence non exclusive permettant a la Bibliotheque et Archives Canada de reproduire, publier, archiver, sauvegarder, conserver, transmettre au public par telecommunication ou par I'lnternet, preter, distribuer et vendre des theses partout dans le monde, a des fins commerciales ou autres, sur support microforme, papier, electronique et/ou autres formats. The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. L'auteur conserve la propriete du droit d'auteur et des droits moraux qui protege cette these. Ni la these ni des extraits substantiels de celle-ci ne doivent etre imprimes ou autrement reproduits sans son autorisation. In compliance with the Canadian Privacy Act some supporting forms may have been removed from this thesis. Conformement a la loi canadienne sur la protection de la vie privee, quelques formulaires secondaires ont ete enleves de cette these. While these forms may be included in the document page count, their removal does not represent any loss of content from the thesis. Bien que ces formulaires aient inclus dans la pagination, il n'y aura aucun contenu manquant. Canada Abstract Endoribonucleases were once thought of as only being key enzymes responsible for the degradation of prokaryotic mRNAs. They are now believed to play critical role in initiating eukaryotic/mammalian RNA decay and hence RNA abundance. To date, only few mammalian endoribonucleases that cleaved mRNA have been identified and studied. It is unknown if mammalian endoribonucleases can control microRNA (miRNA) abundance. The major goal of this MSc thesis was to develop a high-throughput method to identify new human endoribonucleases that cleave miR155. The first objective of this thesis was to develop a high-throughput method to express and purify human recombinant proteins from the hExl human fetal brain library. This was followed by development of a high-throughput fluorescence-based assay to screen purified recombinant proteins for activity against fluorogenic miR155 substrate. Through a series of optimization experiments, we have successfully established a high-throughput procedure and the criteria in selecting a preliminary list of positive candidates. I Table of Contents Abstract I Table of Contents II List of Tables V List of Figures VI Acknowledgements VIII Candidate's Publications Relevant to this Thesis IX Reference List X Chapter 1 - Introduction 1.1 Mechanisms in the control of Eukaryotic gene expression 1 1.2 Key players in the control of RNA degradation 2 1.2.1 Enzymes - decapping enzymes, exoribonucleases, endoribonucleases and RBP..2 1.2.2 Cellular structures involved in mRNA degradation 6 1.2.3 non-coding RNA 8 1.3 Significance of microRNA in the control of gene expression 1.3.1 MicroRNA Biogenesis 1.3.2 Regulatory roles of microRNA 1.3.3 Role of microRNA in cancer 1.3.4 Regulation of microRNA abundance 8 9 9 10 10 1.4 Human endoribonuclease 1.4.1 Human endoribonuclease implicated in cancers 1.4.2 Human endoribonucleases that belong to a complex 1.4.3 Other human endoribonucleases 1.4.4 Summary 14 15 20 21 22 1.5 Research objectives 23 Chapter 2 - Development of a High-Throughput System to express and purify a library of recombinant proteins 2.1 Introduction 2.1.1 Protein Functional Screen 25 25 II 2.1.2 cDNA expression library 2.1.3 Expression and purification of sets 512A, 517A, 523C, 525A and 568D 25 27 2.2 Methodology 2.2.1 Protein expression using two different sets of media 2.2.2 HTS induction optimization 2.2.3 Methods to lyse bacterial cells for use in HTS and individual cell growth 2.2.4 HTS purification optimization 2.2.5 HTS Dialysis Optimization 27 27 29 30 31 34 2.3 Results and discussions 2.3.1 Comparison of protein expression in LB and SB 2.3.2 Optimizing HTS bacterial induction step 2.3.3 Choice of cell lysis method 2.3.4 Optimizing HTS protein purification step 2.3.5 Using Nanodrop spectrophotometer to estimate imidazole concentration 2.3.6 Assessing the minimum time required for dialysis 2.3.7 Determining the amount of protein loss during dialysis 35 35 36 37 38 39 40 42 Chapter 3 - High-Throughput Functional Screen 3.1 Introduction 44 3.2 Methodology 3.2.1 HTS 96 parallel protein expression and purification 3.2.2 HTS dialysis and quantification 3.2.3 HTS protein functional screen 45 45 46 47 3.3 Results and discussions 3.3.1 Protein concentrations 3.3.2 HTS fluorescence-based assay 50 50 50 Chapter 4 - Post Screening Analysis of the Positive Hits 4.1 Introduction 4.1.1 Rationale for carrying out secondary screen 4.1.2 Electrophoretic assay to characterize positive hits 58 58 59 4.2 Methodology 4.2.1 Induction test of positive clones 4.2.2 Expression and purification of positive clones 4.2.3 Secondary screen using fluorescence-based assay 4.2.4 Identity of Positive Hits 4.2.5 Dephosphorylation of miR155 substrates (13nt and 24 nt) for use in electrophoretic assay 61 61 62 64 64 66 III 4.2.6 Standard phenol/chloroform extraction and ethanol precipitation 4.2.7 Production of 5' radiolabeled substrates for use in electrophoretic assay 4.2.8 Assessing the endoribonucleolytic activity of positive hits 4.2.9 Determination of RNA cleavage products 4.2.10 Exoribonucleolytic Reactions 4.2.113'- end radiolabeling of substrates 4.2.12 Reactions with 3'- radiolabeled substrates 4.2.13 Sample loading and gel running 67 68 69 70 71 72 73 73 4.3 Results and discussions 74 4.3.1 Induction test 74 4.3.2 Purity of recombinant proteins 76 4.3.3. Secondary screen using fluorescence-based assay 78 4.3.4 Identity and Integrity of Clones 79 4.3.5 Characterization of candidate enzymes using electrophoretic endoribonuclease assay 80 Chapter 5 - General discussion 5.1 General overview 95 5.2 Significance of finding endoribonucleases that degrade microRNA 95 5.3 The HTS functional screen 96 5.4 From primary screen to secondary screen 98 5.5 Establishment of HTS procedure 101 5.6 Future directions 104 5.7 Concluding Remarks 104 IV List of Tables Table 1. Ingredients of broth used for E.coli culture in this project 28 Table 2. List of reagents used in SDS-PAGE 31 Table 3. Reagents used in HTS protein generation 32 Table 4. Purified protein yields comparison of proteins induced with IPTG for 6 hours at 37°C and 16 hours at 16°C 37 Table 5. Purified protein sample concentrations (mg/mL) of before (A) and after (B) dialysis, and percentage (%) decrease of concentration (C) 42 Table 6. List of substrates used in the fluorescence-based assay and their sequences 49 Table 7. Components in fluorescence-based assay reaction 50 Table 8. Comparison of positive lists from two repeated screens (Trial 1 and 2) 56 Table 9. List of clones exhibiting specificity toward miR155 substrate 57 Table 10. List of reagents used in gravity flow purification 63 Table 11. List of primers and their sequences used in cDNA sequencing 66 Table 12. Comparison of contents in each reaction in the endoribonucleolytic reaction assay. 71 Table 13. Components of reagents used in 3'-radiolabeling procedure 73 Table 14. Inducibility of positive hits that underwent induction test and their ranking 76 Table 15. Identity, protein size and expected protein size of positive clones 80 Table 16. Comparison between results from primary and secondary screen 99 V List of Figures Figure 1. A schematic diagram of conventional exoribonucleolytic decay pathway and hypothetical endoribonucleolytic decay pathway that control stability of mRNA. 1 Figure 2. Schematic diagram summarizing key factors regulating animal microRNA turnover 14 Figure 3. Map of the pQE30NST plasmid 26 Figure 4. Western blots showing the expression of His-tagged proteins in the cells grown in different media 36 Figure 5. SDS-PAGEs showing different fractions of 568F20 in the purification process after cell lysis with three different methods 38 Figure 6. Western blots showing His-tagged proteins from set 517A in cell lysate and after dialysis 39 Figure 7. Position of absorbance peak shifting to the right on wavelength spectrum (increasing in wavelength) as imidazole concentration in the sample increases. 40 Figure 8. Determining the time required to decrease imidazole concentration during dialysis. 41 Figure 9. Principle of fluorescence-based assay used in high-throughput screen 44 Figure 10. Secondary structure of the miR155 substrate used in the fluorescence-based assay, which was designed based on one of the mature miR155 secondary structures 48 Figure 11. Secondary structures of substrates used as controls 48 Figure 12. Kinetics curves of enzymes of diffferent activities 52 Figure 13. Representative results of fluorescence-based assay to screen for activity against the miR155 substrate and Oligo UA 54 VI Figure 14. Secondary structures of electrophoretic assay substrates miR155_13nt and miR155_24nt 60 Figure 15. Secondary structure of electrophoretic assay control substrate Oligo CU-aR.. ..61 Figure 16. SDS-PAGE showing inducibility of positive hit from set 517A 75 Figure 17. Purity of recombinant proteins after purification and dialysis 77 Figure 18. Secondary screen for endoribonuclease activity 79 Figure 19. Electrophoretic endoribonuclease assay and cleavage pattern of TCTP toward 5'-labeled miR155_13nt 81 Figure 20. Electrophoretic endoribonuclease assay of TCTP toward 5'-labeled miRl55_24nt and "None" negative controls with and without 3U RNasin®. ...82 Figure 21. Electrophoretic endoribonuclease assay and cleavage pattern of Caskin-1 and RPS2 toward 5'-labeled miRl55 85 Figure 22. Electrophoretic endoribonuclease assay and cleavage pattern of RPS2 toward 5'labeled miR155_13nt 87 Figure 23. Electrophoretic assay and cleavage pattern of RPS2 toward control substrate Oligo CU 89 Figure 24. Time-dependent experiment and kinetic analysis of RPS2 toward 5'-labeled miR155_13nt 90 Figure 25. Time-dependent experiment of RPS2 toward 3'-labeled miR155_13nt showing weak exoribonuclease activity 92 Figure 26. Weak exoribonucleolytic activity by RPS2 toward 3'-labeled PolyA substrate. 93 Figure 27. Weak exoribonucleolytic activity by RPS2 toward 5'-labeled PolyA substrate.. 94 Figure 28. Schematic diagram summarizing the suggested procedure 103 VII Acknowledgements I would like to thank my MSc. Thesis supervisor Dr. Chow Lee for his guidance, support and encouragement during the course of this thesis. I would also like to thank my supervisory committee members, Dr. Stephen Rader and Dr. Brent Murray for their helpful suggestions and insights in carrying out this research. Special thanks to Maggie Li for her help and guidance in conducting the experiments. Also, I would like to extend my thanks to all members of the Lee lab for their encouragement and technical support, and friends in Prince George for the support and friendship. Most importantly, I would like to thank my parents, Ye Huan-Cai and Tang WeiMin for their unending love, support and encouragement throughout my life, especially during this ten-year student journey in Canada. VIII Candidate's Publications Relevant to this Thesis Abstracts Ye, S., Woodbeck, R., Li, W.M., Lee, C.H. Towards Discovering New Mammalian RNA Cleaving Enzymes Using a High Throughput Screening Method. RiboWest Conference, Prince George BC (2009) IX Chapter 1 Introduction In Eukaryotic cells, gene expression is a tightly regulated process with key players guarding at different steps. This chapter briefly describes the general key players involved in the post-transcriptional control of gene expression with an emphasis on human endoribonucleases and mieroRNAs. 1.1 Mechanisms in the control of Eukaryotic gene expression In eukaryotic cells, gene expression can be controlled at transcriptional and posttranscriptional levels. At the transcriptional level, gene regulatory proteins containing DNA binding motif bind to the regulatory sequence of the target gene and regulate the time and frequency of gene transcription. For example, p53 and GATA-1 recognize two different DNA sequences in target genes in mammalian cell (Alberts et al. 2002). On the other hand, transcription can also be turned off completely upon DNA methylation (Alberts et al. 2002). In the post-transcriptional stage, RNA turnover which dictates mRNA abundance can directly control the abundance of protein (Fan et al. 2002; Raghavan et al. 2002). Conventional mRNA decay pathways such as polyA deadenylation, 3'-5' decay and 5'-3' decays were generally believed to be the major mode of mRNA degradation (Parker and Song 2004). However, this dogma has been challenged by the mRNA specificity of few endoribonucleases (Li et al. 2010) and the recent discoveries of endoribonucleases that demonstrated endoribonuclease activity of a number of proteins known to be involved in exonucleolytic degradation (Tomecki et al. 2010; Lebreton et al. 2008; Schaeffer et al. 2009). Differences and similarities between these two pathways are illustrated in Figure 1. 1 CH 1 INTRODUCTION m7G AAAAAA Exoribonucleolytic Decay Pathway > Endoribonucleolytic Decay Pathway AAA or AAAAAA AAAAAA or AAAAAA Figure 1. A schematic diagram of conventional exoribonucleolytic decay pathway and hypothetical endoribonucleolytic decay pathway that control stability of mRNA. (A) Two major exoribonucleolytic decay pathways. Top: Deadenylase leads to the shortening of polyA tail as the initial step of 3'-5' mRNA exoribonucleolytic decay. Bottom: Removal of 5'-cap by decapping proteins decreases the susceptibility of degradation. (B) Endoribonuclease cleaves mRNA in the middle and accelerates further degradation of target mRNA. (C) Further degradations are executed by 3'-5' and 5'-3' exoribonucleases (Parker and Song 2004; Garneau et al. 2007). 1 CH 1 INTRODUCTION 1.2 Key players in the control of RNA degradation 1.2.1 Enzymes - decapping enzymes, exoribonucleases, endoribonucleases and RBP Decapping enzymes Decapping enzymes are enzymes that remove the 5'- cap structure of transcript (Fillman and Lykke-Andersen 2005). Well studied decapping enzymes include Dcpl/2 and DcpS. Dcp 1/2 is part of a processing body complex that hydrolyzes the cap structure m7Gppp. Removal of the protective cap leads to further degradation of the mRNA. The ability to bind to specific RNA is also essential for the decapping function as exemplified by hDcp2 (Li et al. 2008). Exoribonucleases In the past, exoribonucleases were thought to be the major players in RNA degradation. A number of exoribonucleases have been studied in different mRNA decay pathways. Exoribonucleolytic degradation takes place after the decapping or dephosphorylation of the transcript, from either 5'-3' or 3'-5' direction (Figure 1). Some well known examples of exoribonucleases associated with cancers are CCR4b, PARN and XRN1. CCR4b, a component of human Ccr4-Not complexes, exhibits 3-5' poly(A) exoribonuclease and ssDNA exonuclease activities (Lau and MacRae 2009; Chen et al. 2002; Morita et al. 2007). It regulates the mRNA level of a tumor suppressor gene p27Kipl. Depletion of CCR4b can increase the p27Kipl mRNA level and impair cell growth (Morita et al. 2007). Poly (A)-specific ribonuclease (PARN) is another exoribonuclease that 2 CH 1 INTRODUCTION cleaves from 3'-5' direction. As its name suggests, PARN preferably cleaves the poly (A) tails from 3'-direction. It also interacts with the 5'- end cap for a more efficient degradation of the poly (A) tails (Gao et al. 2000; Martinez et al. 2001). It participates in nonsensemediated mRNA decay and degradation of mRNAs containing AU-rich elements (AREs) in their 3'-UTR (Lai et al. 2003). Besides deadenylase, the 5'-3' exoribonuclease representative XRNlcan also promote mRNA decay. In conventional mRNA decay pathways, upon the decapping of mRNA, XRN1 degrades the decapped mRNA from 5'-3' (Mitchell et al. 1997). It was also found to be an important factor in the spatial regulation of mRNA decay in mouse (Bashkirov et al. 1997). In the absence of XRN1, polyA+ mRNAs accumulate at the processing bodies in mammalian cells (Cougot et al. 2004). The XRN1 gene is also a novel candidate tumour suppressor gene in osteogenic sarcoma (Mullen et al. 2008; Zhang et al. 2002). Endoribonucleases Researchers in the RNA field started to rethink the position of these two types of RNases in term of RNA metabolism after more work on endoribonucleases was done. Compared to exoribonucleolytic decay, endonucleolytic cleavages demonstrate higher sequence specificity (Rodgers et al. 2002, Stevens et al. 2002, van Dijk et al. 2000). One important aspect to consider is that many RNA therapeutic approaches, such as antisense therapy and therapeutic RNA interference, involve specific RNA sequence and endoribonucleolytic activity. 3 CH 1 INTRODUCTION Many of the known endoribonucleases are discovered by chance. A recent review on endoribonuclease concludes that no share protein domains for endonucleolytic RNA cleavage were found in most of the 13 enzymes discussed; structural diversity was found for those that did have identified ribonuclease domains (Li et al., 2010). Regardless of their dissimilarity, these known endoribonucleases have been found to play important roles in essential cellular processes such as RNA interference, ER stress response, viral defence, aberrant RNA surveillance, microRNA biogenesis, tRNA processing, signalling and angiogenesis. Associations between endoribonucleases and cancers have been shown previously by several examples such as Argonaute 2, IREl, APEl G3BP, Drosha and Dicer (Kim et al. 2009). These examples provided evidence that induction or suppression of such enzymes control toward the development of diseases such as cancers. More detailed descriptions of relevant human endoribonucleases are discussed in Section 1.4. RNA-bindlne proteins (RBP) Beside the enzymes that cleave RNA, RBPs also play important role in posttranscriptional control of gene expression. One of the main functions of these proteins are to block the interaction of the target mRNA with other proteins. AU-rich mRNAs are often the target of RNA binding protein regulation. Studies have found one single enzyme, tristetraprolin (TTP), to be the key regulator of a group of 63 genes which have AU-rich mRNA and are implicated in cellular growth, invasion and metastasis (Al-Souhibani et al. 2010). Furthermore, exemplified by transactivation-responsive RBP/ TRBP, RBP is also found to post-transcriptionally control the biogenesis of microRNA (Chendrimada et al. 2005; Melo et al. 2009; Paroo et al. 2009). CH 1 INTRODUCTION RBPs can bind to protect and control stability of the target RNA. For instance, HuR plays an important role in the growth of cancer cells. In MCF7 human breast cancer cell lines, it increases the stability of GATA3 and ER mRNA (Licata et al. 2010; Pryzbylkowski et al. 2008). In human conventional renal cell carcinoma (CRCC), HuR is overexpressed and stabilizes mRNA for proteins that are crucial for human CRCC tumorigenesis (Danilin et al. 2010). The c-myc coding region determinant-binding protein (CRD-BP) is another example of RBP found highly expressed in various types of human cancer including breast, colon, skin, ovary, lung, brain and testicular cancer (Doyle et al., 2000; Ioannidis et al., 2003; Dimitriadis et al., 2007; Ross et al., 2001; Kobel et al., 2007; Hammer et al., 2005; Ioannidis et al., 2005; Elcheva et al., 2008; Ioannidis et al., 2004). CRD-BP has been shown to bind to and stabilize a number of mRNAs implicated in cancers. The mRNA targets of CRD-BP are c-myc, CD44, pTrCPl, IGF-II, P-catenin, GLI1, and MDR1 (Ioannidis et al. 2005; Sparanese and Lee 2007; Vikesaa et al. 2006; Noubissi et al. 2006; Leeds et al. 1997; Gu et al 2008; Noubissi et al. 2009). While HuR and CRD-BP promote the growth of cancer cells, there are RBPs that play the opposite role by suppressing cancer growth. They are exemplified by proteins in the quaking family which is composed of multifunctional mRNA regulators that function as tumour suppressors (Biedermann et al. 2010). 5 CH 1 INTRODUCTION 1.2.2 Cellular structures involved in mRNA degradation Stress granule (SG) SGs belong to a type of the RNA granule groups involved in RNA translational regulation and decay. The SGs rapidly accumulate within 15-30 minutes as a stress response upon environmental stresses (Anderson and Kedersha 2006). The stability of specific mRNAs is regulated by the selective recruitment into the SGs, and the translational rate is subsequentially controlled (Anderson and Kedersha 2002). Components of SG include a wide range of protein classes such as ribosomal, translational proteins, proteins involved in RNA stability, RNA-binding proteins, exonucleases and enzymes from the RNAi pathway (Anderson and Kedersha 2006; Kedersha et al. 2005; Kedersha et al. 2002; Kedersha et al. 1999; Wilezynska et al. 2005; Stoecklin et al. 2004; Gallouzi et al. 2000; Thomas et al. 2005; Hua and Zhou 2004; Tourriere et al. 2003). Processing-body (PB) PB is one of the major compartments involved in mRNA degradation in cytoplasm. They interact closely with the SGs. mRNA marked by destabilizing factors may be transferred from SGs to PBs for degradation (Anderson and Kedersha 2008). It is also a site for RNA interference to take place (Lian et al. 2006; Jakymiw et al. 2005). PBs contain enzymes which carry out the mRNA degradation functions including deadenylation, decapping, exoribonucleolytic and endoribonucleolytic decay (Eulalio et al. 2007; Franks et al. 2008; Parker and Sheth 2007). The degradation function begins with deadenylation of the poly A tail of the target transcript by the Ccr4p/Pop2p/Not complex, which is required for the PB formation (Parker and Sheth 2007; Zheng et al. 2008). After 6 CH 1 INTRODUCTION deadenylation, two major decays take place from either direction of the transcript: 3' to 5' degradation carried by exosome and 5' to 3' degradation by exoribonuclease Xralp following removal of the m7Gppp cap by Dcpl/2 (Gu et al. 2004; Parker and Sheth 2007) or DcpS (Gu and Lima 2005). PBs are also composed of microRNA repression factors such as Argonautes and RNA binding proteins and translation repressors (Parker and Sheth 2007). RNA-Induced Silencing Complex (RISC) The RISC has received a great deal of attention since its discovery in 1985, due to its ability to repress target gene expression by degrading respective mRNA transcript (van den Berg et al. 2008; Rosenberg et al. 1985). It is a multi-protein complex that controls gene expression in translational level. Minimal components of RISC include a main catalytic component Argonaute 2 and a guide strand RNA that is either a microRNA or siRNA. The guide strand is either partially or completely complimentary to the target mRNA. Pairing of these two strands will result in translational repression or mRNA degradation (Huntzinger and Izaurralde 2011). The cleaved mRNA is then released and subjected to further exoribonucleolytic degradations. Exosome The exosome, also called PM/Scl complex, is a well known and characterized complex cellular structure that degrades RNA in 3'-5' direction. Specific exoribonucleases in the exosome are hRrp4p, hRrp40p, hRrp41p, hRrp46p, hMtr3p, Hrrp42p, oIP2, PM/Scl75, PM/Scl-100 and Rppl4 (Raijmakers et al. 2004). Beside its main role in exoribonucleolytic activity, it is recently found that the exosome also contains proteins 7 CH 1 INTRODUCTION with specific endoribonuclease and cytoplasmic mRNA decay activities (Schaeffer et al. 2009). The complex is also composed of proteins with RNA binding, 3'-5' hydrolytic exoribonuclease, 3'-5' phosphorolytic exoribonuclease, RNA helicase and nucleotide binding activities (Raijmakers et al. 2004). In eukaryotes, the exosome is known to contribute to ribosomal RNA and small RNAs processing, pre-mRNA quality control and mRNA turnover (Raijmakers et al. 2004). Exosome mediated RNA degradation is sequence specific. For example, mRNAs with AUrich elements are targeted by the exosome mRNA degradation (Mukheijee et al. 2002). 1.2.3 non-coding RNA Another group of trans-acting factors that control the expression of genes is the non-coding RNAs (ncRNAs). They are abundant in mammalian cells (Mattick and Makunin 2006). RNAs that belong to this group include rRNA, tRNA, snRNA, snoRNA, microRNA and siRNA (Mattick et al. 2006). They fall into two categories according to their function: infrastructural ncRNAs (rRNA, tRNA, snRNA and snoRNA), and small regulatory ncRNA (snoRNA, microRNA and siRNAs) (Mattick et al. 2006). Increasing evidence indicates the association of ncRNAs with different types of cancers, which warrant their possible use as therapeutic target (Mallardo et al. 2008). 1.3 Significance of microRNA in the control of gene expression MicroRNAs are a type of non-coding RNAs with the length of 18 - 24 nt that posttranscriptionally regulate the expression of genes. This group of small RNA has gained 8 CH 1 INTRODUCTION great attention after its discovery in 1993, mainly due to its important role in the regulation of gene expression in the RISC. 1.3.1 MicroRNA Biogenesis MicroRNAs are produced in a highly regulated manner (Siomi and Siomi 2010). They are generated from a microRNA gene or from the intron of a protein coding gene that is also called a mirtron (Chekulaeva and Filipowicz 2009; Kim et al. 2009). Four complexes (the microprocessor complex, the nuclear export complex, the Dicer complex and the RISC) are involved in the biogenesis and action of microRNA. In the nucleus, primary microRNA or pri-microRNA is generated, through the tailoring of Drosha containing microprocessor complex (Han et al. 2004; Gregory et al. 2004; Denli et al. 2004). Pri-microRNA is cropped by Drosha to form the hairpin shaped pre-microRNA. The pre-microRNA is then transported out of the nucleus to the cytoplasm by the nuclear export complex composed of Exportin 5 and RAN. In the cytoplasm, the Dicer complex further cuts the pre-microRNA into its mature form; the mature microRNA is functional as part of the RISC (Ryan et al. 2010; Ahluwalia et al. 2009). 1.3.2 Regulatory roles of microRNA MicroRNAs control gene expression through either translational repression or degradation of mRNA (Cai et al. 2010; Esslinger et al. 2009). They have a huge impact on the global expression of proteins (Baek et al. 2008); about half of all mammalian protein coding genes are predicted to be controlled by these versatile regulatory elements (Krol et al. 2010). Targets of microRNAs include functionally related effector genes, regulators of transcription and regulators of alternative splicing (Makeyev and Maniatis 2008). 9 CH 1 INTRODUCTION MicroRNAs have also been found to be involved in skin morphogenesis (Yi and Fuchs 2010), signal transduction (Inui et al. 2010) and glucose and lipid metabolism (Lynn 2009). 1.3.3 Role of microRNA in cancer MicroRNAs are broadly implicated in various types of diseases, particularly cancer. The role of microRNA in cancer can be placed into two categories - oncogenic and tumour suppressor microRNA (Zhang et al. 2007). Oncogenic microRNAs that capture much of the attention are over-expressed in many types of cancers; this is exemplified by miR155 as the first oncogenic microRNA discovered (Tong and Nemunaitis 2008; Tam and Dahlberg 2002). MiR155 was found to inhibit apoptosis by suppressing Caspase-3 and apoptosis related enzymatic activity (Ovcharenko et al. 2007; Gironella et al. 2007). It is upregulated in several types of cancers including CLL (chronic lymphocytic leukemia), breast cancer, Burkitts lymphoma, Hodgkins lymphoma, B-cell lymphoma, lung cancer, and pancreatic cancer (Iorio et al. 2005; Faraoni et al. 2009; Zhang et al. 2007; Rai et al. 2008; Tong and Nemunaitis 2008; Yue and Tigyi 2006; Wiemer 2007). Because of the widely distributed oncogenic activity of miR155 in a number of malignancies, it serves as an excellent target for cancer therapy. Thus, miR155 was chosen as a target RNA in this research. 1.3.4 Regulation of microRNA abundance Though much evidence has shown the regulatory roles of microRNA in a wide variety of cellular activities, the expression and turnover of microRNAs are also under sophisticated regulations. The microRNA abundance can be controlled in two major ways: through the regulation of microRNA biogenesis and microRNA decay. 10 CH 1 INTRODUCTION Primary microRNAs (pri-miRNA) are generated from microRNA gene transcription by RNA polymerase II (Kim et al. 2009). The transcriptional control of the microRNA gene is quite similar to the protein-coding gene. These two types of gene share similar features in the promoter regions, such as GpG islands, TATA box sequences, initiation elements and histone modifications (Ozsolak et al. 2008; Corcoran et al. 2009). These similarities suggest that transcription factors (TFs), enhancers, silencing elements and chromatin modifications are the key controllers in microRNA gene transcription (Krol et al. 2010). After transcription, pri-miRNAs are cleaved by Drosha in the microprocessor complex in the nucleus (Han et al. 2004; Gregory et al. 2004; Denli et al. 2004). PrimiRNAs become precursor microRNAs (pre-miRNAs) after removal of part of the stem. The pre-miRNAs are then exported from the nucleus. In the cytoplasm, pre-miRNAs are further processed by Dicer to remove the loop to generate a major and a minor mature microRNA. The major strand is loaded onto Argonaute located in the RISC as a guide for gene silencing. Differential expressions of microRNAs in different tissues and during different developmental stages suggest that microRNA biogenesis can be regulated (Davis et al., 2009; Ding et al., 2009; Ambros et al, 2003; Wulczyn et al, 2007). Besides regulation of microRNA biogenesis, the microRNA abundance is also controlled by their turnover. Compared to biogenesis and regulatory control of microRNA, current available information regarding the control of microRNA stability is still very limited. Nonetheless, some progress has been made to find the key players in microRNA degradation. Several studies have found evidence of acceleration and regulation in selected microRNA turnover (Krol et al. 2010; Hwang et al. 2007; Buck et al. 2010; Sethi and 11 CH 1 INTRODUCTION Lukiw 2009; Rajasethpathy et al. 2009). The first two exoribonucleolytic degradation models of microRNA decay were found in studies using model species, Arabidopsis thaliana and Caenorhabditis elegans. In A. thaliana, small RNA degrading nuclease 1 (SDNl) acted directly toward single stranded miR173 in vitro (Ramachandran and Chen 2008). Increased stability of several microRNAs (miR159, miR167, and miR173) after knockdown of the members from this 3'-5' exoribonuclease family indicated that these microRNAs may share a similar RNA decay pathway (Ramachandran and Chen 2008). 53' Exoribonuclease 2 (XRN-2) from C. elegans is the second exoribonuclease discovered (Chatterjee and GroBhans 2009). It is a 5'-3' exoribonuclease that carries out the degradation of mature let-7 microRNA in vivo (Chatteijee and GroBhans 2009). This function of XRN-2 depends on the target availability; the 5'-end of microRNA has to be released from miRISC in order to be accessible for XRN-2 (Chatterjee and GroBhans 2009). The evidence of microRNA degradation by exoribonucleases was also found in human cells. In a recent study conducted with human melanoma cells, miR221 was shown to be degraded by a 3'-5' exoribonuclease, human polynucleotide phosphorylase/Zz/WPase0'^5 (Das et al. 2010). Another recent study using human embryonic kidney 293 cells revealed involvement of the exosome in the degradation of miR382 (Bail et al. 2010). RBP also play a role in the abundance of microRNA. This is exemplified by a human nuclear factor named TDP-43 (Buratti et al. 2010). It can up- or downregulate different microRNAs base on the position it binds to. It stabilizes the let-7b by binding to the mature form sequence, and decreases the abundance of mature form miR663 by binding 12 CH 1 INTRODUCTION to its hairpin precursor (Buratti et al. 2010). Binding to Argonaute proteins in the RISC is also thought to protect the microRNA from degradation (Chatteijee and GroBhans 2009). Furthermore, a small number of cis-acting elements that affect the microRNA stability have also been identified. In animals, 3'-terminal adenylation by GLD-2 was found to stabilize mature miR122, but not pre-miR122 (Katoh et al. 2009). In plants, both 2'-0-methylation and adenylation at 3'- terminal demonstrated stabilizing effect on microRNA (Yu et al. 2005; Lu et al. 2009). Known key players regulating animal microRNA turnover are shown in Figure 2. It is believed that microRNA degradations are likely to be similar to those of mRNAs (Krol et al. 2010). Mature microRNA levels do not always correlate with their pri- or pre-mRNAs. The evidence of selected mature microRNA turnover strongly supports the existence of post-transcriptional mechanisms that control the level of mature microRNA. It is possible that endoribonucleolytic cleavage plays a role in the process of microRNA degradation. However, due to the lack of information in this area, other key players in microRNA decay have yet to be identified and characterized. 13 CH 1 INTRODUCTION (5?)' XRN2 hPNPase old-35 Argonaute 2 TDP-43 exosome B 5' 3'AA GLD2 Figure 2. Schematic diagram summarizing key factors regulating animal microRNA turnover. (A) Binding of RBP (Argonaute 2 or TDP-43) prevents the exoribonucleolytic decay from either ends by exoribonucleases (XRN2 or hPNPaseold-35) or exosome. It may also compete with unknown endoribonucleases and prevent them from cleaving in the middle of microRNA. (B) Adenylation at the 3'-end stabilizes microRNA. 1.4 Human endoribonuclease One of the reasons for our limited knowledge on mammalian endoribonucleases is the lack of tools used to identify them. Most of the recent endoribonuclease discoveries were quite random. A recent review has focused on comparing the different eukaryotic endoribonuclease. It was found that some of the enzymes with known domains demonstrate 14 CH 1 INTRODUCTION structural diversity (Li et al., 2010). Endoribonucleases can be associated with a wide variety of cellular functions; they may have different mRNA targets, and possess different cleavage specificity (Li et al., 2010). In this section, mRNA targets, cleavage specificity and cellular functions of known human endoribonucleases are briefly introduced. 1.4.1 Human endoribonuclease implicated in cancers Areonaute 2 (Ago2) Ago2, also called Eukaryotic translation initiation factor 2C 2 (eIF-2C 2), is an important component in RISC and required for RNA mediated gene silencing (Liu et al. 2004). Ago2 binds to a guide RNA, either a microRNA or siRNA, in the RISC to target a complementary mRNA (Liu et al. 2004; Kim et al. 2009; Carthew and Sontheimer 2009; Jinek and Doudna 2009). The binding of the complementary mRNA with the RISC will then result in gene silencing by inhibiting the translation or cleaving the complementary mRNA (Hutvagner and Zamore et al. 2002; Jinek and Doudna 2009). The interaction of Ago2 with Dicer also suggests a role in microRNA biogenesis (Maniataki and Mourelatos 2005; O'Carroll et al. 2007). Increased level of Ago2 was found in aggressive breast tumours (Blenkiron et al. 2007). Also, increased cell proliferation was observed in Ago2 transfected MCF7 cell line (Adams et al. 2008). Inositol-reauiring enzyme 1 (IRE1) IRE1 is also called endoplasmic reticulum-to-nucleus signalling protein. It is a bifunctional protein that has both endoribonuclease and serine/threonine-protein kinase activity (Tirasophon et al. 1998). In mammalian cells, IRE1 is required for a stress response pathway functioning under ER stress (Tirasophon et al. 1998). It carries out 15 CH 1 INTRODUCTION translational repression by cleaving 28S ribosomal RNA under ER stress (Iwawaki et al. 2001). IRE1 plays both tumorigenic and tumour suppression roles in tumorigenesis through the activation of unfolded protein response by the IREl-XBPls pathway (Kim and Lee 2009). The IRE1 activity was found to increase the cancer survival and progression in several types of cancer, including breast cancer, liver cancer and myeloma (Lin et al. 2007; Gomez et al. 2007; Davies et al. 2008; Shuda et al. 2003; Li et al. 2007; Carrasco et al. 2007). Several observations demonstrating the ability of IRE1 in inducing cancer cell death and inhibition supported the tumor suppression role of IRE1 in cancer (Kim and Lee 2009; Davenport et al. 2007; Guichard et al. 2006; Little et al. 2007; Kraus et al. 2008; Joung et al. 2007; Gao etal. 2008). RNase L RNase L, commonly named 2-5A-dependent ribonuclease, is a 2'-5'oligoadenylate- dependent ribonuclease that cuts single stranded RNA (Liang et al. 2006). It functions as a viral defense following the activation of interferon (IFNs) pathway through a combination of mechanisms by directly degrading viral RNAs and rRNA, and inducing apoptosis and other antiviral genes (Le Roy et al. 2001). RNase L also contributes to tumor suppressor activities such as stress mediated apoptosis cell proliferation and regulation of protein synthesis (Liang et al. 2006, Madsen et al. 2008). Mutated RNase L was related to the increased risk of head and neck, uterine cervix, breast and prostate cancer (Madsen et al., 2008; Carpten et al., 2002). 16 CH 1 INTRODUCTION APE1 Apurinic/apyrimidinic (A/P) DNA endonuclease APE1, also called APEX-1 in humans, is commonly known as a DNA repair enzyme that cleaves A/P DNA in base excision repair mechanism. Other known functions of APE1 include redox activation of transcription factors, 3'-phosphodiesterase, 3'-5' exonuclease, 3' phosphatase, and RNase H activities (Chou and Cheng 2002, Tell et al. 2005). It was recently discovered as an endoribonuclease that binds to a coding region of c-myc mRNA called the coding region determinant (CRD) and preferentially cleaves phosphodiester bonds between UA, CA and UG dinucleotides (Barnes et al. 2009). The cancer types previously reported to be associated with APE1 are myeloma, osteosarcoma, hepatocellularcarcinoma, breast carcinoma, colon adenocarcinoma, lung adenocarcinoma and leiomyoma (Kim and Lee 2009; Tell et al. 2005; Yang et al. 2007; Wang et al. 2004; Pardini et al. 2008; De Ruyck et al. 2007; Orii et al. 2002). The implications of APE1 with cancer may be linked to its ability to control c-myc gene expression level by cleaving its mRNA (Kim and Lee 2009; Barnes et al. 2009). Drosha and Dicer Drosha and Dicer are both members of the RNase III family. Drosha processes primary microRNA (pri-microRNA) as a component of the microprocessor complex in the nucleus (Han et al. 2004; Gregory et al. 2004; Denli et al. 2004). It cleaves double stranded pri-microRNA at the 3'- and 5'- ends and produces precursor microRNA (pre-microRNA) (Lee et al. 2003). Dicer comes in to carry out dsRNA cleavage and generate siRNA or premature microRNA by cleaving the hairpin pre-mRNA (Fortin et al. 2002, Nicholson 17 CH 1 INTRODUCTION and Nicholson 2002, Doi et al. 2003, Zhang et al. 2004). A recent study also suggests that Drosha and Dicer are involved in rRNA biogenesis (Liang, 2011). The major implication of Drosha and Dicer in cancer mainly comes from their functions in microRNA processing and biogenesis by increasing the levels of oncogenic microRNA (Nakamura et al. 2007; Davis et al. 2008; Iorio et al. 2005; Roldo et al. 2006; Slaby et al. 2007; Dillhoff et al. 2008; Markou et al. 2008; Connolly et al. 2008; Chiosea et al. 2006; Chiosea et al. 2007; Chiosea et al. 2008; Flavin et al. 2008; Kaul et al. 2004). G3BP G3BP was identified as a cytosolic protein that binds to Src homology 3 (SH3) domain of GTPase-activating protein (GAP) (Parker et al. 1996). It binds to GAP only in proliferating cells where Ras is in the activated state (Parker et al. 1996, Tourriere et al. 2001). Endoribonucleolytic activity of G3BP occurs at the phosphodiester bond in between CA dinucleotide of 3' UTR of c-myc mRNA (Tourriere et al., 2001). Its high-affinity binding to polyA mRNAs sequence appears to allow the mRNA to decay with specificity as exemplified by the cleavage of 3'UTR of human c-myc mRNA (Tourriere et al. 2001, Gallouzi et al. 1998). Two isoforms of this RNase, G3BP1 and G3BP2 also bind to p53 both in vitro and in vivo, and results of the knockdown experiment suggest their negative regulation of p53 by regulating a p53 regulator MDM2 (Kim et al. 2007). Expression of G3BP is also closely related to lymph node metastasis in esophageal squamous carcinoma (Zhang et al. 2007). 18 CH 1 INTRODUCTION Angiogenin Angiogenin, also known as RNase 5, belongs to the pancreatic ribonuclease family with 65% homology to pancreatic ribonucleases RNase A (Saxena et al. 1992). Compared to RNase A and other known eukaryotic endoribonuclease, Angiogenin has much weaker activity (Li et al. 2009; Kelemen et al. 1999). However, despite its weak enzymatic activity, Angiogenin has great biological significance. It is a cytotoxic RNase that inhibits protein synthesis by degrading cellular tRNAs, and both 28S and 18S ribosomal RNA (Saxena et al. 1992; Lee and Vallee 1989; Shapiro et al. 1986). The level of angiogenin is found to be elevated in prostate cancer, gastric carcinoma and melanoma (Katona et al. 2005; Chen et al. 2006; Vihinen et al. 2007). Flap structure-specific endonuclease 1 (FENl) FEN1, similar to APE1, is an important multi-functional protein in DNA repair and base-excision repair (Robins et al. 1994; Shen et al. 1996; Guo et al. 2008). It also exhibits 5-3' exonuclease activity toward double stranded DNA, RNase H activity and endonucleolytic cleavage of RNA at 5' endogenous stem structures (Robins et al. 1994; Shen et al. 1996; Guo et al. 2008; Stevens et al. 1998). Its association with cancer has been linked to its DNA endonuclease activity (Kim and Lee 2009). However, its endoribonuclease activity has not been associated with cancer. PoMUl-specific endoribonuclease This protein is also called Placental protein 11 (PP11), as it is placental specific (Bohn and Winckler 1980; Bohn et al. 1981). It belongs to the ENDOU family and cleaves single stranded RNAs and results in products containing 2'-3'- cyclic phosphate termini 19 CH 1 INTRODUCTION (Laneve et al. 2008). PP11 is a tumour marker with diagnostic significance (Grundmann et al. 1990), and has been found to express in various type of cancer including mucinous and serous cystadenocarcinoma (Inaba et al. 1982), breast cancer (Inaba et al. 1981), and gastric cancer (Inaba et al. 1980). 1.4.2 Human endoribonucleases that belong to a complex Some endoribonucleases take part in a complex to achieve their functions. The endoribonucleases Argonaute 2, Dicer and Drosha that have already been mentioned in Section 1.4.1 also belong to this group. Both Argonaute 2 and Dicer are components of the microRNA loading complex (miRLC) of RISC and are required for biogenesis and recruitment of microRNA (MacRae et al. 2008), while Drosha is part of the microprocessor complex (Han et al. 2004; Gregory et al. 2004; Denli et al. 2004). The rest of this group is exemplified by CPSF3, SMG6 and Rrp44. CPSF3 CPSF3 is a component of the cleavage and polyadenylation specificity factor (CPSF) complex. It possesses both endoribonuclease and exoribonuclease activities. CPSF3 plays a key role in the histone 3'-end pre-mRNA processing by cleaving the histone pre-mRNAs and carrying out exoribonucleolytic degradation of the downstream cleavage product from the 5' to 3' direction (Ryan et al. 2004; Kolev et al. 2008; Mandel et al. 2006). It also recognizes the AAUAAA signal sequence and interacts with PolyA polymerase and other factors to implement the cleavage and polyA addition (Kaufmann et al. 2004). 20 CH 1 INTRODUCTION SMG6 SMG6, also called Telomerase-binding protein EST1A, is part of the telomerase ribonucleoprotein (RNP) complex. It functions as an endonuclease and cleaves single stranded RNA, but not double stranded RNA or single stranded DNA (Snow et al. 2003; Glavan et al. 2006). It exemplifies the involvement of endoribonucleases in the mRNA surveillance mechanism by cleaving nonsense mRNA in human cells (Eberle et al., 2009). Rrp44 Rrp44 (Ribosomal RNA-processing protein 44, also called Dis3-like 1), is a component of the exosome complex that plays key role in RNA processing and turnover. It has recently been discovered that Rrp44 has both 3'-5' exoribonuclease and endoribonuclease activities (Tomecki etal. 2010). 1.4.3 Other human endoribonucleases RNase K RNase K is a relatively new ribonuclease. The human ortholog of RNase K is a 98 amino acid protein that cleaves specifically at ApU and ApG phosphodiester bonds, and also UpU at a lower rate (Economopoulou et al. 2007). ARD-l/NIPP-1 ARD-1 (Activator of RNA decay) is a site-specific Mg2+ dependent endoribonuclease that has a similar cleavage functionally resembling RNase E from E. coli; it cleaves the same substrate as RNase E in vitro at the same cleavage site. ARD-1 was first 21 CH 1 INTRODUCTION discovered in the expression of human cDNA copy of ARD-1 (Claverie-Martin et al. 1997), and it was later found to be an isoform to NIPP-1 encoded from the same gene by alternative splicing (Chang et al. 1999). Pancreatic RNase The pancreatic ribonuclease family is probably the most extensively studied RNase across different species. The RNase A superfamily is one of the most intensively studied ribonuclease with diverse members from RNase 1-13, which are mainly responsible for host defense (Dyer and Rosenberg 2006). Other biological processes that RNase A contributes to are neurotoxicity, angiogenic activity, immunosuppression and anti-tumour activity (Beintema et al., 1988; Di Donato et al., 1993). 1.4.4 Summary It is believed that endoribonucleases closely regulate the abundance of mRNA or microRNA in cells. However, very little information is known regarding the specific mRNA and microRNA endoribonucleases regulate. Only a few human endoribonuclease described in the earlier sections possess specificity toward a particular mRNA. For instance, IRE1 specifically cleave XBP1 mRNA during splicing to generate a new C-terminus as an unfolded protein response (Iwakoshi et al., 2003; Lee et al., 2002). In addition, IRE1 also cleave non-mRNA, 28S ribosomal RNA. APE1 is shown to cleave c-myc mRNA at its CRD region (Barnes et al., 2009). As mentioned above, G3BP binds and cleaves the 3'UTR of c-myc mRNA (Tourriere et al. 2001, Gallouzi et al. 1998), while Angiogenin possess substrate specificity toward cellular tRNAs, and both 28S and 18S ribosomal RNA (Saxena et al. 1992; Lee and Vallee 1989; Shapiro et al. 1986). 22 CH 1 INTRODUCTION To date, no endoribonuclease has been found to cleave on microRNA directly in cells. For the endoribonucleases that have been shown to carry substrate specificity, it still remains unknown whether these enzymes can act on other RNAs and influence biological functions relevant to the RNA. Therefore, a better understanding of mammalian endoribonucleases is required for us to answer these questions. 1.5 Research objectives There is an increasing interest in the regulatory roles of microRNAs in cancers. To date, characteristics of inhibitory and regulatory targets of microRNAs have been the main focus of microRNA research. In contrast, less effort has been put in finding the key players in the regulation of microRNA abundance, especially the degradation of microRNA. It is already known that exoribonucleases can catalyze the degradation of microRNA as described in Section 1.3.4. No research has been done to find out whether any endoribonucleases play a role in accelerating the decay of microRNAs. One of the major hindrances to identifying endoribonucleases is the difficulty in detecting mRNA or microRNA cleavage products. Hence, despite the increasing importance of mammalian endoribonucleases, a great proportion of information about them still remains unknown. To this end, this thesis was undertaken in an effort to develop a robust fluorescence-based system to identify new human endoribonuclease that can potentially degrade oncogenic microRNA. The first objective of this thesis was to develop a high-throughput fluorescencebased method to screen a library of human recombinant proteins for endoribonclease activity. To achieve this goal, we chose miR155 as the microRNA target in the high23 CH 1 INTRODUCTION throughput screen. Five 96-well sets containing clones of the human fetal brain cDNA library, hExl were randomly chosen. In the first part of this objective, we optimized some experimental conditions in the high-throughput system. The second part of this objective was to obtain a list of positive hits. Following were the questions that we aim to answer as we accomplish the objective: 1. Is the substrate designed suitable based on the screening results? Given that there is no known endoribonuclease that cleaves microRNA, is it feasible to use microRNA as a substrate for endoribonuclease screen? 2. What is the average rate of positive hits from one set? Is it possible that the tested clones are going to be all negative or all positive? 3. How do we distinguish between positive and negative hits? The second objective of this thesis was to verify the positive hits obtained from the primary screen in the first objective. The primary screen step has the potential to pull out endoribonuclease, exoribonuclease and RBP, but our primary goal is to find novel endoribonucleases. The first part of the second objective, which was essentially our secondary screen process, was accomplished by determining the inducibility and purity of clones, by further repeating the fluorescence-based assay to reproduce the data from primary screen, and obtaining identity and integrity of clones. The second part of the second objective was carried out by performing electrophoretic assays using three radiolabeled substrates. Two of these radiolabeled substrates used in the electrophoretic assay resemble the fluorescence-based substrates used in the primary screen. To find out whether the candidate was a potential exoribonuclease, a fourth substrate composed of 15 adenosine monophosphates was used. 24 Chapter 2 Development of a High-Throughput System to express and purify a library of recombinant proteins 2.1 Introduction A high-throughput system (HTS) is a type of experimental design that allows parallel assay with multiple samples. It is commonly used in drug discovery, chemical, biological and biochemical compound discovery. 2.1.1 Protein Functional Screen High-throughput methods that have been proven to be useful for protein functional analysis are in increasing demand. In the past, several in vitro protein functional screens were successfully developed by different labs (Galicia-Vazquez et al. 2009; Kijanka et al. 2009; Mouratou et al. 2002; Woo et al. 2005; Proudfoot et al. 2008). Nucleic acid related enzymatic functions that have been screened for in these studies include endonuclease activity toward DNA substrate, nucleic acid binding and methyltransferase acitvities. In the endonuclease and methyltransferase studies, enzymatic activities were screened using biotin and digoxingenin labeling methods and determined by ELISA (Mouratou et al. 2002; Woo et al. 2005). All of these studies involved substrate labeling. This indicates the common use of substrate labeling in protein functional screen. 2.1.2 cDNA expression library The cDNA expression library, hExl (prefix MPMGp800, Imagenes GmbH), used in this project contains 35,000 clones that express His-tagged human fetal brain 25 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM recombinant proteins (Bussow et al. 2000). It was made by inserting cDNAs generated from human fetal brain tissue directionally into an expression plasmid, pQE30NST, in between restriction sites Sail and NotI (Figure 3). The hExl clones were placed in 384-well plates. Each plate contained 4 sets (A-D). In this thesis, the code of hExl clones is in a format that the first three letter/digits represent position of the clone in the 384 plate and the last three digits represent the plate number. For example, "123512" is clone 123 from plate 512. P-T5 IacO IacO MRGS;His)6 fiamHI P-SP6 fig/ll Wort P-T7 5 Promoter 6xHis MCS-7 >4 Lambda terminator ;l PQE30NST 3494 Figure 3. Map of the pQE30NST plasmid. The hExl library was designed and shown to be suitable for protein characterization of the human proteome (Bussow et al. 2004). Another study also showed the application of hExl recombinant proteins in a high-throughput screen for antibody binding specificity (Kijanka et al. 2009). 26 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM There are several potential advantages in using hExl. First, as some tumor suppressor genes and oncogenes are found to be expressed only during fetal development (Dean 1998), using proteins expressed in human fetal brain may increase the chance of finding endoribonuclease implicated in cancer. Second, as the expression-ready cDNA is already inserted in the plasmid, cloning step is not necessary. Third, it is known that some hExl proteins may be in truncated form (Bussow 2000). Hence, it is possible to produce portions of proteins which are soluble and possess endoribonucleolytic activity. 2.1.3 Expression and purification of sets 512A, 517A, 523C, 525A and 568D We developed a high-throughput method to express and purify 480 hExl clones from selected sets 512A, 517A, 523C, 525A and 568D, through the optimizations of recombinant protein induction, purification, dialysis, and cell lysis. The development of this high-throughput system for generating recombinant proteins used in the functional screen is described in this chapter. 2.2 Methodology 2.2.1 Protein expression using two different sets of media 1) Preparations 2YT and SB broth Inoculation of the E.coli bacterial culture was performed using a 96-pin replicator to transfer a set of frozen hExl clones from 384-well stock plate to four 96-well tissue culture plates. Each of the 96 well contained 100 \iL 2YT with perspective antibiotics (100 (ig/mL ampicillin and 15 jig/mL kanamycin). Glucose (2%) was added to the broth to 27 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM ensure the healthy growth of small size cultures. These starter cultures were incubated in a 37°C shaker at a speed of 200 rpm overnight. On the second day, the 100-jiL starter culture was then transferred to a bigger culture sets containing 900 \iL SB in each well. The SB broth contained Vitamin B1 (20 |ig/mL) and lx KPB to improve the growth of small size culture. Cultures were grown to ODeoo of 0.5 before induction. LB broth The E.coli culture was grown in LB broth under similar conditions as above. Starter culture was grown overnight at 37°C with 100 LB medium containing 100 ng/mL of ampicillin, 15 jog/mL kanamycin and 2% (w/v) glucose. The next day, each clone of a 96clone set was grown in 900 mL LB medium 100 ng/mL of ampicillin, 15 ng/mL kanamycin and 20 ng/mL vitamin B1 at 37°C for 3 hours to a OD600 of 0.5 in 96 deep-well plate microplates (UNIPLATE, Whatman®) at 200 rpm before induction. Table 1. Ingredients of broth used for E.coli culture in this project. Broth / Reagent LB 2YT SB lx KPB Ingredients 1.0 % (w/v) BactoTryptone, 0.5 % (w/v) yeast extract, 1.0 % (w/v) NaCl, pH7.4 1.6 % (w/v) BactoTryptone, 1.0 % (w/v) yeast extract, 0.5 % (w/v) NaCl, pH 7.4 1.2 % (w/v) BactoTryptone, 2.4 % (w/v) yeast extract, 0.4 % (v/v) Glycerol 4.6 % KH2P04,24.3 % K2HP04 Induction All clones were induced with IPTG at final concentration of 1 mM at 37°C for 6 hours. 28 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM Cell lysis After induction, bacterial cell pellets were collected by spinning the deep-well plate at 3000xg for 10 minutes (Beckman Coulter®). Cells were lysed for 5 cycles of complete freezing and thawing. The lysed cells were then subjected to immunoblotting. 2) Western blots Lysed cells were transferred to a piece of nitrocellulose membrane using a 96 Solid Pin Multi-Blot Replicator (408, V&P Scientific). Western blot described previously was performed to visualize the presence of His-tagged proteins in the cell lysate. 2.2.2 HTS induction optimization A 96 Solid Pin Multi-Blot Replicator (VP408, V&P Scientific, San Diego, CA) was used to transfer and inoculate 96 bacteria at a time. It is capable of inoculating 4 sets of clones from one 384-well stock plate. Prior to every application, metal tips of the replicator were dipped briefly in a 10% bleach solution, then into a series of two autoclaved milliQ water baths, followed by a 99% isopropanol bath. Between baths, the liquid on the pin tips was removed by light tapping on a piece of lint-free blotting paper (VP522, V&P Scientific, San Diego, CA). The pins were then air dried. In addition to the sterilization steps, pins were washed with wash buffer provided (V&P Scientific, San Diego, CA). An induction test was performed by comparing expression of the same set of clones induced under two different conditions: incubation for 16 hours at 16 °C and 6 hours at 37°C. Proteins expressed under these two sets of conditions were purified. Four elutions 29 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM were collected and concentrations of proteins in each elution were compared to determine the best condition for small culture parallel purification. 2.2.3 Methods to lyse bacterial cells for use in HTS and individual cell growth A 150 mL E.coli culture of clone F20568 was divided into three parts. Cell pellets from 50 mL cell cultures were each resuspended in 4 mL NPI-10 followed by the following lysis methods: 1) Enzymatic method - cells were incubated in a final concentration of 1 mg/mL lysozyme for 1 hour; 2) Sonication method - resuspended cells were sonicated 5 times at setting 4 (Fishers Scientific Sonic dismembrator Model 100) for 10 seconds each, with 1 minute time period on ice in between each sonication; 3) Freeze and thaw method - the resuspended cells underwent 5 cycles of freezing on dry ice for 5 minutes and thawing in 37°C water bath for 3 minutes. SDS-PAGE Two 12% polyacrylamide gels were typically made together. Resolving gels were made by mixing 3.33 mL 30% acrylamide/0.8% bisacrylamide, 2 mL of 4x lower gel buffer (pH8.8), 2.67 mL of autoclaved water and 16 |*L of 20% ammonium persulfate (APS), and 4.8 jiL of TEMED (Sigma) and pouring into a gel apparatus (Biorad, Hercules, CA) at about 2/3 full immediately after mixing. The rest of the space was filled with water to flatten the top surface of the stacking gel. The water was removed after 30 minutes. The 30 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM 5% SDS-PAGE stacking gel was made by mixing 0.48 mL 30% (w/v) acrylamide/0.8% (w/v)bisacrylamide, 0.75 mL of 4x lower gel buffer (pH 6.9), 1.77 mL of autoclaved water and 7.5 (iL 20% (w/v) ammonium persulfate (APS), and 3 faL of TEMED (Sigma) and quickly pouring it on top of the resolving gel after mixing. Before loading to the SDS-PAGE, each protein sample was processed by adding 16 \iL to 4 (iL of 5x Sample buffer with 5 % (v/v) of p-mercaptoethanol. Mixed samples were then heated in boiling water for 5 minutes. As a reference, 5-10 |iL of molecular weight markers (Bio Basic, Inc., Markham, ON) was loaded to each gel. Gels with loaded samples were then run in lx running buffer at 200V for about 35 minutes until the bromophenol blue dye reached the edge of the gel. Table 2. List of reagents used in SDS-PAGE. Reagents 4 x Lower Gel Buffer 4 x Upper Gel Buffer 5 x Sample Buffer lx Running Buffer Coomassie brilliant blue Destain Buffer Ingredients 1.15 M Tris-HCl, 0.4% SDS, pH 8.8 0.38 M Tris-HCl, 0.4% SDS, pH 6.9 249.75 mM Tris-HCl, pH6.8,10% SDS, 30% glycerol, 0.012% bromophenol blue 20 mM Tris-HCl, 0.1 % SDS, 0.2 M Glycine 50 % methanol, 10% acetic acid, 0.1% Coomassie brilliant blue R-250 20% methanol, 10% glacial acetic acid 2.2.4 HTS purification optimization Unclarified cell lysates were directly applied to a 96 unit HTS parallel His-tag recombinant protein purification system (His MultiTrap™ HP, GE Healthcare). The system was preloaded with 500 \iL of 10% highly cross-linked spherical agarose, precharged with Ni2+ ions (GE Healthcare). Prior to adding the lysates, His MultiTrap™ HP filter plates 31 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM were spun down at 500 x g for 2 minutes to remove the storage solution from the matrices. Five hundred microlitres of autoclaved milliQ water were then added, followed by two rinses with 500 \iL binding buffer containing 20 mM imidazole at 500 x g for 2 minutes each. The unclarified cell lysates were incubated with the matrices for at least 3 minutes, followed by centrifugation for 4 minutes at 100 x g. Unbound proteins were removed by adding 500 nL of wash buffer containing 40 mM imidazole and spinning down at 500 x g for 2 minutes twice. His-tagged recombinant proteins were eluted out in a 96-well collection plate (Corning) by adding 50 \x.h of elution buffer containing 500 mM imidazole to each well and mixing for 1 minute, and were subsequently spun down at 500 x g for 2 minutes. Components of buffers required for the HTS protein purification are listed in Table 3. Table 3. Reagents used in HTS protein generation. Reagents Ingredients HTS lysis buffer 20 ng/mL DNase I, 1 mM MgC12, 1 mM PMSF, 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4 HTS binding buffer 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4 HTS elution buffer 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4 1) Quantification of purified proteins Concentrations of protein samples were tested using the Bradford protein assay. The Dye Reagent Concentrate was diluted 5 times with distilled, deionized (DDI) water and filtered through a Whatman #1 filter paper. Duplicates of 9 dilutions of BSA (Thermo Fisher Scientific Inc., Waltham, MA) protein standard (0.03125, 0.0625,0.125, 0.25, 0.5, CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM 0.75, 1,1.5 and 2mg/mL) and a blank (DDI water) were used to generate the standard curve. Ten microlitres of each protein from the original collection plate were added to a clear 96-well tissue culture plate. Two hundred fiL of the reagent was then added to each well of the microtiter plate. The microtiter plate with a mixture of the purified protein sample and reagent was gently shaken for a better mixing and left at room temperature for about 5 minutes to allow the colour to develop. Absorbance of the colour was then measured in a spectrophotometer (Multiskan, Thermo Scientific) at 600nm. 2) Immunodetection of His-tagged recombinant proteins Western blots were performed to visualize the presence of His-tagged recombinant proteins before and after the purification. A multi-pipettor was used to transfer 1 jiL of each purified protein from the 96-well storage plate to a piece of nitrocellulose membrane with the same order as in the 96-well storage plate. The nitrocellulose membrane was allowed to dry on the bench before putting in lx TBS buffer containing lOmM Tris-HCl (pH 7.4), 0.15 M NaCl and 5% (w/v) skim milk for blocking overnight at 4°C. Following the blocking step, the membrane was incubated with 1° antibody (monoclonal anti-His antibody raised in rabbit, Affinity Bio Reagents, Golden, CO) diluted to 1/1000 in TBST buffer containing lOmM Tris-HCl (pH 7.4), 0.15 M NaCl, 0.1% (v/v) Tween 20 and 1% skim milk for 1 hour at room temperature. The membrane was quickly rinsed once with 20 mL of TBST buffer immediately after the incubation. Each of three additional washes with the same buffer was done at room temperature for 10 minutes. The membrane was then incubated with 2° antibody (anti-rabbit, Promega, Madison, WI) diluted to 1/2000 in TBST with 1% skim milk for 1 hour at room temperature. After the incubation, the membrane was then washed three times as described previously. Blot detection was developed by 33 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM incubating the membrane with SuperSignal West Pico Chemilluminescent substrate (Pierce, Rockford, IL) for 1 minute and visualized under Chemlmager (Alpha Innotech, San Leandro, CA). 2.2.5 HTS Dialysis Optimization Endoribonucleolytic activity of APE1 (44 ng) was shown to be inhibited in the presence of 500 mM imidazole (Oh, 2010). In two separate studies (Lunts 1976; Wolff 1993) imidazole is also found to inhibit various enzymatic activities, and that a thorough dialysis step to exchange the buffer content is therefore necessary. To successfully exchange the buffer content using HTS micro-dialysis, we first need to develop a method to quickly determine the imidazole concentration and the time required for dialysis, and estimate the potential protein loss. (i) Using Nanodrop® spectrophotometer to estimate imidazole concentration A method of using wavelength spectrum peak position to estimate approximate concentration of imidazole present in a solution was discovered unexpectedly during the measurement of protein concentration using the Nanodrop spectrophotometer. Samples with different concentrations of imidazole in elution buffer were tested. The positions of absorbance peak on the wavelength spectrum were measured as a function of imidazole concentration. This test was repeated twice. 34 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM (ii) Minimum time required to remove imidazole using dialysis Minimum time required to remove imidazole from the protein solution was assessed. We used different sample volume sizes (50, 100 and 200 (iL) and tested with and without the presence of 1 mg/mL of bovine serum albumin (BSA) to assess how these two factors affect the dialysis speed. (iii) Test for protein loss during dialysis Pre-dialysis and post-dialysis concentrations of 0.5 mg/mL BSA and purified recombinant proteins were measured to determine the possible protein loss during dialysis. 2.3 Results and discussions 2.3.1 Comparison of protein expression in LB and SB To test whether there are any major differences in protein expression using the commonly employed LB broth and the special broths, 2YT and SB (2YT/SB), the same set of bacterial clones were grown in these media. Using Western blots to detect the presence of His-tagged proteins in E.coli grown in LB and 2YT/SB ( Figure 4), we found that most of the proteins in LB-grown cells were also detectable in 2YT/SB-grown cells. Although some dots were comparatively more intense in cells grown in LB, in general there were no major differences in protein expression in cells grown using these two different broths. Since this test was done after all five sets of clones had been purified, and LB medium is more convenient and common to use in the lab, it was later used as the growth medium for larger cultures, where special broth 2YT/SB were not necessary. Due to the 35 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM fact that this test was done after the high-throughput purification of several sets of clones was already performed, 2YT/SB were used continuously for all clone sets so as to keep the experimental conditions consistent. When growing bigger cultures at the post-screening stage, LB broth was used as it was suitable for growing higher volume of culture. 1 2 3 4 5 6 7 8 9 10 1112 1 2 3 4 5 6 7 8 9 10 1112 Figure 4. Western blots showing the expression of His-tagged proteins in the cells grown in different media. A) LB and B) 2YT/SB. More than 80% of clones show apparent expression of His-tagged proteins. No major differences in expression were found using these two sets of broths. 2.3.2 Optimizing HTS bacterial induction step The average concentration of each elution (elution 1 - 4) and the total average of all elutions are shown in Table 4. Within all elutions, the 16-hour induction at 16°C appeared to produce a higher average of protein concentration. Elution 2 of both induction methods had a higher average protein concentration amongst all elutions. Not all of the proteins induced for 16 hours at 16°C had a higher concentration than the 6-hour induction at 37°C. Most of the clones produced higher protein concentration after 16 hours of induction at 16°C. Another advantage of inducing proteins at a lower temperature and longer time is 36 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM that it allowed a higher chance of proper folding of proteins. Hence, all clones in this project were induced at 16°C for 16 hours. Table 4. Purified protein yields comparison of proteins induced with IPTG for 6 hours at 37°C and 16 hours at 16°C. Condition 6 hours @ 37 °C 16 hours @ 16 °C Average concentration of clone set S17A (mg/mL) Elution 1 Elution 2 Elution 3 Elution 4 0.184 0.231 0.050 0.100 0.236 0.346 0.077 0.126 2.3.3 Choice of cell lysis method Amongst the three cell lysis methods tested, the enzymatic lysis method might be the least vigorous for purifying recombinant proteins. However, when compared with the other two physical cell lysis methods, the protein yield was much lower. Between the two physical cell lysis methods, sonication was the most effective in lysing the E.coli cells as shown by the abundant target protein bands on the SDS-PAGE gel (Figure 5C, Lane 7). Despite its cell lysis effectiveness, the target protein produced under this cell lysis method also appeared to have a lower protein purity. As shown in Figure 5C, unwanted protein bands with various sizes are present in Elution 2. Another reason for the unsuitability of lysing cells using sonication is that some proteins may be sensitive to the vigorous vibration, and hence may be denatured. Taken together, the freezing and thawing method of lysing cells was the best cell lysis method. This method produced sufficient yields and was considered gentle. 37 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM £ £ ~ K * ? k »P a t «P n JV^ A'V S.O S>£ > & £ ^> }> J> «i JS\«* kDa Figure 5. SDS-PAGEs showing different fractions of 568F20 in the purification process after cell lysis with three different methods. A) Enzymatic cell lysis; B) Physical cell lysis using 5 freeze and thaw cycles; C) Physical cell lysis using sonication. A small size band shown in Part (A) indicates the presence of lysozyme in clear lysate, flow through and wash 1. The protein concentrations of elution 2 were estimated with NanoDrop Spectrophotometer. 2.3.4 Optimizing HTS protein purification step To determine whether all clones produced similar His-tagged protein yield, an antiHis-tagged immunoblotting was performed using cell lysates and post-dialysis protein samples. The Western blots show the corresponding His-tagged proteins were present in all cell lysate of set 517A (Figure 6A), but not all were present after purification (Figure 6B). 38 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM This might due to the proteins being in inclusion bodies and therefore not purified under native conditions. A B API1 • 9 •• 6'»# « • I • • *•• • K • • • • # M • O • • * • • • # 1 3 5 7 9 1 1 1 3 Figure 6. Western blots showing His-tagged proteins from set 517A in cell lysate and after dialysis. One microlitre of each protein sample was transferred to the nitrocellulose membrane. (A) All of these clones show expression of His-tagged recombinant protein. (B) Only 34 out of 56 clones were able to produce detectable amount of purified recombinant protein. Data is adapted from unpublished report (Oh 2009). 2.3.5 Using Nanodrop spectrophotometer to estimate imidazole concentration While measuring protein concentration using the spectrum peak position of a Nanodrop spectrophotometer wavelength, we discovered a method to assess the concentration of imidazole present in the solution. The absorbance peak's positions on the wavelength spectrum were measured as a function of the imidazole concentration. This test was repeated twice. The results show that the shift of the absorbance peak's position was consistent with the increased imidazole concentration (see Figure 7). According to this standard curve, 39 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM the concentration of imidazole can be estimated. For example, if the peak is located at around 228nm, imidazole in the solution would be approximately 0.5 M. Identical data from duplicated readings demonstrated the reliability of this method in estimating the imidazole concentration. This quick detection method using NanoDrop spectrophotometer allowed us to measure the concentration of imidazole and assess the effect of dialysis on imidazole concentration in the original buffer. 232 230- _l 5 228- (0 226- 0) Q. & 224< 222- 220 0.5 1.5 Imidazole [c] (M) Figure 7. Position of absorbance peak shifting to the right on wavelength spectrum (increasing in wavelength) as imidazole concentration in the sample increases. 2.3.6 Assessing the minimum time required for dialysis Figure 8 shows the effect of dialysis on the concentration of the imidazole, with and without BSA. In both conditions, the imidazole concentration dropped significantly after 30 minutes, and remained constant after 120 minutes of constant buffer exchange with all sample volumes. Smaller sample volumes tend to have better chances of removing 40 CH 2 DEVELOPMENT OF A HIGH-THROUGHPUT SYSTEM imidazole from the buffer as indicated by a lower imidazole concentration of all time points. Hence, 50 |il dialysis sample size and 120 minute dialysis time were chosen for the HTS dialysis system. Another phenomenon was observed during the experiment. When the protein concentration was increased, the detected concentration of imidazole using the NanoDrop method was also increased at the same time. Two explanations are possible for this observation. First, the BSA proteins were partially blocking the membrane and subsequently decreasing the exchanging rate of buffers. Second, the presence of BSA was interfering with the absorbance measurement. This could be due to the similarity of ring structures present in both imidazole and some amino acids such as histidine. A. B. BSA 0 mg/ml BSA 1 mg/ml U.3] 0.5, 0.4- 0.4A 200 Ul 31 0.3- average RFU of each set +2x standard deviation; Medium = last RFU > average RFU of each set + 1 x standard deviation. Average RFU for the set was calculated using the last RFU values from all the clones except the clones that produced obviously high signal in the assay (eg. 119). The list of positive hits is longer than what we anticipated. Average rate of positive hit for one set was about 23 %. It is very possible that a considerable portion of these "hits" are false positives due to contamination. There are two hypothetical approaches to eliminate the false positive. The first approach is to compare the reproducibility of screening results. Screens of sets 517A, 525A and 568D were repeated at least one more 54 CH 3 HIGH-THROUGHPUT SCREEN time. When the different screen was compared, only some hits were reproducible in both lists. As shown in Table 8, not all hits are reproducible and the number of reproducible positive hits only covered less than half of all hits from two combined trials. However, repetitive screen was not performed with sets 512A and 523C due to the insufficient amount of protein samples. Although this approach was not applied to all clone sets and hence was not used to eliminate false positive, we were aware of this problem. In the second approach used to eliminate the false positives we focused on hits that were positive only toward miR155 but not Oligo CG and CU or Oligo UA (Oh 2010; Woodbeck 2010). We reasoned this may be a suitable way to quickly eliminate false positives based on the possibility that some bacterial RNase may contaminate the protein samples. This method can also help us focus on clones that may possess substrate specificity toward miR155. A total of 25 positive candidates selected for all sets (Table 9) were subjected to further post-screening or secondary screen analysis. These clones appeared to be positive in at least one trial. 55 CH 3 HIGH-THROUGHPUT SCREEN Table 8. Comparison of positive lists from two repeated screens (Trial 1 and 2). 517A Trial 1 Trial 2 A01 A05 A07 A17 A17 A21 A21 C07 C17 C19 C19 E17 E19 E19 105 105 109 119 119 K07 K17 M01 M17 005 019 019 525A Trial 1 Trial 2 A01 A01 A03 A05 A09 A09 A15 A15 A17 A17 A19 A19 A21 C09 C15 C15 E01 E15 E15 E17 E23 G03 G03 105 105 115 115 123 123 M01 M09 Mil Mil M15 M15 M23 001 568D Trial 1 Trial 2 B04 B06 B08 B16 B18 B22 D18 D22 D22 D24 F06 F08 F08 F20 F20 F22 H02 H04 H06 H12 H14 H18 H20 H22 J10 J16 J16 J20 L20 L20 N10 N12 N12 N14 N20 N22 P02 P16 P22 Note: Reproducible positive hits are bolded and shown in both columns (e.g. A17 and A21 in set 517A). In set 517A, 7 positive hits were reproducible; in set 525A, 13 positive hits were reproducible; in set 568D, 6 positive hits were reproducible.) 56 CH 3 HIGH-THROUGHPUT SCREEN Table 9. List of clones exhibiting specificity toward miRISS substrate. Clone MPMGp800.. Signal Ranking * Reproducibility** N/A Medium C19512 N/A E15512 Medium N/A 123512 Medium N/A Medium 013512 N/A Medium 023512 N Medium A01517 N Medium E17517 N/A Medium B01523 N/A High F01523 N/A High L01523 N/A High NO1523 N/A P07523 Medium N/A High P09523 R High A15525 R Medium CI5525 R High E15525 R Medium G03525 N High B06568 N Medium D24568 N F06568 Medium R High F20568 N Medium H06568 N Medium J10568 R J16568 Medium N N10568 Medium Note: * RFU signals in fluorescence assay: High = last RFU > average RFU of each set +2x standard deviation; Medium = last RFU > average RFU of each set + 1 x standard deviation. ** R- reproducible hit, N- not reproducible, N/A- data not available. 57 Chapter 4 Post Screening Analysis of the Positive Hits 4.1 Introduction 4.1.1 Rationale for carrying out secondary screen Post screening analysis (secondary screen) is necessary because the positive hits obtained from the high-throughput screen (primary screen) might contain false positives, especially with such a high percentage of total positive hits (23%) initially identified in Chapter 3. Several factors may contribute to the occurrences of false positives. First, cross contamination amongst clones in 96-well plates during the bacterial growth is possible and can introduce a second recombinant protein into adjacent protein samples during the handling of clones in 96-well plates. In this case, an inactive clone can be contaminated with an adjacent active clone and be selected as a positive hit. Second, since the recombinant protein is grown in the E.coli cells, the bacterial host itself can be a source of contamination. Third, during the high-throughput cell lysis, a batch of DNase I (Roche, Switzerland) was added to the lysis buffer according to the manufacturer's protocol (Table 5). We later found out that this batch of DNase I powder contained RNase activity. The RFU was increased with the increased amount of this DNase I (data not shown). Although this contaminant may not remain with the purified protein, this finding cautioned us about the possibility of false positive signal in the screen when this batch of DNase I was included in the primary screen. Lastly, it was also possible that some of the positive hits selected were not endoribonucleases. Exoribonuclease and RNA binding protein could also alter the stem loop structure of miR155 substrate (Figure 10A) and 58 CH 4 POST SCREENING ANALYSIS subsequently cause the Cy3 to be distant from BHQ1. Therefore, post screening analysis must involve electrophoretic assay which can distinguish these possibilities. 4.1.2 Electrophoretic assay to characterize positive hits The fluorescence-based screen was designed for quick detection of potential ribonuclease activity, but it could not provide details of the activity. The electrophoretic assay was used to access the RNA cleaving activity and the RNA cleavage sites generated by candidate enzymes. Electrophoretic assays with four different substrates were carried out to further understand and characterize the possible enzymatic activity of the selected proteins. By radiolabeling the substrate at either end, the nature of the cleavage products can be determined when the substrate was cleaved. Two substrates were designed based on the mature miR155 sequences, and they differed by nucleotide length. The 13 nucleotide substrate (miR155_13nt, Figure 14A) was identical to the substrate sequence used in the screen, except that it had no fluorophore and quencher attached. As mentioned in Chapter 3, the 13-nt substrate was an artificial substrate derived from the mature form of miR155 and fits the technical criteria of fluorescence-based assay. After all, it is not the actual mature miR155 itself. Therefore, it is deemed important to include the second 24-nt full length (miR155_24nt) which more closely mimics the biological structure. The most stable predicted secondary structure of miR155_24nt is shown in Figure 14B. Substrate Oligo CU-aR (Figure 15) was used as a control for two reasons. First, it was used as a criterion to eliminate false positives in the screening step. Second, using a different substrate could provide more RNA cleavage information and possibly answer the 59 CH 4 POST SCREENING ANALYSIS question as to whether enzymatic activity was sequence specific. The Oligo CU-aR substrate used in electrophoretic assay was composed entirely of ribonucleotides instead of the hybrid form used in the primary screen. The suffix "aR" indicates ribonucleotide composition in the substrate, distinguishing it from the fluorogenic substrate Oligo CU. To further determine whether the candidate enzyme possesses exoribonucleolytic activity, Poly (A)15 substrate was used. To be consistant with the primary screen, RNase A was used as a positive control. Clone E01517 was include as a negative control, because it did not cleave miR155 in the primary screen and had a relatively high protein yield. G— U B / G \ i 20 i 1 u A } 11 1 A — U 11 II A A | | 1 1 u G I1 11 c G— I1 11 G G j i 1 1 U — G | 11 I A — U 11 u— > - \ .<$• A ,<& <& ^<$> ^ A* S'n^ '• v , Clone (Batch 1) ^ A> & ^ ^ SV?_^ S' S+fstft>4?y*p <5 J? Clone (Batch 2) <& «S^ <& & v? 0?1 /* r T v -13G TCTP -12A -11U -10A Q 5ii£ X , C "" r ^ u \V / u G < A 9GA A \ 1 \ A u -11 UA •12 AG C 8 9 10 11 32p Figure 19. Electrophoretic endoribonuclease assay and cleavage pattern of TCTP toward 5'-labeled miR155_13nt. (A) Increasing amount of TCTP and negative clone E01517 were tested against miR155_13nt for 20 minutes at 37°C in a standard electrophoretic endoribonuclease assay. (B) The RNA secondary structure of miR155_13nt shows the location of cleavage by TCTP. 81 CH 4 POST SCREENING ANALYSIS • <5> ^<*r B TCTP Strong Weak + *• Removal of phosphate Addition of-OH • — • With Without RNasin RNasin In 5' 20 13 GU 1iC6 I 14JJLG —24U N , G-U ^ • 17U -8U -7C -6G 10 u 1 a a U I -5U - 4A slsc -3A • 2U 2 3 4 S 6 16 au "14U 17 ua •11U a a I U I c |< G 18 ag |<, 19 gg G *| \< 20 20 gg G G SJIS—•! l4 u G 22 gu | auu 3 IS — 8U 216s 1., a- U »pu — U — a — u 1 15 ga i 2U 7 Figure 20. Electrophoretic endoribonuclease assay of TCTP toward 5'-labeled miR155_24nt and "None" negative controls with and without 3U RNasin®. (A) Increasing amount of TCTP and 1 fig of negative clone E01517 were tested against miR155_24nt for 20 minutes at 37°C in a standard electrophoretic endoribonuclease assay. (B) The RNA secondary structure of miRl 55_24nt shows the location of cleavage by TCTP. (C) No difference is found between "None" with and without 3U of RNasin®. The substrate miR155_24nt appeared to be unstable in the electrophoretic assay as shown in Figure 20 A (Lanes 1 and 2), C and Figure 21 (Lanes 1 and 2). If the miR155_24nt folded as the most stable predicted structure (Figure 14B), 19G, 21G and 82 CH 4 POST SCREENING ANALYSIS 22G should have been paired as shown in Figure 14B. However, it was observed that the cleavage product at these G sites appeared in the RNase T1 ladder reaction. This observation led us to suspect that the substrate did not stay in one secondary structure constantly when the reaction took place. To determine the possibility of RNase A contamination in our assay, a time-dependent test was performed by incubating 1 \ih of labeled miR155_24nt in each reaction mixture with or without RNasin® Plus RNase Inhibitor (Promega, Madison, WI). One unit of RNasin® was shown previously to be able to inhibit the activity of 5ng of RNase A by 50% (Promega, Madison, WI). As observed in Figure 20C, miR155_24nt incubated with or without 3U RNasin® were significantly degraded at all of the U sites. The band intensities of these two conditions were almost identical. Addition of RNasin did not change the appearance of cleavage at U sites. Furthermore, the U site is a known natural self-cleaving site. Taken together, the cleavage product at U sites was not a result of contamination from common ribonuclease sensitive to RNasin®, such as RNase A, but maybe due to the unstable nature of the substrate. Caskin-1 (clone C195I2) Clone CI9512, which was identified to be caskin-1 or CASK interacting protein, did not exhibit strong expression (Figure 21, Lane 8). It also appeared to have extra bands being purified at 72, 60, 31 and 29 kDa along with the major band at 15 kDa. The 15 kDa band appeared to be more intense than the rest of the bands in Figure 17; it was more likely to be the recombinant protein bands. If this 15 kDa band was the recombinant Caskin-1, with the complete sequence of Caskin-1 being 157 kDa (1433 AA), the truncated form of caskin-1 that we purified was only 1/10 of the full length protein. 83 CH 4 POST SCREENING ANALYSIS Based on the four fluorescence-based assay replicates, this truncated caskin-1 was able to generate an increase in RFU signal in all 4 trials of fluorescence assay comparing to the negative control E01517 (Figure 18). Caskin-1 appeared to cleave full length miR155 substrate strongly at 4AU and 5UG (Figure 21). However, these strong cleavage bands may be due to the higher amount of substrate present as the intensity of the uncleaved substrate appeared to be higher in Lane 4 of Figure 21. We are aware of the uneven radioactivity presented in Figure 21, even though the amount of radiolabeled substrate for each sample was normalized before loading to the gel. 84 CH 4 POST SCREENING ANALYSIS A • • o Caskin-1 RPS2 \ 10 Addition of -OH / U A I I -A — U I I A A I | U G I C — G— mm 5U 5 UG 4AU ! I .G G i I U G I I 20 A — U 3 AA I Q | »PU _ u — A — 2U I U— 1 2 3 4 5 6 7 8 9 Figure 21. Electrophoretic endoribonuclease assay and cleavage pattern of Caskin-1 and RPS2 toward 5'-labeled miR155_24nt. (A) Increasing amount of Caskin-1 and RPS2, and 1 fig of negative clone E01517 were tested against miR155_24nt for 20 minutes at 37°C in a standard electrophoretic endoribonuclease assay. Stronge cleavage and removal of the phosphate group at 5U are shown on the gel. (B) The RNA secondary structure of miR155_24nt shows the location of cleavage by Caskin-1 and RPS2. 85 CH 4 POST SCREENING ANALYSIS 40S ribosomal protein S2 (RPS2. Clone 123512) As seen on the SDS-PAGE (Figure 17, Lane 8), RJPS2 had distinctive single bands with notably high purity. The size of this clone as estimated from the SDS-PAGE was about 15 kDa which was half of its predicted full length size of 32 kDa (293 AA). Based on the fluorescence assay in the secondary screen, RPS2 had the strongest activity to cleave the miR155 substrate by generating the highest percentage change of RFU and highest last RPU value in four trials. RPS2 was fiirther tested in vitro for both endoribonuclease and exoribonuclease activities using the miR155_13nt and Poly (A)is substrate. 1) RPS2 cleaves 5' radiolabeled miR155_13nt As shown in Figure 22A, two different batches of RPS2 consistently cleaved sites at 4AU and 5UC. To further assess the endoribonucleolytic activity of the stronger batch of RPS2 (Lane 3-4 of Figure 22A), we repeated the concentration-dependent electrophoretic endonuclease assay with this batch of RPS2 twice. A representative of the repeated experiment is shown in Figure 22B. A cleavage product at 5UC intensifies as the amount of protein in the reaction increases from 0.1 jig to 1 jxg (Figure 22B, Lane 3-4). A smaller and weaker cleavage product is shown at a slightly lower position of 4AU. It is suspected that this cleavage product is generated by a cleaving in between 4AU dinucleotide followed by a removal of the phosphate group. When compared with TCTP that cleaves strongly at 5UC and weakly at 7GU, 9GA, 11UA and 12AG (Figure 19), RPS2 appeared to have a slightly different cleavage pattern. This evidence supports the argument that the cleavages seen in these two reactions were generated by two different enzymes. 86 CH 4 POST SCREENING ANALYSIS Strong Weak Removal of phosphate Figure 22. Electrophoretic endoribonuclease assay and cleavage pattern of RPS2 toward 5'-labeled miR155_13nt. (A) Increasing amount of two batches of RPS2 and 1 jig of negative clone E01517 were tested against miR155_13nt for 20 minutes at 37°C in a standard electrophoretic endoribonuclease assay. Both batches of RPS2 showed cleavage, but the first batch exhibit stronger activity. (B) Representative gel of repeated electrophoretic endoribonuclease assay using first batch of RPS2. (C) Cleavage locations at the predicted secondary structure of miR155_13nt. In the primary screen, RPS2 was selected as a positive hit that had cleaving activity towards thel3-nt miR155 substrate but not toward Oligo CU and CG. To further assess the substrate specificity observed in the screen, RPS2 was subjected to electrophoretic assay using a control substrate Oligo CU-aR. As anticipated, no cleavage appeared at 10CU site of the Oligo CU-aR (Figure 23). This was consistent with the primary screen observation that RPS2 was not able to cleave the CU dinucleotide. Interestingly, two very weak products were observed in between 5G 87 CH 4 POST SCREENING ANALYSIS and 4G, and 4G and 3A. It is possible that the phosphate groups were removed from the cleaved products at 5GU and 4GG. It is interesting to note that RJPS2 cleaved between UC dinucleotide in miR155_13nt substrate but not in the Oligo CU aR substrate. Conversely, RPS2 cleaved between GU dinucleotide in Oligo CU_aR but not in miR155_13nt. Both of these sites are in the loop region of both substrates. Taken all together, it is difficult to discern any sequence specificity of RPS2. Further experimentation is needed to investigate the ability of RPS2 to cleave in a sequence-specific manner. Unlike the electrophoretic assay using miR155_13nt, there was no distinctive cleavage pattern of RPS2 on the full length substrate miR155_24nt (Figure 21). Although RPS2 were able to generate cleavage at 4AU and 5UG, this pattern is identical to those generated by Caskin-1. 88 CH 4 POST SCREENING ANALYSIS Strong Weak Removal of phosphate Figure 23. Electrophoretic assay and cleavage pattern of RPS2 toward control substrate Oligo CU. Weak cleavage and removal of the phosphate group at 5G and 4G are shown on the gel. (A) Increasing amount of RPS2 was tested against Oligo CU for 20 minutes at 37°C in a standard electrophoretic endoribonuclease assay. (B) Cleavage locations at the predicted secondary structure of Oligo CU. 2) Time dependent effect of RPS2 on 5'-radiolabeled miR155_13nt To further study the ability of RPS2 to cleave miR155, the miR155_13nt was used as a substrate to investigate the enzyme kinetics, as it is more stable than miR155_24nt. A representative picture of the time-course experiment, which was repeated three times, is shown in Figure 24. Over a period of 40 minutes, a significant increase of cleavage product cut at 5UC was observed. Along with the 5UC cleavage site product, another timedependent cleavage was observed just one nucleotide shorter at 4AU. It was observed that 89 CH 4 POST SCREENING ANALYSIS an extra band gradually appeared below the 4AU band as the incubation time increased. According to this observation, RPS2 appeared to be able to cut at 4AU weakly, and remove the phosphate group from the 3'-end of the cleavage product. We used the cleavage product at 5UC which was the most distinct decay product to determine the time course of the enzymatic activity which is shown in Figure 24A. A relative increase in radioactivity was obtained by measuring the band intensity of 5UC against a selected background. As observed, the enzymatic activity represented by the 5U band was increased over a period of 60 minutes. id^ -c& None RPS2 0.5 H9 ^ ^ ^^ _ ^ <5 b * _ 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20 30 40 SO Time point (min) Figure 24. Time-dependent experiment and kinetic analysis of RPS2 toward 5'labeled miR155_13nt. (A) 0.5 \ig of RPS2 were tested against miR155_13nt for increasing time period at 37°C in a standard electrophoretic endoribonuclease assay. B) The relative increase of radioactivity was indicated by the increase of band intensity at 5UC cleavage site. The intensity of band was measured using OptiQuant Software. The increase of band intensity was calculated by using the band intensity at a particular time point against the intensity of a randomly selected background. Results presented represent averaged data ± standard deviation from three experiments. 90 CH 4 POST SCREENING ANALYSIS 3) Effect of RPS2 on 3'- radiolabeled miR155_13nt To further investigate the cleavage pattern observed in electrophoretic assays with the 5'-labeled miR155_13nt was in fact due to the endoribonuclease activity of RPS2; the assay was repeated using a 3'-labeled miR155_13nt substrate (England et al. 1980). If RPS2 did possess only endoribonuclease activity, the cleavage product from 3'-labeled substrate would match the cleavage pattern observed in Figure 22. As shown in Figure 25, the corresponding 5UC dinucleotide cleavage was not observed in the 3'-labeled substrate. Interestingly, a laddering pattern of the substrate was observed, possibly indicating exoribonuclease activity in the 5'-3' direction. The experiment with 3'-labeled substrate was conducted only once to evaluate whether the cleavage patterns of 5'- and 3'-labeled substrates match with each other. Therefore no reliable conclusion could be drawn from this single experiment. Nonetheless, this experiment with the 3'-labeled substrate warrants future investigations on exoribonucleolytic activity of RPS2. 91 CH 4 POST SCREENING ANALYSIS A >*xN v* J^ None RPS2 0.5 |jg •—m ^ B ^ Strong Weak * «• RPS2 —2U 7GU —4A 5UC V 4AU —10A _8SA A A -11U -12A 1 2 3 4 5 6 7 8 9 1 0 11 ma/ u *VlOAU 2UA „ 11UA U — C -— A G MP- pCp Figure 25. Time-dependent experiment of RPS2 toward 3'-labeled miR155_13nt showing weak exoribonuclease activity. (A) 0.5 fig of RPS2 were tested against 3'labeled miR155_13nt for increasing time period at 37°C in a standard electrophoretic endoribonuclease assay. Nucleotide labelling corresponds to the 3'-end of the cleavage site. "FL" uncleaved full length substrate. (B) Cleavage locations at the predicted secondary structure of miR155 13nt. 4) RPS2 showed weak exoribonucleolytic cleavage toward 3'- labeled Poly(A)i5 substrate To further investigate the potential that RPS2 exhibits 5'-3' exoribonuclease activity, a 3'-labeled Poly (A)15 substrate was used in the enzymatic assay. As observed in Figure 26, the 3'-radiolabeled Poly(A)is appeared to be degraded from the 5'-end in the presence of 0.5 ng RPS2 (Lane 5-7). All sample lanes with RPS2 (Lane 4-7) had the appearance of several bands with light intensity at the bottom of the gel, indicating that the 92 CH 4 POST SCREENING ANALYSIS substrate was degraded in the 5'-3' direction. This suggests that RPS2 may have weak exoribonuclease activity. • J^None RPS2 0.5 pg SVP A-* & A A A A A A A A A A A A A A A32p.pcp 1 2 3 4 5 6 7 8 9 1 0 1 1 Figure 26. Weak exoribonucleolytic activity by RPS2 toward 3'-labeIed PoIyA substrate. 0.5 (xg of RPS2 were tested against 3'-labeled Poly (A)i5 substrate for increasing time period at 37°C in a standard electrophoretic assay. Light intensity cleavage bands reveal the exoribonucleolytic activity of RPS2 toward 3'-labeled Poly A substrate. 5) RPS2 showed weak exoribonucleolytic cleavage toward 5' labeled Poly(A)j5 substrate To determine if RPS2 has 3'-5' exoribonuclease activity, we challenged recombinant RPS2 with 5'-radiolabeled Poly (A)15 substrate (Figure 27). However the activity was detectable but very weak as compared to the strong cleavage by snake venom protein (SVP) that cleaved Poly A from the 3'-end. Directional exoribonucleolytic cleavage 93 CH 4 POST SCREENING ANALYSIS was indicated by the ladder patterns of bands at different time points. SVP cleaves from the 3'-end. The increase in the amount of shorter cleavage products was more pronounced 10 minutes after the start of the reaction. RPS2 started to show visible pattern at 5 minutes; this pattern remained constant at 10 and 20 minutes, while the negative control E01517 did not generate any cleavage even at 20 minute time point. Thus, this observation suggests that RPS2 may have weak 3'-5' exoribonuclease activity towards Poly(A)i5. None RPS2 0.5 pg SVP /* 0 5 ng 32Pa A A A A A A A A A A A A A A 1 2 3 4 5 6 7 8 9 10 11 12 Figure 27. Weak exoribonucleolytic activity by RPS2 toward 5'-labeled PolyA substrate. 0.5 ng of RPS2 and negative control clone E01517 were tested against 5'labeled Poly (A)15 substrate for increasing time period at 37°C in a standard electrophoretic assay. Light intensity cleavage bands reveal the exoribonucleolytic activity of RPS2 toward 5'-labeled Poly (A)i5 substrate. 94 Chapter 5 General discussion 5.1 General overview MicroRNAs have been receiving increasing attention as an important factor that controls expression of many genes including those implicated in cancer. Just like mRNAs, microRNA turnover is likely to be a crucial step in regulating their function (Parker and Song, 2004; Krol et al, 2010). Degradation of both mRNA and microRNA has been thought to be carried out mainly by exoribonucleases (Krol et al, 2010). However, exoribononucleases do not exhibit the specificity that endoribonucleases possess. Since very few human endoribonucleases have been characterized as compared to other model species such as E.coli, we hypothesize that there are many yet unidentified human endoribonucleases that can cleave mRNAs and the functional mature form of microRNAs. To date, no functional screen has been developed for endoribonuclease activity established outside our laboratory. The main goal of this MSc thesis was to develop a highthroughput functional screening method to identify new human endoribonucleases that cleave miR155. Overall, this study has established a high-throughput experimental protocol to screen library of human recombinant proteins for possible ribonuclease activity. Using this protocol, we have identified a list of new candidate human ribonucleases. 5.2 Significance of finding endoribonucleases that degrade microRNA The fate of a particular microRNA has great influence on the regulatory ability, as a single type of microRNA can control the expression of up to thousands of mRNA. For 95 CH 5 GENERAL DISCUSSION example, the number of gene targets of human miR155 is approximately 5445 predicted by miRanda program (John et al. 2004). Many of these target genes also play essential roles in cancer. The oncogenic microRNA and tumour suppresser microRNA regulate the abundance of enzymes implicated in cancer by controlling the number of mRNA copy. In mammalians cells, microRNA acts on target mRNA very specifically by degrading only the complimentary target (Su, 2009). This unique feature of microRNA has been the key to its regulatory function. Thus, if we understand what the determining factors for selective microRNA degradation are (e.g., in specific tissue or in cancer cells), we can better understand the global regulation of gene expression. Studies on microRNA have been largely focused on the function and targets of microRNAs. To date, there has been limited information on how microRNAs are degraded. Only a handful of factors have been identified as the key players in microRNA turnover. They are exoribonucleases, RBP and adenylase (Das et al. 2010; Bail et al. 2010; Ramachandran and Chen 2008; Chatteijee and GroBhans 2009; Buratti et al. 2010). However, these enzymes do not possess sequence specificity, and thus can not explain the precise specificity of microRNA regulation. Therefore, there likely exist other key players such as endoribonucleases that are responsible for the highly regulated characteristic of microRNA turnover. 5.3 The HTS functional screen The fluorescence-based assay, originally developed in our laboratory, has proven to be valid for the detection of the endoribonuclease activity of APE1 (Kim et al. 2010). To extend its further use, we have developed a functional screening-based method on the 96 CH 5 GENERAL DISCUSSION principle of the previous fluorescence-based assay. An HTS functional screen using microRNA as the substrate has been developed during the course of this thesis. At the same time, we are aware of some potential problems using miR155_13nt for the sensitive fluorescence-based screen. Compared to the substrate design of Oligo UA, the miR155_13nt differs in several respects that may help us understand the potential problem in the screen (Kim etal. 2010). Using miR155_13nt, several improvements have been made during this project. First, the substrate used in the primary and secondary screen, miR155_13nt, is an all RNA design, whereas in Oligo UA there is only one ribonucleotide. There are advantage and disadvantage in having only one possible cleavage site in the substrate. Making a hybrid DNA-RNA substrate may help eliminate hits that have exoribonuclease activity. However, by doing this, potential proteins that cleave the double stranded part of the substrate will be eliminated. Moreover, if we limit the possible cleavage sites to the one or two particular ribonucleotide sites, we may fail to detect other potential endoribonucleases. Second, the secondary structure of miR155_13nt has a free energy of 9.2 kJ / mol, while it is - 9.1 kJ / mol for the Oligo UA based on the folded DNA structure. The lower the free energy, the more stable the structure is. The original rationale of using miR155 was the wide association of this microRNA in various cancers. As long as we continue to use miR155 as a template, the problems we encountered will still exist. Third, the Oligo UA has a stronger stem than miR155_13nt, by having more CG base pairs on a longer stem. An extra nucleotide was artificially added to the 5'- and 3'- 97 CH 5 GENERAL DISCUSSION end to create an extra CG pair at the end and a more stable structure. If we continue using the same microRNA, miR155, additional CG complimentary pair at the end may increase the stability. Nonetheless, we were able to obtain a list of positive hits despite the imperfect design of the original substrate miR155_13nt. We used the combination of selection method by last RFU (method 2 as described in Section 3.3.2) and elimination method that omit non-unique clones toward miR155 (Section 3.3.3). Eliminating mutual positive hits with other oligonucleotides may not be the most suitable elimination method. As it may exclude true positive clones that cleave in between CG, CU or UA, only a portion of the potential true positives was covered and survived from the elimination. Furthermore, most of the positive hits that were selected using this elimination method generally had weak positive signals in fluorescence-based assay (Table 9). Therefore, future studies focusing on the positive hits using other selection and elimination methods can help us to determine which one is the best to eliminate false positives. 5.4 From primary screen to secondary screen As summarized in Table 16, clones that showed positive results in both primary and secondary screen did show cleavage on RNA substrates in the electrophoretic assay (i.e. RPS2 and TCTP). Negative control, clone E01517, which did not appear to be cleaving the fluorogenic substrate in the secondary fluorescence-based assay, also did not show noticeable cleavage products in electrophoretic assay. The 24-nucleotide full length microRNA substrate was problematic because of its sensitivity to general degradation as observed at the U sites (Figure 20 and 21). Although 98 CH 5 GENERAL DISCUSSION miR155_24nt required less free energy for forming the three possible secondary structures than the 13-nucleotide substrate (miR155_13nt), it was more sensitive to the natural cleavage sites. This also provides an answer as to why the full length substrate is not suitable for both fluorescence-based assay and electrophoretic assay. Table 16. Comparison between results from primary and secondary screen. Clone MPM Gp 800.. C19512 Primary Screen Post screening analysis Secondary Electrophoretic screen Assay 13nt 24nt + + N/A Identity Signal Repeatable CASK interacting N/A M protein (Caskin-1) + + + N/A 123512 RPS2 M + + + Yes F20568 TCTP H Yes E01517 Negative control Note: non-detectable level of activity; "N/A" data is not available or inconclusive. - - - - According to the results herein, both remaining candidates RPS2 and TCTP were positive from the primary screen to electrophoretic assay. As none of these clones have previously been shown to have ribonuclease activity, the results obtained in this study may be of importance and warrant father investigation. In Eukaryotes, RPS2 belongs to the 40S small subunit of 80S ribosome. The ribosome is a primary place for protein synthesis, where the 40S small subunit is responsible for holding the mRNA for translation. An in vitro study has found the correlation of RPS2 with increased cell proliferation (Kowalczyk etal. 2002). RPS2 is known to bind to mRNA as a ribosomal protein; it also binds to microRNA (Wang et al. 2011). Recent study shows that RPS2 binds to pre-let-7a-l to prevent the expression of tumour suppressor microRNA let-7a in human prostate cancer (Wang et al. 2011). Future 99 CH 5 GENERAL DISCUSSION studies can focus on finding out the RNA binding ability of RPS2 toward miR155 to add more information to its microRNA binding characteristic. Results shown in Chapter 4 indicate that RPS2 is possibly a ribonuclease with both endoribonuclease and exoribonuclease activity. Combining these results together, it is suggested that RPS2 may preferably cleave after unpaired U and A closer to the 5'-end, and carry out weak exoribonuclease activity from both 5'-3' and 3'-5' directions after the endoribonucleolytic cleavage. These activities may play a surveillance role in keeping any aberrant mRNA from being translated. In the past, TCTP has been linked to cell growth and malignant transformation, and is generally more abundant in tumours (Bommer and Thiele 2004; Li et al. 2010; Tuynder et al. 2002). Interestingly, it is involved in a very rare cellular event called tumour reversion (Marce, 2004). The molecular functions of TCTP that have been identified so far include calcium-binding activity (reviewed in Bommer et al. 2002) and tubulin-binding activity (Kim et al. 2000). Although, the previous findings of RPS2 and TCTP suggest that they may be oncogenic and not involved in the turnover of oncogenic miR155, their identification in this project may suggest a more complex role they play in regulating miR155 function. Since RPS2 and TCTP had different cleavage patterns, it was very likely that they were two different enzymes. Thus, more careful work is still required to establish their specific function toward miR155. Some shared cleavage sites that RPS2 and TCTP generated led us to suspect that it might be a bacterial ribonuclease that caused the appearances of the same bands at 3 AA, 4AU and 5UC of miR155_13nt. However, comparison of the purity of these three proteins (Figure 17) with the lower purity of the negative clone (E01517) made us question about 100 CH 5 GENERAL DISCUSSION this speculation. If the recombinant RPS2 and TCTP were contaminated, why would these cleavages not appear in the reaction using a less pure E01517? It would be worth retesting RPS2 toward 3'-labeled miR155 and confirm the results seen in Figure 27. Since TCTP has shown exoribonuclease-like activity toward miR155_24nt (Figure 20 A and B), conducting an electrophoretic assay with Poly (A)15 substrate would help us to clarify this. In addition, tests like Electromobility Shift Assay (EMSA) would be helpful to determine whether RPS2 and TCTP have RNA binding ability toward miR155. 5.5 Establishment of HTS procedure Compared to the original designed procedure, the established procedure to discover new human endoribonucleases has been slightly modified to maximize the chance of capturing novel enzymes. To summarize the modified HTS experimental procedure, a schematic diagram is shown in Figure 28. Several changes made to the initial design of the experiment (Figure 28). First, increasing the culture volume can improve the low yield in high-throughput protein purification. Second, repeating the primary screen and checking the reproducibility of positive hits may be a good tool to eliminate false positive. As shown in Chapter 3 and 4, reproducibility of a few clones supported this statement. Positive status of clone El 7517 was not repeatable in the primary screen and did not survive the secondary screen. Clone F20528 with repeatable positive signal in primary screen survived in the secondary screen and showed enzymatic activity in the electrophoretic assay. The negative signal of negative control E01517 was repeatable in primary screen and was confirmed in the electrophoretic assay. However, since primary screen of some clones was conducted once, there was not enough evidence to prove that reproducibility can predict true positive. Therefore, for the future screen, repeating the primary screen step can provide 101 CH 5 GENERAL DISCUSSION more insights. Third, other selection methods still remain untested. Subjecting positive hits by other selection method to further analysis can provide information on choosing the best selection method. Fourth, substrate miR155_24nt is excluded due to it being unstable. Several further improvements can be made in future studies. First, using another library which has more complete information and full length clones will help to speed up the process of identifying positive clones. Second, now that this HTS procedure is established, screening for a higher number of clones will increase the chance of capturing true positive endoribonuclease. Third, using more than one type of microRNA as substrate to compare instead of Oligo CG, CU and UA can increase the chance of identifying new ribonucleases against selected microRNAs. The results presented in this thesis also allow us to know what to expect when conducting HTS screen using clones in hExl library and miR155 as a substrate, and difficulties that one may encounter when conducting future screen. In general, the established HTS procedure has allowed us to get one step closer to finding novel human endoribonucleases in vitro. 102 CH 5 GENERAL DISCUSSION Inoculation in 96 wells 4x 1ml E.coli culture HTS Cell lysis by freeze and thaw GE His-Trap HP purification 96 well dialysis 3x HTS Functional Screen | Q) Positive Hit Selection |^) Cnegative signaD Cjnedium signaC> CThiqh signal Tests for inducibility .visible band Individual purification using Gravity Flow method with 200 ml culture Post Screening Analysis Vidualize Protein Purity with SDS-PAGE Secondary screen with Fluorescence Based Assay cDNA sequencing for clone identities and integrity using primers pQE-F and pQE-Nhe1 -R Not inframe •(eliminated^ Concentration dependent 5'-labelled substrate resembling substrate in the screen C7 cleavage Cjo cleavage^ Time dependent Electrophoretic Assay 5'-labelled PolyA Time dependent 3 -labelled substrate resembling substrate in the screen Time dependent ISxoRNase activity*!? no exoRNase activity C3ornplimentory^> Plot comDlimentorv"""> potential endoRNase EMSA Figure 28. Schematic diagram summarizing the suggested procedure. Modifications: 1. Culture volume increase from 2x ImL to 4x ImL; 2. Repeat the high-throughput primary screen three times to collect statistic data and determine if reproducibility is an effective tool to select true positive; 3. Testing positive hits using other untested selection methods can provide more information on choosing the best methods; 4. cDNA sequencing using primers pQE-F and pQE-Nhel-R instead of SP6 and T7; 5. Additional EMSA to find out whether positive hit possess RNA binding activity if ribonuclease activity is not observed. 103 CH 5 GENERAL DISCUSSION 5.6 Future directions This MSc project has led to several future research directions. First, the endoribonuclease activity of RPS2 and TCTP must be retested by repeating experiments with 3'-labeled substrate. Second, the combination of selection method using last RFU (second method as described in Section 3.3.2 (1)) and elimination method (eliminating non-unique hits toward miR155_13nt) may have not been the most effective way of selecting strong endoribonucleases as the fluorescent signal for both RPS2 and TCTP did not appear to be very high. Although we might have eliminated false positive hits by selecting unique hits, we may also have eliminated potentially highly active enzymes that cleave between CG, CU or UA. It is highly recommended to investigate the clones which exhibited high increases in fluorescence signal in future studies. In terms of screening for microRNA specific enymes, it may be of value to design a microRNA substrate with a deoxyguanosin monophosphate at one end and a deoxycytidine monophosphate at the other to stabilize the substrate and prevent capturing exoribonuclease since endoribonuclease is the main target of interest. 5.7 Concluding Remarks This MSc project was intended to develop and validate a high-throughput procedure to screen a library of human recombinant proteins for endoribonucleases that cleave a microRNA substrate. Three major achievements and observations have been made during this project. First, a high-throughput functional screen has been successfully developed through a series of optimization experiments. Potential positive candidates were obtained from the screen. 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