The Functional Role of NRAP in the Nucleolus

Author Inder, Kerry

Published 2006

Thesis Type Thesis (PhD Doctorate)

School School of Biomolecular and Biomedical Sciences

DOI https://doi.org/10.25904/1912/3452

Copyright Statement The author owns the copyright in this thesis, unless stated otherwise.

Downloaded from http://hdl.handle.net/10072/367738

Griffith Research Online https://research-repository.griffith.edu.au

THE FUNCTIONAL ROLE OF NRAP IN THE NUCLEOLUS

Presented by

Kerry Inder, B Biomed Sci. (Hons)

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

School of Biomolecular and Biomedical Science And Natural Product Discovery Faculty of Science, Griffith University, Brisbane, Australia

Submitted March, 2006

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STATEMENT OF ORIGINALITY

The material presented in this thesis has not previously been submitted for a degree in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

Kerry Inder.

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PUBLICATIONS AND ABSTRACTS ARISING FROM THIS STUDY

PUBLICATIONS

Inder, K.L., Utama, B., Gan, Y., Wang, X., and Kennedy, D. Nrap/Nol6 is involved in rRNA processing through the regulation of B23/NPM and p19ARF. (submitted)

ABSTRACTS

Inder, K.L., Utama, B., Gan, Y., Wang, X., and Kennedy, D. Nrap/Nol6 is involved in rRNA processing through the regulation of B23/NPM and p19ARF., ComBio Combined Conference, Adelaide, Australia, 2005. (oral presentation)

Hartmann, B., Aitken, K.L., Utama, B., Kennedy, D. Nrap, a novel nucleolar protein which interacts with B23/Nucleophosmin during ribosome biogenesis. East Coast Protein Meeting, Coffs Harbour, Australia, 2003.

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ACKNOWLEDGEMENTS

For the opportunity to undertake my PhD and the financial support to complete it, I must thank Ron Quinn from Natural Product Discovery, Griffith University. I must also mention Rama Addepalli and Sandra Duffy (also of Natural Product Discovery) for their encouragement to commence my PhD and support, both scientific and emotional throughout.

To all the staff and students in the school of Biomolecular and Biomedical Science at Griffith University, I also give my sincere thanks. Of these, special mention must be made to fellow students Jamie Nourse and Emily Dunner for their endless advice. Also, I acknowledge my supervisor Derek Kennedy and all the past and previous members of the RNA Metabolism Laboratory for their support. A special mention must go to Belinda Hartmann for her scientific advice, but more importantly her support and friendship through the fluctuations of a PhD. This advice extended beyond her time as part of the RNA Metabolism group and I wish her all the best in her knew found direction. I also thank Matthew Walker Brown, for endless patience while working with me through the most taxing times. Finally, of the RNA Metabolism Laboratory students, I would like to thank Renee Stirling. Renee provided me with endless advice towards my study. Renee deserves thanks for her patience and understanding in some of the most difficult times.

To Associate Professor Xing Li Wang, and all the members of his laboratory at the Department of Molecular Virology and Microbiology, Division of Cardiothoracic Surgery, Baylor College of Medicine, Houston, Texas, USA, my sincere thanks. Your support was very much appreciated and surpassed any expectations one could hope for. The time in this laboratory renewed my enthusiasm for research, and gave me the self- confidence and drive needed to achieve my objectives. Most of all I must acknowledge Budi Utama, research officer at Baylor College of Medicine. His interest and enthusiasm towards my research was greatly appreciated but most of all I am grateful for his scientific advice which continued after leaving Texas and well into the final stages of my studies. His advice and assistance was imperative in the completion of my PhD and extended beyond that of a friend or colleague to the level of mentor.

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I would like to thank Dr Charles Sherr, St. Judes Children’s Research Hospital, Tennessee, USA, for providing the p19ARF construct used as a template in this project. Also to Kienan Savage, Queensland Institute of Medical Research, who kindly donated some anti-p53 antibody. In addition, I thank all those colleagues I have failed to mention but in some way have assisted with protocols and reagents.

Finally I wish to acknowledge all my friends and family who have supported me throughout my PhD. My biggest thanks go to my husband, Shannon. Your patience, love and understanding did not go unnoticed.

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ABSTRACT

The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored.

Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody against the N-terminal region of Nrap. The anti-Nrap antibody was found to detect two Nrap bands in mouse fibroblast cells, possibly corresponding to the two mouse Nrap isoforms, α and β. In addition, mammalian expression vectors containing the full Nrap sequence as well as deletion constructs were created. The subcellular localisation of each construct was observed by fluorescent microscopy. It was revealed that recombinant Nrap did not localise to the nucleolus, possibly because it was exported to undergo degradation by the 26S proteasome. Two putative NLSs were found to be responsible for directing Nrap to the nucleus but a region accountable for nucleolar localisation was not identified. The data indicated that multiple domains working together are likely to direct Nrap to the nucleolus.

Nrap was also observed to co-localise with nucleolar proteins B23 and p19ARF. Moreover, it was shown by reciprocal immunoprecipitation that these three nucleolar proteins existed in a complex in unsynchronised mouse fibroblast cells. Recent reports demonstrated a complex relationship between B23 and p19ARF although the functional significance remained unclear. Nrap’s in vivo association with B23 and p19ARF indicated a specific functional role in the nucleolus. Nrap knockdown using siRNA significantly increased B23 protein levels in a dose-dependent manner and down- regulated p19ARF protein levels at higher siRNA concentration. Preliminary studies also implicated Nrap in cell proliferation through these novel interactions. Both endogenous and recombinant Nrap were found to be highly unstable suggesting that Nrap might regulate B23 and p19ARF through its own tightly regulated stability.

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Finally, the role of Nrap in rRNA processing was investigated by northern blot analysis. Nrap knockdown was found to affect the levels of 45S, 32S and 28S rRNAs. The changes found may be a consequence of the concurrent perturbation in the levels of B23 and p19ARF caused by Nrap knockdown. As the results were not consistent with previous reports, it was likely that changes to rRNA processing could be contributed to Nrap loss of function. This study demonstrated for the first time a functional role of Nrap in rRNA processing possibly through its association with B23 and p19ARF.

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TABLE OF CONTENTS

Title ...... I Statement of Originality ...... II Publications and Abstracts Arising from this Study...... III Acknowledgements ...... IV Abstract...... VI Table of Contents ...... VIII List of Figures...... XIII List of Tables...... XV List of Abbreviations...... XVI

CHAPTER ONE: General Introduction...... 1

1.1 INTRODUCTION...... 2 1.2 Nucleolus...... 3 1.3 Nucleolus During Mitosis ...... 4 1.4 rDNA Transcription ...... 5 1.5 rRNA Processing and Assembly ...... 8 1.6 snoRNAs and snoRNPs...... 10 1.7 Nucleolar Proteins ...... 11 1.8 Nuclear and Nucleolar Transport ...... 14 1.9 Emerging Nucleolar Functions...... 16 1.10 Nrap...... 18 1.11 Conserved Domains ...... 19 1.12 Nrap Expression and Distribution ...... 20 1.13 Cytotoxic Drug Studies ...... 21 1.14 AIMS...... 24

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CHAPTER TWO: Materials and Methods...... 25

2.1 MOLECULAR BIOLOGY MATERIALS AND METHODS...... 26 2.1.1 Transformation of DNA into Competent Cells ...... 26 2.1.2 Plasmid DNA Preparation...... 26 2.1.3 Determination of DNA/RNA Concentration...... 26 2.1.4 Restriction Endonuclease Digest...... 27 2.1.5 Agarose Gel Electrophoresis...... 27 2.1.6 Denaturing Agarose Gel Electrophoresis...... 27 2.1.7 Polymerase Chain Reaction...... 28 2.1.8 Gateway Cloning...... 28 2.1.9 PCR primers ...... 29 2.1.10 Sub-cloning Mouse Nrap Constructs into pFLAG-CMV-4...... 29 2.1.11 Sub-cloning p19ARF into pFLAG-CMV-4...... 30 2.1.12 PCR Screening of Recombinant Plasmids ...... 30 2.1.13 Sequencing of DNA ...... 30 2.1.14 Isolation of Total RNA...... 31 2.1.15 Reverse Transcription PCR...... 31 2.1.16 Real-Time Quantitative PCR...... 31 2.1.17 Generation of DIG Labeled DNA Probes ...... 32 2.1.18 Northern Blot...... 33 2.1.19 Probe Detection ...... 33 2.2 PROTEIN CHEMISTRY MATERIALS AND METHODS...... 34 2.2.1 Expression, Isolation and Purification of GST Fusion Protein ..... 34 2.2.2 Polyclonal Antibody Production ...... 35 2.2.3 Clearing GST Antibodies from Rabbit Serum ...... 35 2.2.4 Generation of CNBr Separose 4B Activated Column...... 35 2.2.5 Purification of Antibody by CNBr Activated Separose ...... 36 2.2.6 Generation of HiTrap NHS-activated HP Column...... 36 2.2.7 Purification of Antibody by NHS-activated HP Column...... 37 2.2.8 Cross-linking Nt-GST to Glutathione Sepharose...... 37 2.2.9 Purification of Antibody Using Coss-linked Glutathione Beads .. 38 2.2.10 Purchased Antibodies ...... 38

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2.2.11 Cell Lysis...... 39 2.2.12 BCA Protein Estimation...... 39 2.2.13 SDS PAGE...... 40 2.2.14 Staining of SDS PAGE...... 40 2.2.15 Western Blotting...... 40 2.2.16 Development of Blots...... 41 2.2.17 Co-immunoprecipitation...... 41 2.3 CELLULAR BIOLOGY MATERIALS AND METHODS...... 43 2.3.1 Mammalian Cell Culture...... 43 2.3.2 Transfections of Mammalian Cell Lines...... 43 2.3.3 Immunoflourescence...... 43 2.3.4 Cell Treatment with Proteasomal Inhibitor MG132...... 44 2.3.5 Nrap siRNA Knockdown ...... 44 2.3.6 Cell Proliferation Assay ...... 45 2.3.7 Cycloheximide and DRB Treatment of Cells...... 45

CHAPTER THREE: Properties of Nrap and its domains ...... 46

3.1 INTRODUCTION...... 47 3.2 MATERIALS AND METHODS...... 49 3.3 RESULTS...... 51 3.3.1 Production of anti N-terminal Nrap antibody...... 51 3.3.2 Cloning of Mouse full length Nrap ...... 57 3.3.3 Nrap is an unstable protein that may undergo proteasomal degradation...... 58 3.3.4 Recombinant Nrap localises to the nucleolus...... 61 3.3.5 Multiple domains are required for nucleolar localisation of Nrap 64 3.4 DISCUSSION...... 69

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CHAPTER FOUR: Nrap associates with nucleolar proteins B23 and p19ARF 72

4.1 INTRODUCTION ...... 73 4.2 MATERIALS AND METHODS...... 75 4.3 RESULTS...... 76 4.3.1 Nrap associates with the nucleolar protein B23 ...... 76 4.3.2 Nrap is in complex with B23 and p19ARF tumour suppressor...... 79 4.3.3 Knockdown of Nrap using siRNA ...... 83 4.3.4 Knockdown of Nrap affects the protein levels of B23 and p19ARF 86 4.3.5 Nrap may play a role in cell proliferation through its association with B23 and p19ARF ...... 88 4.4 DISCUSSION...... 92

CHAPTER FIVE: Nrap is involved in rRNA processing...... 96

5.1 INTRODUCTION...... 97 5.2 MATERIALS AND METHODS...... 99 5.3 RESULTS...... 100 5.3.1 Generation of probes for northern blot analysis...... 100 5.3.2 Northern blot analysis detects cytotoxic drug induced changes in rRNA processing...... 103 5.3.3 Over expression of Nrap affects rRNA processing...... 107 5.3.4 Knockdown of Nrap inhibits rRNA processing ...... 109 5.4 DISCUSSION...... 111

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CHAPTER SIX: General Discussion and Future Directions ...... 115

6.1 GENERAL DISCUSSION ...... 116 6.2 FUTURE DIRECTIONS ...... 122 6.2.1 Functional differences between Nrap-α and Nrap-β ...... 122 6.2.2 Further analysis of Nrap degradation ...... 124 6.2.3 RNA targets of Nrap...... 125 6.3 MODEL FOR NRAP ACTIVITY ...... 127

REFERENCES ...... 130

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LIST OF FIGURES

Figure 1.1 Schematic of the mouse rDNA repeating unit ...... 6 Figure 1.2 Schematic representation of the mammalian rRNA processing pathway ...... 9 Figure 1.3 Distribution of Nrap expression in mouse ...... 20 Figure 1.4 Effect of cytotoxic drugs on Nrap distribution ...... 23 Figure 3.1 SDS-PAGE of Nt-GST ...... 51 Figure 3.2 Western analysis using serum from Nt-GST immunised rabbits 52 Figure 3.3 Coomassie stained SDS-PAGE of serum and eluant from CNBr column ...... 54 Figure 3.4 Western blot analysis of the purified anti-Nrap antibody ...... 56 Figure 3.5 Western blot analysis of full length Nrap expression ...... 59 Figure 3.6 Western blot analysis of MG132 treatment on Nrap-flag transfected cells...... 60 Figure 3.7 Sub-cellular localisation of Nrap-flag...... 62 Figure 3.8 Nrap-flag localises to sub-nuclear structures...... 63 Figure 3.9 Schematic of the regions of Nrap sequence subcloned into pFLAG-CMV-4 ...... 65 Figure 3.10 Western blot analysis of Nrap deletion construct expression ...... 66 Figure 3.11 Sub-cellular localisation of Nrap deletion constructs ...... 68 Figure 4.1 Nrap associates with B23...... 78 Figure 4.2 B23 and p19ARF are located in the nucleolus ...... 80 Figure 4.3 Nrap associates with p19ARF ...... 82 Figure 4.4 Melting curve analysis of primers in real time quantitative PCR 84 Figure 4.5 siRNA mediated knockdown of Nrap does not affect the levels of B23 and p19ARF mRNA ...... 85 Figure 4.6 siRNA mediated knockdown of Nrap affects the protein levels of B23 and p19ARF...... 87 Figure 4.7 The effect of Nrap in cell proliferation ...... 91 Figure 5.1 Schematic of the region of mouse rDNA that is transcribed as the primary transcript...... 100 Figure 5.2 Evaluation of PCR labelled probes by agarose gel...... 101

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Figure 5.3 Northern blot analysis of rRNA transcripts by DIG labelled probes...... 102 Figure 5.4 Northern blot analysis of rRNA processing from DRB treated cells ...... 104 Figure 5.5 Northern blot analysis of rRNA processing from cycloheximide treated cells ...... 106 Figure 5.6 Northern blot analysis of rRNA processing after Nrap over expression ...... 108 Figure 5.7 Northern blot analysis of rRNA processing after Nrap siRNA treatment ...... 110 Figure 5.8 Image J analysis of rRNA transcripts affected by Nrap knockdown...... 111 Figure 6.1 Potential Model of Nrap Actvity...... 128

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LIST OF TABLES

Table 2.1 Primer sequences ...... 29 Table 2.2 Real-time quantitative PCR primer sequences ...... 31 Table 2.3 Primer sequences used to generate probes...... 32 Table 5.1 DNA probes for northern blot analysis...... 101

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LIST OF ABBREVIATIONS

ATP Adenosine triphosphate bp Base pair BSA Bovine serum albumin °C Degrees celsius cAMP 3′-5′-cyclic adenosine monophosphate CB Cajal Body cDNA Complementary DNA CNBr Cyanogen Bromide CPE Core promoter element Ct Threshold cycle C-terminal Carboxy terminal DAPI 4,6-diamidino-2-phenylindole DFC Dense fibrillar component dH2O Distilled water DIC Differential interference contrast DIG Digoxigenin-11-dUTP DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide DNA Deoxyribose nucleic acid dNTP 2′-deoxynucleoside 5′-triphosphate (N = A, C, G or T) DRB 5,6-dichloro-β-D-ribofuranosyl-benzimidazole EST Expressed sequence tags ETS External transcribed spacer FC Fibrillar center g Grams GAR Glycine/arginine rich GC Granular component GFP Green fluorescent protein GST Glutathione S-transferase hr Hours HRP Horse radish peroxidase

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IGS Intergenic spacer IPTG Isopropyl-1-thio-β-D-galactopyranoside ITS Internal transcribed sequence kb Kilobase pairs L Litre M Molar mAb Monoclonal antibody mg Milli grams min Minutes mL Milli litre mM Milli Molar

MQ H2O Milli Q water mRNA messenger RNA NDF Nucleolus-derived foci NES Nuclear export sequence ng Nano grams NLS Nuclear localisation signal NOR Nucleolar organiser region NPC Nuclear pore complex Nrap Nucleolar RNA associated protein N-terminal amino terminal OAS 2′-5′oligoadenylate synthetase OD Optical density ORF Open reading frame pAb Polyclonal antibody PAGE Polyacrylamide gel electrophoresis PAP Poly (A) polymerase PBS Phosphate buffered saline PCR Polymerase chain reaction PIC Protease inhibitor cocktail PMSF Poly-methyl sulfonyl fluoride PNB Pre-nucleolar bodies pre-rRNA Precursor ribosomal RNA

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RACE Rapid amplification of cDNA ends RBD RNA-binding domain rDNA Ribosomal DNA RNA Ribose nucleic acid RNase Ribonuclease RNP Ribonucleoprotein RRM RNA recognition motif rRNA Ribosomal RNA RT-PCR Reverse transcription – polymerase chain reaction SDS Sodium dodecyl sulphate sec Seconds siRNA Small interfering RNA SL-1 Selectivity factor one snoRNA Small nucleolar RNA snRNA Small nuclear RNA SRP Signal recognition particle SSU Small-subunit processome TBP TATA-binding protein TBS Tris buffered saline TFIIIA Transcription factor III A TTF-1 Terminator factor for polymerase I UBF Upstream binding factor UPE Upstream promoter element UTR Untranslated region w/v Weight per volume μg Micrograms μL Micolitres

XVIII Chapter One Introduction

CHAPTER ONE

GENERAL INTRODUCTION

1 Chapter One Introduction

1.1 Introduction

The novel nucleolar protein Nrap (nucleolar RNA associated protein) was recently found to be present in all eukaryotes. Nrap was later designated as Nol6 (in accordance with the Guidelines for Human Gene Nomenclature and authorised by the Human Genome Organisation Gene Nomenclature) but will be referred to as Nrap throughout this thesis. Nrap homologues have been identified in a number of species (Utama et al., 2002). In Saccharomyces cerevisiae, the homologue of Nrap (YGR090w/Utp22) has been associated with components involved in early processing of pre-rRNA (Bernstein et al., 2004; Grandi et al., 2002; Schafer et al., 2003). Preliminary characterization of this protein in mammalian cells also suggests a role in ribosome biogenesis (Utama et al., 2002). Further analysis of mammalian Nrap is essential to elucidate its functions and importance to synthesis of ribosomes. Not only will this information lead to a more complete characterization of a novel protein, but may also give further insight into the complex mechanisms involved in ribosomal biogenesis.

Biosynthesis of eukaryotic ribosomes is a major disbursement of cellular resources with actively proliferating cells generating tens of thousands of ribosomes per minute. Ribosomes play an indispensable role in mRNA translation by assisting in correct positioning and catalyzing peptide bond formation, ultimately making them essential for protein synthesis within cells. Mature ribosomes are composed of two subunits, the large 60S subunit (containing rRNAs 28S, 5.8S and 5S) and the small 40S subunit (containing 18S rRNA), formed by the association of many ribosomal proteins and the appropriate rRNA (Fatica and Tollervey, 2002).

By the mid 1960s it had been well established that the nucleolus was directly affiliated with the process of ribosome biosynthesis. The genes that encode ribosomal RNA (rRNA) were found exclusively in the nucleolus and often exceed 100 copies per cell. Transcription of ribosomal DNA (rDNA) has accounted for 50% of total cellular transcriptional activity (Olson et al., 2002). Subsequently, the transcribed RNA must be processed into mature RNAs prior to their incorporation into ribosomal subunits, an event dependent on the recruitment of many ribosomal and non-ribosomal proteins to the nucleolus. Many of the nucleolar proteins involved in the process remain

2 Chapter One Introduction uncharacterized. This thesis reviews information known about theses processes and examines the function of mammalian Nrap in relation to nucleolar activity.

1.2 Nucleolus

The nucleolus, a membraneless sub-organelle of the nucleus, was first identified by microscopy in the 19th century. It has since been determined that the major function of the nucleolus is ribosome biogenesis. Transcription of the ribosomal genes and their assembly into ribosomes is important in maintaining nucleolar structure. The structure of the interphase nucleolus is organized around the rRNA genes, located at special chromosomal sites referred to as nucleolar organizing regions (NOR) (Scheer, 1999). Nucleoli therefore vary in appearance depending upon the demands of the cells. Quiescent cells display much smaller ring shaped nucleoli in contrast to highly proliferating cells, such as Leydig cells, which display large complex nucleoli referred to as compact or reticulate (Carmo-Fonseca et al., 2000).

The typical nucleolus consists of three distinct and highly conserved substructural features, the fibrillar centre (FC), the dense fibrillar component (DFC), and the granular component (GC). The FC is recognized by fine fibril regions often located in the central portion of the nucleolus. The number and size of FCs varies depending on the demands of the cell. The DFC forms a network surrounding the FCs and in some cases will also extend towards the periphery. It is characterized by densely packed fine fibrils. Lastly, the GC can be identified by its granular appearance generally situated in the peripheral regions (Schwarzacher and Mosgoeller, 2000). Studies showed that the FC consisted of hundreds of copies of the rRNA genes with only a subset ever actively transcribed at the one time. The exact location of transcription of the rRNA genes has yet to be elucidated and is currently a controversial issue. Studies have shown that the genes being actively transcribed appear to have a more peripheral location than those lying dormant leading to the popular belief that transcription occurs on the border between the FC and the DFC (Derenzini et al., 1990; Thiry et al., 1991). It is widely accepted that ribosome biogenesis follows a vectorial pattern. Newly synthesized pre-rRNA transcripts have been detected in the DFC whereas later processing events are known to occur in the GC

3 Chapter One Introduction before they enter the nucleoplasm and are finally exported to the cytoplasm (Huang, 2002).

1.3 Nucleolus During Mitosis

Throughout mitosis, the transcription of rDNA is inactivated and the nucleolus disassembles, dispersing components to daughter cells before reforming at the end of mitosis. The disintegration of nucleoli occurs in various stages with the DFC disappearing first during prophase, followed by the GC (Dundr and Olson, 1998). Reassembly of the nucleolus begins at early telophase and coincides with the initiation of rDNA transcription (Dousset et al., 2000). The mechanisms controlling this cycle are currently largely unknown. Studies have shown one point of regulation occurs by RNA polymerase I initiation. Associated transcription factors have been shown to be susceptible to CDK1-cyclin-B activity. Phosphorylation by CDK1 results in abrogation of transcription while dephosphorylation restores the original function (Heix et al., 1998).

Nucleolar components disperse to varying locations during mitosis. The components of the FC remain associated with the rDNA in the NORs when the chromatin condenses (Weisenberger and Scheer, 1995). Members of this group include RNA polymerase I and its associated transcription factors, nucleolin and DNA topoisomerase, lending to the conclusion that basic transcriptional machinery remain together and intact during mitosis. On the other hand, rRNA processing factors target two major locations. These elements either partially distribute over the surface of chromosomes where they remain until telophase or alternatively scatter into the cytoplasm where they accumulate into nucleolus-derived foci (NDF) (Dundr et al., 1997). Less well defined is the fate of pre- rRNA transcripts. A study by Dundr and Olson (1998) presented evidence showing partially processed transcripts also distributing to NDF. These are most likely to be in association with the relevant processing factors. Results suggest that processing of transcripts is also repressed during mitosis and the association with the processing

4 Chapter One Introduction factors in NDFs is simply a method of protecting them from degradation (Dundr and Olson, 1998).

Upon reformation of the nucleolar structure and reactivation of transcription, RNA processing factors begin to assemble into prenucleolar bodies (PNBs) (Scheer, 1999). Interestingly, the composition of PNBs is similar to NDF. Significant differences between the two bodies exist though, with PNB being smaller in size and less mobile (Dundr et al., 2000). It was originally thought that PNBs migrate toward transcription within the NORs where they form the DFC followed by the GC. Although studies into the lifetimes of PNBs have revealed an ordered recruitment of their content (Savino et al., 2001), the migration of PNBs has recently been questioned. Results using fluorescently tagged proteins showed a directed flow between the PNBs and newly forming nucleoli although the precise mechanism of delivery of PNB components still remains unclear (Dundr et al., 2000). Nucleolar structure is complete after the nucleolar domains have reformed around the active NORs.

1.4 rDNA Transcription

The first step in ribosome synthesis is the transcription of rRNA genes. The resultant rRNA is neither capped or polyadenylated and is subsequently further processed into the mature 18S, 28S and 5.8S rRNAs. Efficient transcription of rDNA requires RNA polymerase I, a large complex enzyme with multiple subunits. Its function is the specialized transcription of rDNA with the assistance of many essential associated factors (Hannan et al., 1998).

RNA polymerase I can potentially interact with over 150 copies of mammalian rRNA genes, distributed over several chromosomes. The rRNA genes are arranged in tandem repeats with each set of genes separated by intergenic space (IGS). Close examination of the rDNA reveals short 5′ and 3′ external transcribed spacers (5′ ETS and 3′ ETS) located immediately before and after the rRNA genes while two Internal Transcribed Spacers (ITS-1 and ITS-2) separate the genes within the primary transcript. The rRNA

5 Chapter One Introduction genes are located in the order 18S, 5.8S and 28S and are transcribed as a single precursor rRNA (pre rRNA) designated as the primary transcript (figure 1.1).

Figure 1.1. Schematic of the mouse rDNA repeating unit. The rRNA genes 18S, 5.8S and 28S are flanked by ETSs and separated by two ITSs. This region is transcribed as a 47S pre- RNA primary transcript. The repeating unit is separated by the intergenic spacer (IGS). The rDNA promoter is located upstream of the rRNA genes. The IGS contains Sal boxes (termination sequences) and enhancers (Grozdanov et al., 2003).

Initiation of transcription of rRNA genes relies upon a promoter element located upstream of the genes. The vertebrate rDNA promoter consists of two domains, the core promoter element (CPE) and the upstream promoter element (UPE). Two auxiliary transcription factors, selectivity factor one (SL-1), a multiprotein complex containing TATA-binding protein (TBP) and TBP associated factors (Comai et al., 1992), and upstream binding factor (UBF), are also necessary for initiation, although the involvement of UBF varies amongst organisms (O'Sullivan et al., 2002). Although the precise mechanism of in vivo transcription remains to be elucidated, many studies have been performed in vitro. From these it is thought that SL-1 is involved in binding to the core promoter, a process facilitated by UBF and possibly other factors (Kwon and Green, 1994), forming a stable pre-initiation complex capable of multiple rounds of transcription (Kato et al., 1986). This initial complex causes the recruitment of RNA polymerase I and other associated factors to form a second complex. RNA polymerase I proceeds past the initiation complex, leaving it intact, while continuing on to transcribe the rDNA (Schnapp and Grummt, 1991).

6 Chapter One Introduction

Termination of transcription primarily relies upon the binding of transcription terminator factor for polymerase I (TTF-1) to a terminator element (Grummt et al., 1985; Grummt et al., 1986). Large variation exists between the terminator protein and the terminator sequence between species (Evers and Grummt, 1995; Reeder and Lang, 1997). Mammalian terminator sequences comprise of a motif called the Sal box (so called because it contains a Sal I restriction enzyme recognition sequence) (Grummt et al., 1985). Located at the 3′ end of the primary transcript are several copies of the Sal box, but interestingly, another Sal box, designated the promoter proximal terminator, can be found at the end of the IGS. Hence, transcripts can terminate immediately after the primary transcript or hundreds of base pairs downstream. From recent studies, it appeared that the proximal terminator might serve another purpose. Promoter occlusion is a phenomenon by which transcription through a promoter disrupts the semi-stable pre-initiation complex. Therefore TTF-1 binding to the proximal terminator prevents SL-1 and UBF from being displaced from the DNA by the transcribing machinery (Henderson et al., 1989).

Termination sequences are located in the IGS, but this DNA was originally considered junk or non transcribed DNA. It is now known that the IGS is transcribed and plays an important role in transcription enhancement and termination. Although large variations in the sequence and organization of the IGS exist between organisms, the underlying mechanisms are all similar. Generally, IGS regions contain an assortment of repetitive elements. While many are yet to be functionally defined, others have been elucidated. Mouse IGS contains simple sequence repeats, transposable elements, and long sequence blocks (Grozdanov et al., 2003). Repeats regularly consist of functional promoters. Recent reports implicated these regions in transcription enhancement, termination and replication (Grozdanov et al., 2003).

7 Chapter One Introduction

1.5 rRNA Processing and Assembly

Transcription of rDNA yields the initial 47S RNA transcript that undergoes multiple endonuceolytic and exonucleolytic cleavages, and modifications, to be processed into the mature 18S component of the 40S subunit and the 5.8S and 28S components of the 60S subunit. The initial steps in the RNA processing pathway involve three rapid sequential cleavages. The 5′ETS is trimmed to generate a slightly smaller 46S intermediate which is subsequently cleaved to eliminate the 3′ETS (45S transcript), followed by excision of the remaining 5′ETS, generating the 41S transcript (Bowman et al., 1983; Bowman et al., 1981; Mishima et al., 1985). Further processing at the ITS1 yields the mature 18S transcript, and a 36S precursor containing the 5.8S and 28S sequences (Raziuddin et al., 1989; Shumard et al., 1990). Cleavage at the 5′ end of the 36S intermediate gives rise to the 32S intermediate, which is again cleaved to generate the mature 28S transcript and 12S RNA (Hadjiolova et al., 1984). The 12S precursor is finally processed to form the 5.8S rRNA (figure 1.2).

The fourth rRNA, 5S RNA, is also incorporated into the large 60S ribosomal subunit. This RNA is unique in that it is not transcribed from the same rDNA as the other rRNAs. 5S RNA is transcribed by polymerase III from independent genes located in the nucleus. After transcription, 5S RNA is exported to the cytoplasm upon binding with transcription factor IIIA (TFIIIA) as a RNP particle (Pelham and Brown, 1980). In the cytoplasm TFIIIA is release allowing the 5S RNA to interact with the protein L5, forming a new complex which translocates back to the nucleus, to the nucleolus and into the 60S sunbunit (Allison et al., 1991; Murdoch and Allison, 1996).

8 Chapter One Introduction

Figure 1.2. Schematic representation of the mammalian rRNA processing pathway. The transcribed precursor transcript undergoes multiple cleavages to remove the 5′ETS, 3′ETS, ITS1 and ITS2, yielding the mature 18S, 5.8S and 28S rRNA (Strezoska et al., 2002).

During rRNA processing, ribosomal proteins start to assemble with the appropriate rRNA to form pre-ribosomal subunits. Early stages of ribosomal maturation occur in the nucleolus, but as they mature the pre-ribosomal particles migrate out to the nucleus and are independently exported to the cytoplasm for the final stages of assembly. The earliest pre-ribosome, the 90S particle, includes the U3 ribonucleoparticle (snoRNP) and other ribosomal (permanent component of ribosome) and non ribosomal (participate in processing or maturation of ribosome) factors. These are required for the early cleavages of the 47S primary transcript, separating the rRNA into the pre 40S and pre 60S subunits (Grandi et al., 2002; Venema and Tollervey, 1999). Many of the components of the 90S particle are also involved in synthesis of the 40S subunit, while processing of the 60S subunit predominately lacks any similarity (Grandi et al. 2002). Maturation of the small 40S subunit is comparatively much simpler than that of the larger 60S subunit. The pre 40S subunit is exported to the cytoplasm where it undergoes

9 Chapter One Introduction the final steps of maturation and assembly. Assembly of the large 60S subunit involves maturation of the 28S and 5.8S rRNA along with importing 5S into the nucleolus to associate with the complex, and finally export to the cytoplasm for the last stages (Fromont-Racine et al., 2003). Export of ribosomal subunits to the cytoplasm depends on the CRM1 export pathway through NPCs (Thomas and Kutay, 2003). Greater detail is known about the export of the 60S subunit. Reports demonstrated that its export is facilitated by Nmd3, a NES containing adaptor protein (Ho et al., 2000; Trotta et al., 2003).

1.6 snoRNAs and snoRNPs

Crucial to the rRNA processing pathway is the myriad of small nucleolar RNAs (sno RNAs). Although a few snoRNAs orchestrate cleavage of RNA, they primarily act as guides for covalent modifications of highly conserved and functional sites of rRNA. In mammalian systems, snoRNAs do not act alone on the rRNA but rather in a complex, termed small nucleolar ribonucleoproteins (snoRNP), consisting of many non-ribosomal proteins (Pinol-Roma, 1999; Tollervey and Kiss, 1997; Weinstein, 1999). Very few of the associated snoRNP proteins have been characterized. Recent advances have been successfully made in yeast using various genetic and biochemical analysis (Girard et al., 1992; Hughes and Ares, 1991; Tollervey et al., 1991). Discovery of proteins involved in mammalian ribosome biogenesis, in comparison to yeast, requires significantly more research.

A great deal more is known about snoRNAs with over 150 identified. The snoRNA component of the snoRNP binds by complementarity of short conserved regions to the pre-rRNA at the appropriate sequence, thus allowing proteins to perform the required modification. SnoRNAs can be categorized into two distinct classes, the C/D box and H/ACA box snoRNAs. 2′-O-ribose methylation of pre-rRNA requires the guidance of C/D box snoRNAs, while H/ACA box snoRNAs participates in the formation of pseudouridine (ψ) on the pre-rRNA (Balakin et al., 1996). U3 snoRNA, the most extensively studied snoRNA and also the most abundant, is classed as a C/D box snoRNA and participates in early cleavage steps of the primary transcript.

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1.7 Nucleolar Proteins

Proteomic research has recently advanced our understanding of nucleolar proteins, identifying over 350 in human nucleoli (Andersen et al., 2005; Andersen et al., 2002; Scherl et al., 2002). Remarkably, the genes that encode up to 30% of these proteins were previously uncharacterised (Andersen et al., 2002; Scherl et al., 2002). Proteins responsible for building pre-ribosome/ribosome subunits are referred to as ribosomal proteins with many being well characterised. Many nucleolar proteins or non-ribosomal proteins are also essential in ribosome biogenesis with their functions including transcription of rDNA, rRNA processing, and assembly, maturation and transport of subunits (Reeder, 1990; Sollner-Webb and Mougey, 1991). Fewer proteins from this second group are well defined suggesting that much is still to be learnt about nucleolar structure and function. Further examination into proteins residing in the nucleolus has revealed a third group of proteins that do not appear to be involved in the classical functions of the nucleolus. Interestingly, a small percentage of proteins have been related to cell cycle regulation and DNA repair just to name a few (Scherl et al., 2002).

One of the most well characterised nucleolar proteins is nucleolin. This protein was first described by (Orrick et al., 1973) and originally called C23 but is now mainly referred to as nucleolin. This highly abundant protein makes up to 10% of the total nucleolar protein in Chinese hamster ovary cells with a high percentage located in the DFC of the nucleolus, although a small portion can also be identified in the granular component (Biggiogera et al., 1990). The localization of nucleolin suggests a role in ribosome biogenesis. Many studies have confirmed this theory with nucleolin being implicated in a range of steps including maturation and assembly of ribosomes. Nucleolin is involved in the early 5′ ETS cleavage of newly synthesized rRNA. It has been shown to bind to the nascent rRNA and is also responsible for the recruitment of other factors such as U3 snoRNP (Ginisty et al., 1998).

Nucleolin has several domains responsible for its properties. The N-terminus of the protein consists of alternating acidic and basic domains. These domains are highly phosphorylated and the acidic domains are also proposed to be associated with binding of histone H1. Located immediately after the acidic/basic domains is a bipartite nuclear

11 Chapter One Introduction localization signal (NLS). Studies on the NLS demonstrated it was responsible for nuclear localization but not sufficient for targeting to the nucleolus (Schmidt-Zachmann and Nigg, 1993). In addition, the compilation of a nucleolar database has not resulted in the identification of a common motif for nucleolar targeting. Four RNA-binding domains (RBD), also known as RNA recognition motifs (RRM) are situated in the central portion of the protein. These domains direct RNA binding specificity. Individually each RBD binds insignificantly, but together the domains confer correct RNA binding. This insight suggested that nucleolin might bind different RNA targets, as there are multiple RBDs. The C-terminal is unknown in function. It contains a glycine/arginine-rich (GAR) domain, spanning approximately 70 amino acids. This domain contains several RGG motifs (arginine/glycine/glycine repeats) with asymmetrical dimethylation of the arginines (Pellar and DiMario, 2003). This domain has been shown to mediate protein-protein interactions but further research is required to fully understand this domain in more detail. The sub-localization of nucleolin in the nucleolus is dependent on the GAR and RBD being functionally intact with the acidic/basic region appearing to be redundant in this property (Schmidt-Zachmann and Nigg, 1993).

Protein B23, also termed nucleophosmin, NO38 or numatrin, is another major nucleolar phoshoprotein proposed to participate in ribosome biogenesis (Okuwaki et al., 2002; Savkur and Olson, 1998; Wang et al., 1994). B23 is primarily located in the granular component of the nucleolus where it exhibits a range of functions including nucleic acid binding, intrinsic ribonuclease, and molecular chaperone activity. B23 has also been implicated in shuttling nucleolar proteins to the nucleolus from the cytoplasm, possibly by binding to nuclear and nucleolar localization signals (Li, 1997; Valdez et al., 1994).

B23 is alternatively spliced from a single gene to be expressed as two isoforms termed B23.1 and B23.2. The isoforms differ only by the absence of 35 amino acids in the C- terminal end of B23.2 with B23.1 the predominant isoform expressed. B23 consists of three important functional domains, a non polar N-terminal domain, two highly acidic regions in the central portion, and a basic C terminal region. The central and C-terminal segments of the protein are responsible for the ribonuclease activity of B23 (Hingorani et al., 2000). Studies by Savkur and Olson, 1998, indicated that B23 ribonuclease

12 Chapter One Introduction preferentially cleaves pre-rRNA at a specific site in the ITS2 region. Another major property, nucleic acid binding, resides in the C terminus end of B23 (Wang et al., 1994). The non polar and acidic regions in the N-terminal half of the protein are necessary for the molecular chaperone properties of the protein (Hingorani et al., 2000). Chaperone activity has been demonstrated by comparing properties of B23 with the small heat shock proteins with known chaperone activity, and by testing with a variety of substrates such as carboxypeptidase and citrate synthase, commonly used to test for chaperone function (Hingorani et al., 2000; Szebeni and Olson, 1999). As the nucleolus contains extremely high concentrations of macromolecules, the crowded environment results in protein denaturation and aggregation unless a molecular chaperone such as B23 is present. It is only upon binding of the protein kinase CK2 to B23 that the substrate can be released (Szebeni et al., 2003).

Another important function of B23 is its role in shuttling proteins between the cytoplasm and the nucleolus. B23 has been reported to interact with nucleolar protein p120, nucleolin and the viral protein Tat (Li, 1997; Valdez et al., 1994). This interaction is likely to be via the nucleolar localization signals within these proteins. Evidence suggested that B23 might bind to nucleolar targeted proteins and act as a carrier protein to deliver them to the nucleolus, possibly being an important mechanism in nucleolar import of proteins.

Like B23, fibrillarin is a nucleolar protein with multiple functions in ribosome biogenesis including pre-rRNA processing, pre-rRNA modification, and ribosome assembly (Tollervey et al. 1993). It is also known to be associated with snRNA U3, U8 and U13. Fibrillarin is localised to the DFC of nucleoli (Biggiogera et al., 2001) but is also found in cajal bodies (CB), sub-nuclear structures containing components of RNA transcription and RNA processing pathways. Studies have revealed a link between CB and nucleolar architecture and RNA polymerase I activity but their relationship with nucleoli needs to be further elucidated. The role of fibrillarin in CB is also unclear. Fibrillarin is highly conserved from human through to yeast (termed NPO1) in its sequence and function (Jansen et al., 1991). Human fibrillarin is composed of 321 amino acids and has a predicted molecular mass of 36 kDa. It consists of three major structural domains. The N-terminus incorporates a GAR domain possibly involved in increased efficiency of nucleolar targeting (Snaar et al., 2000). The central portion of 13 Chapter One Introduction the protein resembles and RNA binding domain while a domain capable of forming alpha helices is situated at C-terminus of the protein. This third domain has been proposed to target fibrillarin to CBs (Snaar et al., 2000).

1.8 Nuclear and Nucleolar Transport

Proteins targeted to the nucleolus must first gain entry into the nucleus. The nuclear membrane is a phospholipid bilayer only permeable to small non polar molecules. Macromolecular exchange across the nuclear envelope occurs through nuclear pore complexes (NPC), large 125 MDa multiprotein structures spanning from the cytoplasm to the nucleoplasm. Cellular trafficking is controlled by the NPC by allowing small molecules (less than 50 kDa) to freely diffuse the NPC, while larger proteins such as ribosomal subunits and ribonucleoproteins (RNPs) rely on signal mediated transport (Feldherr and Akin, 1997).

The majority of proteins are imported into the nucleus by recognition of a nuclear localization signal (NLS). Three classes of signal have been identified to date. The first one discovered was a stretch of basic amino acids (KKKRK) from the SV40 large T- antigen (Kalderon et al., 1984) now classified as a classical or monopartite sequence. Another common NLS is the bipartite sequence, characterized by a stretch of basic residues interrupted by a five to twenty amino acid spacer region (Robbins et al., 1991). Import of most NLS containing proteins occurs via the importin family of proteins which function as adaptor and receptor molecules for transport through the NPC. To date, six importin α and twelve importin β proteins have been identified. The protein hnRNP A1 contains a third class of NLS, termed M9, which mediates nuclear import via a thirty-eight amino acid domain. This sequence differs significantly from the more classical NLS, as it does not contain any basic amino acids. M9 also functions as a bi- directional signal and uses the protein transportin rather than one of the importin family members to mediate nuclear transport of hnRNP A1 (Siomi et al., 1997).

Nuclear export sequences (NES) have also been identified on many proteins, characterized by a leucine rich amino acid sequence. The export protein, Crm1 or

14 Chapter One Introduction exportin, an importin β related protein, forms an export complex with the cargo protein via recognition of the NES. Other export mechanisms that are not Crm1 mediated also exist, however these are outside the scope of this review.

As the nucleolus is a membraneless sub-organelle, it would be logical to assume that nuclear bound proteins would be capable of diffusing in and out of the nucleolar compartment. Interestingly, many proteins targeted to the nucleolus have been shown to contain sequences necessary for nucleolar accumulation rather than simple diffusion.

Early discoveries of nucleolar targeting sequences were identified in viral proteins (Dang and Lee, 1989). In the HIV protein Tat, a region of multiple basic amino acids was found to be responsible for nucleolar accumulation. However, this domain alone fused to reporter proteins was unable to localize to the nucleolus. Nucleolar targeting was achieved by the incorporation of a NLS to the fusion protein (Dang and Lee, 1989). This pattern was also described more recently in Betanodavirus greasy grouper nervous necrosis viruses (GGNNV) protein α with an arginine rich sequence identified as the nucleolar localization sequence. Again, individually this sequence is incapable of targeting the nucleolus (Guo et al., 2003).

Viral proteins cannot be considered a normal nucleolar component. Much less is known about targeting of cellular proteins to the nucleolus. Recent studies have indicated that co-operation between different domains within the protein and their interactions with nucleolar components are required for nucleolar targeting. The major nucleolar protein nucleolin is imported into the nucleus via a bipartite NLS and the accumulation into the nucleolus is dependent upon two different protein domains. The RNP domain, implicated in binding to ribosomal RNA (Schmidt-Zachmann and Nigg, 1993) and the GAR domain are both necessary for nucleolar localisation. Interestingly, both of these domains are common to other nucleolar proteins as well.

Emerging evidence strongly suggests that nucleolar localization is more complicated than a single linear sequence, with protein-protein interactions playing an important role. A study into nucleolar localization of the protein p120 has demonstrated that the domain necessary for nucleolar accumulation binds to the nucleolar shuttling protein

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B23 (Valdez et al., 1994). Several other nucleolar proteins have been shown to bind to B23 including the viral protein Tat. Tat interacts with B23 via its nucleolar localization domain (Li, 1997). These results suggested that B23 might function as a carrier protein from the cytoplasm to the nucleolus. Nucleolar localization signals may be part of a bigger picture that includes interactions with shuttling proteins, nucleolar components, and functional domains within the proteins themselves.

1.9 Emerging Nucleolar Functions

Ribosome biogenesis has been known as the primary function of the nucleolus for over four decades now, but recently evidence has emerged to suggest that it may also be the site of other important cellular activities. Early clues to these non traditional roles came about by the identification of a number of proteins known not to participate in ribosome biogenesis localising to the nucleolus. A recent study has identified over 350 different nucleolar proteins localised to the human nucleolus. Preliminary functional analysis of these proteins suggested a proportion of these were not involved in ribosome biogenesis, reinforcing the concept of a plurifunctional nucleolus (Scherl et al., 2002).

One example of a multifunctional nucleolus is evident by data that suggests the nucleolus may be the site of signal recognition particle (SRP) assembly. In higher eukaryotes, the SRP consists of a 300 nt RNA component as well as six proteins (Walter and Blobel, 1980; Walter and Blobel, 1982) that together functions as a translational arrest machine (Walter and Johnson, 1994). The significance of the SRP in the nucleolus is not entirely understood but interestingly, it was shown that localisation of SRP RNA within the nucleolus is in a different region to ribosome synthesis (Politz et al., 2002). These results suggested that certain regions of the nucleolus might be specific for functions other than ribosome biogenesis. The nucleolus has also been implicated in the modulation of telomerase, a ribonucleoprotein responsible for synthesis of telomeric DNA repeats at the ends of chromosomes (Pederson, 1998). The RNA component of telomerase has been shown to localise to nucleoli (Etheridge et al., 2002; Yang et al., 2002). In addition, the nucleolus has been associated with mRNA production and processing of tRNA.

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To date, many of the non traditional roles of the nucleolus remain poorly understood. In contrast, the idea that the nucleolus may act as a cellular stress sensor in conjunction with p53, is an area receiving considerable research and generating much interest. Upon conditions of cellular stress, p53 levels are elevated leading to inhibition of cell growth and possibly apoptosis of the cell (Ryan et al., 2001). In normal growth condition, synthesis of p53 continues, but its accumulation is inhibited by its rapid degradation by the 26S proteasome. The protein Mdm2 mediates this process by acting as an E3 ubiquitin ligase, accelerating the nuclear export and ubiquitination of p53, marking it for degradation (Haupt et al., 1997; Momand et al., 1992). The other key player in this unique regulatory process is the tumour suppressor p19ARF (p14ARF in human). Oncogenic signals cause the release of p19ARF from the nucleolus to the nucleoplasm where it combines with Mdm2 to inhibit its activities upon p53. This allows for increased stability of p53 levels, enabling it to serve as a transcription factor upon a range of genes involved in cell recovery or cell apoptosis (Sherr, 2001).

The most obvious observation linking the p53 pathway with the nucleolus is that p19ARF is normally localised to the nucleolus (Weber et al., 2000; Zhang and Xiong, 1999). p19ARF has since been implicated in ribosome biogenesis by inhibiting the processing of the primary transcript (Sugimoto et al., 2003). It was also found to interact with the 5.8S rRNA (Ayrault et al., 2004) as well as a number of nucleolar proteins including B23 (Itahana et al., 2003). It has been suggested that p19ARF may also serve to activate a ribosome biogenesis checkpoint in response to hyperproliferative signals (Itahana et al., 2003; Weber et al., 2000).

In addition to the nucleolar role of p19ARF, the p53 pathway has also been linked to the nucleolus in a number of other ways. An interesting observation was made by disrupting the nucleolus with a variety of different p53 stress inducing agents, resulting in elevated p53 levels. This data suggested that nucleolar integrity may mediate p53 stability (Rubbi and Milner, 2003). p53 also regulates RNA polymerase I activity within the nucleolus. This occurred by p53 interfering with the assembly of the transcriptional initiation complex on the promoter of rDNA (Zhai and Comai, 2000).

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1.10 NRAP

Systematic screening for RNA binding proteins in mouse identified a novel transcript (Kennedy et al., 1996) later termed Nucleolar RNA associated protein (Nrap). Preliminary characterization of Nrap was performed by (Utama et al., 2002) and the summarized results follow.

Nrap homologues have been identified in a number of species including Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharmyces pombe. A high degree of sequence conservation was discovered amongst fungi, plants and animals. This data indicates that Nrap is a novel protein highly conserved between species, possibly possessing an essential role in eukaryotic cellular functions. This later observation was further supported by experiments in yeast where knockout studies of the yeast Nrap gene has shown to be essential (Winzeler et al., 1999).

Additional protein isoforms of Nrap have also been identified. The mouse cDNA sequence was obtained by performing 5′ rapid amplification of cDNA ends (RACE) resulting in the discovery of two isoforms. Alternate splicing produced a longer isoform (mNrap-α) consisting of 1141 amino acids giving a predicted molecular mass of 128kDa. The alternate isoform (mNrap-β) was found to be shorter with only 1054 amino acids and a predicted molecular mass of 118kDa. Mouse cDNA homologous to human EST (expressed sequence tags) was aligned to identify human Nrap. Three alternate splice variants were found designated hNrap- α (1146 amino acids), hNrap-β (1007 amino acids) and hNrap-γ (699 amino acids). Comparisons between mouse and human Nrap amino acid sequence revealed 88% conservation (Utama et al., 2002).

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1.11 Conserved Domains

Analysis of the Nrap amino acid sequence revealed the protein had very little similarity with other known proteins. Extensive blocks of homology were identified between species though, suggesting Nrap may be a new class of protein with highly conserved domains.

Nrap has been found to have weak homology to a specific domain known as the PAP/25A core domain. This domain is represented in three other proteins, poly (A) polymerase (PAP), 2′-5′oligoadenylate synthetase (OAS), and yeast topoisomerase I. PAP plays a crucial role in 3′ poly A tail synthesis in mRNAs (Martin and Keller, 1996; Martin et al., 2000; Raabe et al., 1991). DNA topoisomerase proteins relieve torsional stress during DNA transcription (Sadoff et al., 1995). OAS converts ATP into 2′-5′- linked oligomers when activated by double stranded RNA. Its only known function is to bind and activate endoribonuclease responsible for degradation of viral and cellular RNA (Rebouillat and Hovanessian, 1999; Rebouillat et al., 1999). A recent study by (Rogozin et al., 2003) have found amino acids 324-453 of Nrap aligns with the domain in the C-terminus from a large subset of OAS proteins. This domain corresponds to the PAP/25A core domain. Comparative analysis from this study reported this domain consists of an aspartate residue conserved in nearly all proteins identified. Predominately the domain has a helical structure with homology to another domain termed the ATP-cone, a regulatory nucleotide binding domain. Interestingly, Nrap was the only protein with the PAP domain that did not also contain a nucleotidyltranserase domain. Proteins comprising of a PAP/25A core domain appear to encompass diverse functions, yet still share the common feature of interacting with nucleotides. Hence, it has been speculated that the PAP domain may contribute to these interactions. More research needs to be performed to further clarify the functions of this domain.

The only other identified domain within Nrap was the nuclear import sequences, common to many nucleolar proteins. Two basic mono-partite NLSs were located at amino acid positions 29-32 and 98-101 in the mouse homolog. To confirm their activity, the N-terminal 311 amino acids of Nrap were cloned into a GFP vector and transfected

19 Chapter One Introduction into NIH3T3 cells. The fusion protein displayed florescence in the nucleus indicating the NLSs were functional in vivo.

Potential phosphorylation sites were also located within the Nrap sequence, however none of these have been explored further. These sites are for a range of proteins including cAMP-dependent kinase, protein kinase C and casein kinase II. Functionality of these sites still remains to be shown.

1.12 Nrap Expression and Distribution

Nrap protein expression levels were examined in a variety of different mouse tissue. Immunoblot results displayed expression in a wide range of tissue types including brain, lung, heart, muscle, liver, stomach, small intestine, colon, kidney, testis and spleen. Interestingly, the highest expression was observed in spleen tissue and the lowest expression in muscle tissue. The molecular mass of the expressed Nrap was shown to be approximately 130kDa indicating the larger isoform is predominately expressed. Low levels of the smaller isoform were also detected in spleen, small intestine and lung tissue (figure 1.3).

Figure 1.3. Distribution of Nrap expression in mouse. Immunoblot analysis of various adult mouse tissue with anti Nrap antibodies (Utama et al., 2002).

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After expression levels were determined for Nrap its sub-cellular distribution was studied and established to be in the nucleolus. This result was repeated in a wide range of cell lines, all exhibiting uniform distribution of Nrap throughout the interphase nucleolus. These results were confirmed by double labelling immunofluorescence studies with anti-B23 monoclonal antibodies. So far the sub-nucleolar location of Nrap has not been resolved.

The dispersal of mouse Nrap during mitosis could potentially provide insight into the role of Nrap in the nucleolus and was subsequently studied by immunofluorescence. Nrap was found to redistribute largely to the inner face of the condensed chromosomes as they separated during anaphase (Utama et al., 2002). The intensity of the Nrap signal was also thought to increase during telophase, coinciding with nucleolar reformation. Interestingly, the distribution pattern of Nrap during mitosis resembled that of B23 which also localizes to the periphery of chromosomes (Dundr et al., 1997).

1.13 Cytotoxic Drug Studies

Further insight into the role of Nrap within the nucleolus was obtained by studies with cytotoxic drugs. 5,6-dichloro-β-D-ribofuranosyl-benzimidazole (DRB), cycloheximide and actinomycin D all affect ribosome biogenesis at different stages and were employed to investigate Nrap’s role in these functions.

DRB, an adenosine analogue, inhibits mRNA synthesis by interfering with RNA polymerase II transcription. DRB results in disorganization of the nucleolus into its individual units visualized by unravelling of the nucleolus. After treatment with 25μg/ml DRB Nrap was observed to remain associated with the unravelled nucleolar structure suggesting a stable association is maintained with the sub-compartments of the nucleolus (figure1.4 A-D). Interestingly, this effect has been observed in other nucleolar proteins associated with rRNA transcription such as RNA polymerase I and UBF (Schofer et al., 1996).

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Actinomycin D is a DNA intercalater that preferentially targets G/C rich sequences. It is capable of specifically inhibiting RNA polymerase I transcription at concentrations less than 0.01 μg/mL, also causing the nucleolus to segregate into its components. RNA polymerase II can also be blocked at higher concentrations (Perry and Kelly, 1970). Actinomycin D has previously been shown to translocate nucleolar proteins B23, DNA topoisomerase 1, and PAP from the nucleolus. High concentration of actinomycin D caused dispersion of Nrap into the nucleoplasm (figure 1.4 I-L). A similar effect was observed with low concentration of actinomycin D (0.01 μg/mL and 0.0025 μg/mL) (figure1.4 E-F). Surprisingly, the pattern of dispersion did not follow that of known nucleolar proteins such as B23, which instead was granular in appearance (Utama et al., 2002). Nrap was therefore distinguished from transcriptional machinery such RNA polymerase I as they do not translocate from the nucleolus after Actinomycin D treatment.

The third drug, cyclohexidmide, was used to clarify the results obtained with actinomycin D. Cycloheximide is an antibiotic produced by S.griseus responsible for inhibition of translation in eukaryotes, leading to a lack of protein synthesis while still allowing transcription of the primary transcript. No gross changes were observed in Nrap localization after treatment with cycloheximide. Even after prolonged periods only a slight enlargement of nucleoli was observed (Utama et al., 2002). Results from the cytotoxic drugs all indicate that Nrap most probably associates with rRNA in the early processing steps.

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Figure 1.4. Effect of cytotoxic drugs on Nrap distribution. Panels (A) to (D) show Nrap immuno-fluorescence in asynchronous NIH3T3 cells treated with 25 µg/mL DRB: (A) before DRB treatment; (B) 3 hr after DRB treatment; (C, D) 30 and 90 min, respectively, after the removal of DRB. Nrap remains attached to the unravelled nucleoli during DRB treatment (B) and throughout the nucleolar reconstruction (C,D). Panels (E) to (H) and (I) to (L) show a time course of the effects of two concentrations of actinomycin D (0.0025 µg/mL and 0.2 µg/mL, respectively) at 0 hr (E, I), 2 hr (F, J), 4 hr (G, K) and 7 hr (H, L) (Utama et al., 2002).

Studies implicate a role for Nrap in ribosome biogenesis and hence any association with RNA was of interest. Treatment of permeabilised cells with RNaseA and DNaseI resulted in the dispersion of Nrap by RNaseA and no observed effect from DNaseI. These results imply an interaction with RNA either directly or indirectly.

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1.14 AIMS

The goal of this thesis was to establish the function of the protein Nrap in mammalian cells. It had previously been elucidated that Nrap is located in the nucleolus and results suggested it might play a role in ribosome biogenesis most likely through rRNA biosynthesis. A more complete characterization of Nrap would not only provide important functional data about this novel protein, but may also give further insight into the complex mechanisms involved in mammalian ribosomal biogenesis and nucleolar function.

The first aim was to create a range of tools to enable the study of Nrap in a mammalian system. The main objective of this aim was to raise an antibody against Nrap. This would enable endogenous Nrap protein levels to be examined. Moreover, the generation of a functional antibody would expand the repertoire of techniques available to investigate Nrap and expression and subsequent biological activity. In addition to the antibody, another vital tool was to generate a mammalian expression vector containing the Nrap sequence, thus allowing manipulations to Nrap levels by expression of a recombinant Nrap protein. To investigate potential functional domains within Nrap, the collection of expression vectors was extended to include deletion constructs of Nrap. Used in conjunction, the tools generated proved extremely useful in studying Nrap expression and function.

The second aim was the identification of protein binding partners of Nrap. It was hoped that knowledge of the proteins that Nrap interacted with would give further clues to the function of Nrap. Using the newly generated anti-Nrap antibody and expression vector, Nrap was found to associate with nucleolar proteins B23 and p19ARF. Analysis of these associations by Nrap knockdown revealed a regulatory relationship. Indeed, this data provided clues into the biological role of Nrap.

The final aim of the project was to investigate the involvement of Nrap in rRNA processing. The association found between Nrap, B23 and p19ARF suggested a role for Nrap in rRNA processing. Therefore, the effect of Nrap knockdown and over expression on ribosome biogenesis was investigated by northern blot analysis.

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CHAPTER 2

MATERIALS AND METHODS

25 Chapter Two Materials and Methods

2.1 MOLECULAR BIOLOGY MATERIALS AND METHODS

2.1.1 Transformation of DNA into Competent Cells

Competent E.coli cells, strain DH5α, (ECOS) were transformed by incubating with the recombinant plasmid and using the heat shock method. Typically, approximately 500 ng of plasmid was added to 20 μL of competent cells, placed on ice for 30 min before heat shocking at 42°C for 1 min and returning to ice for 5 min. 500 μL of warm LB media

(10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl made up to 1 L with dH20 water, pH 7) was then added to the cells and incubated on a shaker for 1 hr at 37°C. 100 μL was plated onto LB agar plates (15 g bacto-agar per litre LB media) containing the appropriate antibiotic (Kanamycin at 25 or 50 mg/L or Ampicillin at 50 mg/L). The agar plates were then incubated at 37°C for 16 hr.

2.1.2 Plamid DNA Preparation

Plasmids were prepared using the QIAprep Spin Miniprep Kit (Qiagen) as per manufacturer’s instructions. Plasmid DNA preparations for mammalian cell transfections were prepared using the QIAfilter Midiprep kit (Qiagen) as per manufacturer’s instructions.

2.1.3 Determination of DNA/RNA Concentration

Estimations of DNA/RNA sample concentrations were determined by spectrophotometric analysis. Samples were diluted 1:100 in MilliQ water and the OD (optical density) value at 260 and 280 nm determined. The concentration was then calculated according to the following equation: DNA concentration (μg/mL) = A260 x dilution factor x 50 μg/mL. The quality of the sample was determined by calculating the A260/ A280 ratio. A ratio between 1.5 and 2 indicated a good quality sample.

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2.1.4 Restriction Endonuclease Digests

Restriction endonucleases were obtained from New England Biolabs and Fermentas. Restriction digests were carried out as recommended by the manufacturer. In general, 0.5 μg of DNA was incubated at 37°C for 3 hr with 1-10 units of enzyme and the appropriate buffer in a final volume of 50 μL.

2.1.5 Agarose Gel Electrophoresis

Electrophoresis of DNA was carried out at room temperature using 1-2 % (w/v) agarose gels. Samples were run concurrently with λHindIII/EcoRI molecular weight markers (MBI Fermentas). Agarose gels were prepared by heating and dissolving the required amount of agarose (Progen) in 1 x TAE buffer (40 mM Tris-acetate (pH8.3), 1 mM diamino ethanetetra-acetic acid, disodium salt (EDTA)). After the solution was cooled to approximately 50oC, 0.3μg/mL of ethidium bromide was added and the gel poured. The DNA samples were mixed with 6 X loading buffer (Fermentas) and electrophoresis carried out in 1 X TAE buffer at 100V for 20-45 minutes. The DNA bands were then visualised under UV transillumination at 300nm.

2.1.6 Denaturing Agarose Gel Electrohphoresis

Formaldehyde based agarose gels (1%) were used to analyse RNA samples. 1g of molecular biology grade agarose (Progen) was dissolved in 85.2 mL DEPC treated water by heating until the solution boiled. Upon the solution cooling to approximately 60°C, 9.4 mL of 10 x MOPS (200 mM MOPS, 50 mM NaAc, 20 mM EDTA, pH 7) was added, followed by 5.2 mL 37% formaldehyde. The gel was poured and allowed to set. The gel was pre-run for 30 min at 60V. The RNA samples were mixed with loading buffer (50% formamide, 6% formaledyde, 10% glycerol, 0.05% bromophenol blue in 1 x MOPS) and heated to 60°C for 10 min. The RNA samples were loaded onto the gel

27 Chapter Two Materials and Methods and run at 60V for 3 hr in 1 x MOPS. Prior to northern blotting, gels were rinsed twice for 15 min in 20 x SSC (Sigma).

2.1.7 Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) was carried out in 20-50 µL volume containing 200

μM dNTP, 2.5 mM MgCl2, 1 x polymerase buffer, 50 μM of forward and reverse primers, 0.5 units of DNA polymerase Fermentas Taq (Fermentas) or Failsafe (EPICENTRE)) and 100 ng to 500 ng of template DNA. Reactions were performed in a Thermal iCycler PCR machine (BioRad). Typically cycling conditions included a pre- amplification denaturation cycle at 94oC for 3 min, 25 amplification cycles consisting of denaturation at 94oC for 30 sec, annealing at 55-65oC for 50 sec, extension at 72oC for 1 to 4 min followed by a final extension at 72oC for 10 min. Samples were then cooled and stored at 4oC. To identify any non-specific amplification products negative control reactions were included with every PCR. All PCR product sizes were confirmed by agarose gel electrophoresis.

2.1.8 Gateway Cloning

Primers were designed to incorporate the necessary DNA recombination sequences, and the pFLAG-CMV-4 incorporated a Gateway cassette (Invitrogen). Recombination was achieved following manufacturers instructions.

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2.1.9 PCR Primers

All primers were synthesised by Proligos. Table 2.1 Primer sequences Primer Name Primer Sequence Nrap forward 5′-ggggacaagtttgtacaaaaaagcaggctctGCTGTGAAGATGGGACCG-3′ Nrap reverse 5′-ggggaccactttgtacaagaaagctgggtgAACTCGACAAGGAATAATCGC-3′ NLS forward 5′-ggggacaagtttgtacaaaaaagcaggctccGGCATGGACAAAGAGAAGGC-3′ NLS reverse 5′-ggggaccactttgtacaagaaagctgggtcCTACTGGATCCGCTTGGTAACCT-3′ PAP forward 5′-ggggacaagtttgtacaaaaaagcaggcttcGAGACCCACATGCACTTGCT-3′ PAP reverse 5′-ggggaccactttgtacaagaaagctgggtcCTATCTCAGGACCTGGTAGCCG-3′ C-t forward 5′-ggggacaagtttgtacaaaaaagcaggcttcAGTGTCCTGCAGTTTCTGGC-3′ ARF forward 5′-ggggacaagtttgtacaaaaaagcaggcttcAGCATGGGTCGCAGGTTC-3′ ARF reverse 5′-ggggaccactttgtacaagaaagctgggtcCTAACATGCTAGACACGCTAGC-3′ Note: Lower case sequence represents Gateway recombination site introduced into primer.

2.1.10 Sub-cloning Mouse Nrap Constructs into pFLAG-CMV-4

The complete sequence for the mouse Nrap-β isoform had previously been cloned into the pBluescript vector (provided by Budi Utama, Baylor College of Medicine). This construct was used as a template to amplify mouse Nrap and the Nrap subsections. Primers Nrap forward and Nrap reverse were used to amplify full length Nrap (base pairs 1-3196), NLS forward and NLS reverse to amplify the NLS region (base pairs 75- 345), PAP forward and PAP reverse to amplify the PAP domain (base pairs 907-1039), NLS forward and PAP reverse to amplify the N-terminal (base pairs 75-1039), Ct forward and Nrap reverse to amplify the C-terminal (base pairs 1040-3196), and PAP forward and Nrap reverse to amplify the PAP plus the C-terminal (base pairs 907-3196). PCR products were verified using agarose gel electrophoresis. Sub-cloning each PCR product into the pFLAG-CMV-4 expression vector was achieved using the Gateway cloning system. All clones were verified by DNA sequencing.

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2.1.11 Sub-cloning p19ARF into pFLAG-CMV-4 p19ARF cloned into the vector MSCV-IRES-GFP (provided by Dr Charles Sherr, St. Judes Children’s Research Hospital, Tennessee, USA) was used as a template to amplify the DNA. Primers ARF forward and ARF reverse were used to amplify the entire p19ARF sequence. The PCR product was verified using agarose gel electrophoresis. Sub-cloning of the PCR product into the pFLAG-CMV-4 expression vector was achieved using the Gateway cloning system.

2.1.12 PCR Screening of Recombinant Plasmids

Recombinant clones were screened for those that contained the desired insert using a PCR based approach as follows. Five colonies were picked per transformation and used to inoculate 5 mL of LB medium containing the appropriate selective antibiotic. Following overnight incubation with shaking at 37ºC, 500 μL aliquots of the cultures were taken and centrifuged at 10 000 rpm for 2 min. The pellet was then washed three times with 100 μL of MilliQ water before being resuspended in 100 μL MilliQ water. The samples were boiled for 5 min, vortexed briefly and then centrifuged at 10 000 rpm for 2 min and the supernatant collected. 10 μL of the supernatant was then used as a template in the PCR reaction.

2.1.13 Sequencing of DNA

DNA sequencing reactions were performed using the ABI Prism dye terminator cycle sequencing reaction kit according to the manufacturer’s instructions and sequencing reactions were analysed by the Griffith University DNA Sequencing Facility (Griffith University, QLD Australian). Assembled sequence data was analysed using the AssmeblyLIGN (Oxford Molecular Group).

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2.1.14 Isolation of Total RNA

Total RNA was isolated from NIH3T3 and NS47 cells using the Micro-to-Midi Total RNA Purification System (Invitrogen) according to the manufacturer’s protocol.

2.1.15 Reverse Transcription PCR

Reverse transcription PCR was used to obtain first stand complementary DNA (cDNA) from 1 μg total RNA isolated from NIH3T3 and NS47 cells. First strand synthesis was performed with random primers (Progen) and RevertAid M-MuLV Reverse Transcriptase (Fermentas) according to the manufactures instructions.

2.1.16 Real-Time Quantitative PCR

Real-time quantitative PCR was performed with cDNA using primers outlined in Table 2.2. Table 2.2 Real-time quantitative PCR primer sequences Primer Name Primer Sequence Nrap Forward 5′-TGTGCCGATGTTACAGCTTCCACT-3′ Nrap Reverse 5′-TGTCCAGCAACGCCATAGACAGTT-3′ Actin Forward 5′-GGTCATCACTATTGGCAACG-3′ Actin Reverse 5′-TCCATACCCAAGAAGGAAGG-3′ B23 Forward 5′-TCGGCTGTGAACTAAAGGCTGACA-3′ B23 Reverse 5′-AGGAGCAGATCGCTTTCCAGACAT-3′ p19ARF Forward 5′-GGTCACTGTGAGGATTCAGC-3′ p19ARF Reverse 5′-CCTCTTCTCAAGATCCTCTCTAGCCT-3′

The real-time PCR was performed by iCycler iQ real-time PCR detection system (Bio- Rad) with a SYBR Green I Supermix kit (Bio-Rad) and run for 40 cycles at 95°C for 20 s and 60°C for 1 min. The PCR efficiency of the primers was examined by serially

31 Chapter Two Materials and Methods diluting the cDNA templates. The melting curve analysis was performed over the range 55°C to 95°C by monitoring SYBR Green fluorescence with increasing temperature (0.5°C increment changes at 10 sec intervals). PCR specific products were determined by clear single peak on the melting curves. Real-time PCR was performed in triplicate for each cDNA sample. Each gene’s mRNA level was acquired from the value of threshold cycle (Ct) of the real-time PCR relative to the level of actin using the formula 2ΔCt (ΔCt = actin Ct - gene of interest Ct).

2.1.17 Generation of DIG Labelled DNA Probes

DNA probes complementary to different regions of the mouse rRNA primary transcript were amplified from 10 ng of mouse cDNA (primers – Table 2.3). During the PCR reaction, probes were labelled with Digoxigenin-11-dUTP (DIG) (Roche) with the concentration of each nucleotide being 70 μM DIG-11-dUTP, 130 μM dTTP and 200 μM dATP, dGTP, dCTP. Successful generation of probes was analysed on a 2% agarose gel.

Table 2.3 Primer sequences used to generate probes. Primer Name Primer Sequence 5′ETS For 5′-TACTGGCTTGGGTCTGTCGC-3′ 5′ETS Rev 5′-ACCACAGCTGGCTCCACCAT-3′ 18S For 5′-TTCGATGGTAGTCGCCGTGC-3′ 18S Rev 5′-CCCGCTCCCAAGATCCAACT-3′ 5.8S For 5′-CTTAGCGGTGGATCACTCGG-3′ 5.8S Rev 5′-TCAACCGACGCTCAGACAGG-3′ 28S For 5′-GGGCCGAAACGATCTCAACC-3′ 28S Rev 5′-GGGCTAGTTGATTCGGCAGG-3′ 3′ETS For 5′-CTGCGGGCTTTCCCGTCGCA-3′ 3′ETS Rev 5′-CGCCGAGGATGGGGATCCCA-3′

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2.1.18 Northern Blot

3 µg of total RNA was separated on a 1% formaldehyde agarose gel in MOPS running buffer, and using a downward nucleic acid transfer system as described by (Chomcynski, 1992), was transferred onto Hybond-N+ membrane (Amersham Biosciences). The membrane was baked for 2 hr at 80°C and then stained with methylene blue (0.03% methylene blue in 0.3M sodium acetate pH 5.2) for 1-2 min or until bands were visible. Excess stain was removed by successive changes of water until the background was sufficiently reduced to assess equal loading. Staining was permanently removed by washing with 100% ethanol. The membrane was pre- hybridised with DIG Easy Hyb (Roche) at 50°C for 30 min. Probes were diluted to 2 μL/mL in DIG Easy Hyb and allowed to hybridise to the membrane at 50ºC overnight. Membrane was then washed twice with 2 x SSC (30 mM sodium citrate, 0.3 M NaCl, pH 7.0), 0.1% SDS at room temperature for 5 min, followed by two washes with 0.1x SSC, 0.1% SDS at 68ºC for 15 min.

2.1.19 Probe Detection

DIG luminescent detection kit (Roche) was used to detect the probes. Briefly, the membrane was washed for 5 min in washing buffer (0.1 M Maleic acid, 0.15 M NaCl; pH 7.5, 0.3% Tween 20) followed by 30 min incubation in blocking solution (Roche). Anti-digoxigenin-AP antibody was diluted 1/10 000 in bocking solution and incubated on the membrane for 30 min. The membrane was then washed 2 x 15 min with washing buffer, equilibrated for 5 min in detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5) and visualised with CSPD substrate (Roche).

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2.2 PROTEIN CHEMISTRY MATERIALS AND METHODS

2.2.1 Expression, Isolation and Purification of GST Fusion Proteins

Bacteria were transformed with recombinant pGex-N-terminal Nrap (pGex-Nt) plasmid and cultured overnight on LB-ampicillin agar. A single colony was picked, used to inoculate 5 mL of LB-ampicillin medium and was incubated overnight at 37°C with shaking. The overnight culture was then used to inoculate 500 mL of fresh LB- ampicillin medium which was incubated with shaking at 37°C until an OD600 of approximately 0.7 - 0.9 was reached. Expression of the GST fusion protein was then induced by the addition of 5 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) and 2% glucose and allowed to shake overnight. All steps from this point were performed at 4°C unless otherwise stated. The cells were pelleted at 5000 rpm for 30 min, washed by resuspending in 10 mL of PBS containing 1 mM PMSF (Sigma) and bacterial PIC (protease inhibitor cocktail) (Sigma) and pelleted again by spinning at 5000 rpm for 15 min. The cells were then resuspended in 20 mL of PBS containing 1 mM PMSF and bacterial PIC and lysed by sonication (on ice, 30% duty cycles, 20 seconds each for five times). To aid in the solubilization of the fusion protein, Triton-X 100 was added to the supernatant to a final concentration of 1%. The sample was then rocked for 30 min, centrifuged at 5000 rpm for 20 mins and the supernatant was transferred to a fresh tube. 200 μl of 50% Glutathione Sepharose (Sigma) bead slurry was added to the supernatant and rocked for 2 hr. The glutathione beads were pelleted and washed two times with 5 mL PBS containing 1 mM PMSF and bacterial PIC. Five volumes of elution buffer (20 mM Glutathione (Sigma), 0.1% Triton X-100 in 50 mM Tris-HCl, pH 8.0) were added and the solution rocked for 2 hr. The beads were then pelleted and the supernatant, which contained the expressed fusion protein, was collected. Any glutathione in the sample was removed by buffer exchange against PBS containing 1 mM PMSF, bacterial PIC and 0.1% Triton-X 100. A BCA Protein Assay was then performed to determine the protein concentration of samples. The quality of the expressed fusion protein was determined by SDS-PAGE analysis.

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2.2.2 Polyclonal Antibody Production

Polyclonal antibodies were raised in two rabbits immunized with 500 μg of the purified expressed N-terminal Nrap GST (Nt-GST) fusion protein emulsified in MPL + TDM + CWS Adjuvant System (Sigma), prepared according to the supplier’s instructions. Intradermal injections were performed on weeks 1, 4 and 6. Booster intradermal injections were given on weeks 8 and 10 using 300 μg of protein without adjuvant. Serum was collected from each of the rabbits at weeks 7 and 8, and a final bleed was performed by heart puncture on week 12. After collection of serum, the blood was incubated at 37°C for 1 hr. The blood was then stored at 4°C overnight. Samples were spun down at 2000 rpm at 4°C for 5 min, the serum was carefully collected and then the step repeated. The serum was filtered through 0.45 μm filter, aliquoted and stored at - 70°C.

2.2.3 Clearing GST Antibodies From Rabbit Serum

5 mL of serum was diluted with 5 mL of PBS and filtered through a 0.22 μM filter. 200 μg purified GST was added to the serum and incubated for 2 hr at 4°C with inversion. 100 μL of a 50% glutathione bead slurry was added to the serum and incubated for a further 2 hr at 4°C with inversion. The beads were then spun at 5000 rpm at 4°C for 5 min and the supernatant collected. This procedure was repeated three times. The serum was analysed to confirm that all of the unwanted GST had been removed by probing a western blot of the supernatant with anti-GST antibodies.

2.2.4 Generation of CNBr Sepharose 4B Activated Columns

Polyclonal antibodies against the N-terminal region of Nrap were affinity purified from rabbit sera by chromatography using CNBr sepharose 4B activated columns. A suspension of 0.7 g cyanogen bromide (CNBr) sepharose 4B activated beads (Amersham) in 6 mL of 1 mM hydrochloric acid (HCl) were loaded into a chromatography column (Pierce) and allowed to stand for at least 15 min to pack the

35 Chapter Two Materials and Methods beads. The columns were then washed with 300 mL of 1 mM HCl. 10 mL of coupling buffer (0.5 M NaCl, 0.1 M NaHCO3 pH 8.3) was passed through the column. 5-10 mg/ml of purified Nt-GST was prepared in 3 mL of coupling buffer and loaded into the column. Coupling was achieved by sealing the column and inverting it overnight at 4°C. Following this, the columns were allowed to stand for 30 min until all the beads were packed then washed with 10 mL of coupling buffer. The column was loaded with 10 mL blocking buffer (0.5 M NaCl, 0.1 M tris Cl pH 8.0), sealed and rocked gently at room temperature for 2 hr. After this incubation, the columns were washed three times with 5 mL blocking buffer, then 5 mL wash buffer (0.5 M NaCl, 0.1 M Na acetate pH 4.0) and finally with 50 mL PBS. A small volume of PBS containing 0.01% sodium azide was left to cover the surface of the beads to preserve the columns.

2.2.5 Purification of Antibody by CNBr Activated Sepharose

10 mL GST free serum was passed through the protein-bound column four times, keeping the eluant for analysis. Antibody bound to the column was washed with 50 mL PBS, followed by 3-4 mL of elution buffer (50 mM citrate buffer pH2.6). The eluant was collected in a tube containing 2 mL neutralising buffer (0.1 M tris pH 9.5) and kept on ice until the protein concentration was determined. The eluant was dialysed by stirring with 20% glycerol in PBS at 4°C overnight. The column was regenerated by adding 4 mL of blocking buffer and 4 mL of wash buffer respectively, repeating three times. This was then followed by 2 mL of blocking buffer and then a final wash with 50 mL PBS. A small volume of PBS containing 0.01% sodium azide solution was added to preserve the column.

2.2.6 Generation of HiTrap NHS-activated HP Column

A drop of chilled 1 mM HCl acid was added to the top of a HiTrap NHS-activated HP column (Amersham) to help air bubbles to be avoided. Three times 2 mL of 1 mM HCl, ice cold, was added to the 1 mL HiTrap column to wash out the isopropanol. 5-10 mg of

Nt-GST was dissolved in 1 mL coupling buffer (0.2 M NaHCO3, 0.5 M NaCl pH 8.3)

36 Chapter Two Materials and Methods and injected into the column. The column was sealed and allowed to stand for 1 hr room temperature. Washing and deactivation of the excess ligand unbound to the column was achieved by adding three times 2 mL buffer A (0.5 M ethanolamine, 0.5 M NaCl pH 8.3), three times 2 mL buffer B (0.1 M acetate, 0.5 M NaCl pH 4.0), and again three times 2 mL buffer A. The column was allowed to stand for 30 min before adding three times 2 mL buffer B, three times 2 mL buffer A, and then three times 2 mL buffer B. Finally 2 mL of PBS with 0.1% sodium azide was injected into column.

2.2.7 Purification of Antibodies by HiTrap NHS-activated HP Column

The column was washed with 3 mL of start buffer (0.05 M tris HCl, 0.15 M NaCl pH 7.5) followed by 3 mL elution buffer (0.5 M acetic acid). The column was then equilibrated with 10 column volumes of start buffer. Serum free of GST was applied to the column and recirculated four times. The column was then washed with 5-10 column volumes of start buffer before bound antibody was eluted with 1-3 column volumes of elution buffer. The eluant was neutralised using neutralising buffer (2 M tris HCl pH 9.0). The column was re-equilibrated by washing with 5-10 column volumes of start buffer.

2.2.8 Cross-linking Nt-GST to Glutathione Sepharose.

Nt-GST was expressed and captured with 200 μL of 50% glutathione beads as previously described. Instead of washing and eluting the fusion protein from the glutathione beads, Nt-GST was cross-linked to the beads for purification of the antibody. The beads were spun at 5000 rpm for 15 min at 4°C. The beads were resuspended in 5 mL of 200 mM hepes pH 8.5 and then spun for 2 min at 2000 rpm at 4°C. This wash step was repeated another two times. An aliquot was then removed for later analysis. 15 mg Dimethyl pimelimidate dihydrochloride (DMP) (Pierce) was dissolved in 2 mL of 200 mM hepes pH 8.5 and added to the protein bound beads. This was incubated for 30 min at room temperature rocking gently to allow cross-linking of the primary amine groups. DMP was removed after the incubation by spinning the

37 Chapter Two Materials and Methods slurry for 2 min, 3500 rpm at room temperature. 5 mL 0.2 M ethanolamine was then added and incubated for 30 min at room temperature, rocking gently, to terminate the cross-linking reaction. The slurry was again spun for 2 min at 3500 rpm at room temperature to remove the ethanolamine. The slurry was washed with 5 mL glycine buffer and then received two final washes with TBS + 0.5% sodium azide and stored until further use.

2.2.9 Purification of Antibody using Cross-linked Glutathione Beads

Glutathione beads cross-linked with Nt-GST were added to serum previously cleared of GST antibodies. This was incubated overnight at 4°C with inversion. The beads were then spun for 15 min at 5000 rpm at 4°C. Bound antibody was eluted from the cross- linked beads by adding 3 ml 2 M glycine buffer and inverting for 30 min at 4°C. The beads were spun for 15 min at 5000 rpm at 4°C. The supernatant containing the eluted antibody was retained and neutralized with 2 M Tris pH 9.0. The sample was buffer exchanged against PBS and concentrated by spin column. A BCA Protein Assay was performed to determine the protein concentration of the sample as previously described. The purified antibody was analysed on SDS-PAGE and probed against Western Blots of cell lysate.

2.2.10 Purchased Antibodies

The following primary antibodies were purchased for western blotting, co- immunoprecipitations or immunofluorescence applications: anti-GST polyclonal antibody (pAb), anti-Flag monoclonal antibody (mAb) (Sigma), anti-Flag pAb (Sigma), anti-B23/NPM mAb (Zymed), anti-p19ARF pAb (Calbiochem), and anti-actin pAb (Sigma). Anti-p53 mAb was a generous gift from Kienan Savage, Queensland Institute of Medical Research. Mouse- and rabbit-IgG (Santa Cruz) were used as control IgGs in co-immunoprecipitations. Secondary antibodies for western blotting were horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) and horseradish peroxidase- conjugated goat anti-mouse IgG (BioRad). Secondary antibodies used in

38 Chapter Two Materials and Methods immunofluorescence were Alexa Fluor 488 goat anti-mouse IgG (γ1) (Molecular Probes) and goat anti-rabbit texas red (Molecular Probes).

2.2.11 Cell Lysis

Mammalian cells were harvested fresh and pelleted by centrifugation at 200 rpm for 10 min at 4°C. The cell pellet was washed 3 times with ice cold PBS (138 mM NaCl, 2.7 mM KCl, 0.1 M Phosphate buffer, p7.4) and resuspended in 2-3 volumes of fresh, ice cold Lysis Buffer B (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EGTA, 2 mM EDTA,

25 mM β-glycerol phosphate, 0.1 mM Na4VO3, 25 mM NaF, 0.2% Triton X-100, 0.3% NP-40, 0.1 mM PMSF, 0.1 mM protease inhibitor cocktail (PIC) (Sigma), prepared fresh). The lysate was then rocked on ice for 30 min, cleared by centrifugation at 13000 g for 15 min at 4°C and the supernatant transferred to a clean tube.

2.2.12 BCA Protein Estimation

BCA protein assays (Pierce) were performed as per manufacturer's instructions. Briefly, a working reagent was prepared by mixing one part reagent B to 50 parts of reagent A. The reactions were performed in a 96 well microtitre plates by mixing 10 μL of each standard or unknown sample with 200 μL of the working reagent. The plates were then incubated for 30 min at 37°C and cooled to room temperature. The absorbance of the reaction product was measured at 562 nm on a plate reader and sample concentrations were then extrapolated from the BSA standard curve.

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2.2.13 SDS PAGE

SDS polyacrylamide gel electrophoresis (PAGE) was performed as described in (Laemmli et al., 1970). Samples for electrophoresis were prepared by mixing 1:1 in 1 x SDS sample buffer (10% glycerol, 2% SDS, 0.2% bromophenol blue, 100 mM Tris- HCl, (pH 6.8), 100 mM DTT added fresh) and boiling for 5 min just prior to loading. The percentage of acrylamide in gels (10-15%) used was dependent on the size of the proteins being analysed. Gradient gels (4-20%) were purchased from Invitrogen. The gels were run in a mini ProteanII cell (BioRad) at 15-40 mA for 1.25 hrs in PAGE buffer (25 mM Tris, 200 mM glycine, 0.1% SDS). Upon completion of electrophoresis gels were either stained or used for western blotting.

2.2.14 Staining of SDS PAGE

Gels were immersed in staining solution (50% methanol, 10% acetic acid, 0.1% coomassie brilliant blue in dH20) for 3-16 hr at room temperature and were destained by gentle agitation in Destaining solution (5% methanol, 7% acetic acid in dH20) until the background was removed and the bands were clearly visible.

2.2.15 Western Blotting

Transfer of proteins from SDS-polyacrylamide gels onto PVDF membranes (Immombilon-P, Millipore) by electrophoretic transfer was performed in a trans blot electrophoretic transfer cell (BioRad) using a procedure modified from (Towbin et al., 1979). Briefly, a sheet of PVDF membrane and 2 pieces of absorbent filter paper (Whatman 3MM) were cut to the same size as the gel. The membrane was briefly soaked in 100% methanol to activate the membrane and left to equilibrate in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 15% (v/v), pH 8.3), along with the gel and filter paper. The transfer unit was assembled by stacking a fibre pad, then a piece of 3MM filter paper, the gel, the membrane, another piece of 3MM filter paper, and finally another fibre pad. The unit was then submerged in transfer buffer in the electroblotting

40 Chapter Two Materials and Methods tank and the transfer allowed to proceed for 1 hr at a constant voltage of 100 V. The membrane was blocked in 5% Blotto (5% (w/v) skim milk powder in PBS) at room temperature for 1 hr, followed by incubation with the primary antibody (anti-GST at 1/2000, anti-Nrap pAb at 1/500, anti-flag mAb at 1/5000, anti-flag pAb at 1/3000, anti- B23 mAb at 1/10000, anti-p19ARF pAb at 1/1000, and anti-actin pAb at 1/5000), diluted in Blotto, for 2 hours at room temperature. The blot was then washed in PBS-Tween (0.1% Tween-20 in PBS) (3 x 10 min) and then incubated for 2 hours at room temperature with the secondary antibody (anti-mouse HRP and anti-rabbit HRP both at 1/3000), also diluted in Blotto. After removal from the second antibody solution, the blot was washed in PBS-Tween (3 x 10 min) and developed.

2.2.16 Development of Blots

The components of the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) were activated by mixing them 1:1, and applied to the membranes. The membranes were placed in the LAS-3000 Imager (Fujifilm) and digitalised images were generated. Fujifilm software (Image Gauge, Image Reader) was used to optimise the images captured by the LAS-3000.

2.2.17 Co-immunoprecipitation

Total protein lysate was first obtained using the lysis buffer B lysis procedure. Where appropriate, lysates were incubated at 30°C for 10 min with and without 100 μg/mL RNase A (Sigma) and insoluble material was remove by centrifugation at 15 000 rpm for 10 min at 4°C. For each co-immunoprecipitation reaction 1-2 mg of total protein lysate was used and 1 μg of antibody (anti-Nrap pAb, anti-flag pAb, anti-B23 mAb, and or anti-p19ARF pAb). 50 μL of a 50% slurry of protein A/G agarose beads (Amersham) pre-washed in lysis B buffer was conjugated to normal anti-mouse and rabbit IgG (control IgG used to remove non-specific binding to antibodies) and used to pre-clear the lysate by rocking for 90 min at 4°C. The beads were pelleted by centrifugation at

41 Chapter Two Materials and Methods

13 000 rpm for 15 sec at 4°C and the pre-cleared lysate was transferred to a fresh centrifuge tube. The beads from this pre-clearing step were washed 3 times in 750 μL lysis buffer B and kept for western blot analysis. An aliquot of the pre-cleared lysate was also taken for analysis by western blotting to track the proteins of interest throughout the procedure. 1 μg of the appropriate antibody was then conjugated to 50 μL beads and rotated at 4°C for 2 hr. The beads with the bound antibody were washed three times in 750 μL lysis buffer B. The pre-cleared lysate was then added and rocked at 4°C overnight. The bound immune complexes were pelleted by centrifugation at 13 000 rpm for 15 sec at 4°C, washed two times with 750 μL lysis buffer B, and then two times with 750 μL PBS. 30μL of 1 x SDS sample buffer was added to the samples, boiled and analysed by western blotting.

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2.3 CELLULAR BIOLOGY MATERIALS AND METHODS

2.3.1 Mammalian Cell Culture

NIH3T3, 293T and COS-7 cells were grown in Dulbecco's Modified Eagle's medium (DMEM) (Life Technologies), and NS47 cells (mouse skin fibroblasts, kindly donated by Dr. Hao Wang, Baylor College of Medicine, Houston, USA) were grown in RPMI (Life Technologies). Complete media was supplemented with 10% fetal bovine serum (Sigma), penicillin (100 U/mL), streptomycin (100 μg/mL) and glutamine (4 mM). NS47 growth media also contained 10 mM Hepes (Sigma). All cells were maintained in 5% CO2 at 37°C in a humidified incubator. Cells were harvested either by trypsin digestion or by using a cell scraper.

2.3.2 Transfection of Mammalian Cell Lines

Cells were grown to approximately 80% confluency in antibiotic-free media. Cells were transfected with the appropriate plasmid using Lipofectamine 2000 (Invitrogen), as per manufacturer’s recommendations. Cells were harvested and analysed 24 hr after transfection.

2.3.3 Immunofluorescence

Transfected cells grown on glass cover slips were washed with PBS (three times) and then fixed in 4% paraformaldehyde (PFA) for 30 min. The slides were washed again three times in PBS. The cells were permeabilised and blocked in Blocking Solution (PBS containing 1% BSA, 1% goat serum, 0.1% Triton X-100) for 1 hr. Primary antibody (anti-flag mAb at 1/1000, anti-flag pAb at 1/500 and or anti-B23 mAb at 1/2000) was diluted in Blocking Solution, added to the cells and incubated for 1 hr at room temperature. After washing three times with PBS, the cells were incubated with the appropriate secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG at 1/1000 and or goat anti-rabbit texas red at 1/1000) diluted in PBS containing 1% BSA for 1 hr

43 Chapter Two Materials and Methods at room temperature and then washed three times again. Finally, for nuclear counter staining, cells were incubated with DAPI stain (1/10000 dilution in PBS) for 10 min followed by a final wash three times with PBS. Cover slips were mounted onto the glass slides using glycerol:PBS (1:1, v:v) and the slides visualised using a Olympus BX50 microscope. The images were then optimised and adjusted for presentation using V++ software in conjunction with Adobe Photoshop 7.0.

2.3.4 Cell Treatment with Proteasome inhibitor MG132

After NS47 cells reached 70% confluency, the media was removed and fresh media containing 5 μg/mL MG132, an inhibitor of proteasome activity, was added to the cells. The MG132 was allowed to incubate with the cells for 6-16 hr before being removed and the cells harvested.

2.3.5 Nrap siRNA Knockdown

The Nrap siRNA oligonucleotide targeting exon 2 of mouse Nrap/Nol6 was (sense 5′- GGCAUGGACAAAGAGAAGGTT-3′, antisense 5′-CCUUCUCUUUGUCCAUGCCT T-3′). Blast analysis of this sequence showed no similarity with any other gene apart from Nrap itself. siRNA targeting the luciferase gene was used as a non-related gene control (sense 5′-GUCUGACAGUUACCAAUGC-3′, antisense 5′-GCAUUGGUAAC UGUCAGAC-3′). Both control and the Nrap siRNAs (at concentrations ranging from 0- 100 nM) were transfected using Lipofectamine 2000. Cells were harvested 24 hr after transfection.

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2.3.6 Cell Proliferation Assay

BrdU incorporation was measured using the BrdU Cell Proliferation Assay Kit (Calbiochem) following the manufacturers instructions. Briefly, 100 μL of cells at 2 x 105 cells/mL were seeded into a 96 well culture plate. Cells were transfected with either siRNA or Nrap constructs. Controls consisted of wells containing media only and cells present in the well but no BrdU label was added. All treatments were performed in replicate. The BrdU label was diluted 1/2000 and incubated with cells for 24 hr. Media was then removed from the wells and cells were fixed with 200 μL fixative/denaturing solution for 30 min. The contents of wells were removed by inversion and tapping on a paper towel. Anti-BrdU antibody was diluted 1/100 in antibody dilution buffer and 100 μL was added to each well and incubated for 1 hr at room temperature. Wells were washed three times with wash buffer by completely filling each well and then removing contents by. 100 μL of filtered Peroxidase Goat Anti-Mouse IgG HRP diluted in conjugate diluent (dilution depended on batch) was incubated with cells for 30 min. Wells were washed three times before the plate was flooded with distilled water and then removed. 100 μL of substrate solution was then added to each well and incubated in the dark for 15 min. In the same order, 100 μL of stop solution was added to the substrate solution in the wells. The absorbance was measured at dual wavelengths of 450-550 nm.

2.3.7 Cycloheximide and DRB Treatment of Cells

After NS47 cells reached 70% confluency, the media was removed and fresh media containing 25 μg/mL of either cycloheximide or 5,6-dichloro-β-D-ribofurano-syl- benzimidazole (DRB) was added to the cells. Cycloheximide was incubated for 24 hr and DRB was incubated for 4 hr before the total RNA was isolated.

45 Chapter Three Properties of Nrap and its domains

CHAPTER THREE

PROPERTIES OF NRAP AND ITS DOMAINS

46 Chapter Three Properties of Nrap and its domains

3.1 INTRODUCTION

Nrap is a novel nucleolar protein found to be present in all eukaryotes. It was first identified by Utama et al., (2002) while screening for RNA binding proteins. Two isoforms were detected in mouse, and termed α and β. The larger α isoform was identified by database searches of mouse ESTs that encoded portions of Nrap and then compiled in silico. The second isoform, Nrap-β, was compiled physically to obtain a full-length cDNA construct. Analysis of the mouse Nrap sequences revealed Nrap-α to be 1141 amino acids in length (pI 6.71) and a 3′UTR of a least 889 bp (ESTs covering this region did not extend to a poly A tail). Nrap-β was found to be slightly smaller in length, 1054 amino acids (pI 6.99), and contained 114 bp in its 3′UTR. The ORF of each isoform was found to be identical except for a small region at the extreme C- terminal end of the sequence where they are alternatively spliced.

Along with many other nucleolar proteins, the functional role of Nrap in mammalian cells is still largely unknown. Preliminary analysis of Nrap has identified it as a nucleolar protein likely to participate in ribosome biogenesis (Utama et al., 2002). To further elucidate the role of Nrap in the mammalian nucleolus, a number of different tools were developed. First, an antibody against Nrap was required. Utama et al., (2002) identified the first 170 amino acids of mouse Nrap to be an ideal antigen for raising antibodies and proved to be hydrophilic. This second property would be important in aiding in the preparation and isolation of the peptide to be used in raising the antibody. A polyclonal antibody against the N-terminal region of Nrap was raised in rabbits. Presented in this chapter are the results of the expression of the fusion protein used to generate the antibodies, purification, and evaluation of the specificity of the antibody. Downstream applications with the Nrap antibody are presented in the following chapters.

Analysis of the Nrap amino acid sequence revealed the protein has very little similarity with other known proteins. A putative NLS, and weak homology to the PAP/25A core domain were the only potential domains identified. Investigation of the speculated domains was vital to further characterise Nrap. Therefore, mouse Nrap was sub-cloned

47 Chapter Three Properties of Nrap and its domains into a mammalian expression vector to study recombinant Nrap in mammalian cell lines. In addition, deletion constructs were generated to determine functional domains within Nrap. Of particular interest were the domains responsible for nuclear and or nucleolar localisation of Nrap. Preliminary analysis of the functionality of the different regions of Nrap was performed using fluorescent microscopy.

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3.2 MATERIALS AND METHODS

The materials and methods performed in this chapter have been described in detail in Chapter Two sections:

• 2.1.1 Transformation of DNA into Competent Cells

• 2.1.2 Plasmid DNA Preparation

• 2.1.3 Determination of DNA/RNA Concentration

• 2.1.4 Restriction Endonuclease Digest

• 2.1.5 Agarose Gel Electrophoresis

• 2.1.7 Polymerase Chain Reaction

• 2.1.8 Gateway Cloning

• 2.1.9 PCR primers

• 2.1.10 Sub-cloning Mouse Nrap Constructs into pFLAG-CMV-4

• 2.1.12 PCR Screening of Recombinant Plasmids

• 2.1.13 Sequencing of DNA

• 2.2.1 Expression, Isolation and Purification of GST fusion proteins

• 2.2.2 Polyclonal Antibody Production

• 2.2.3 Clearing GST Antibodies from Rabbit Serum

• 2.2.4 Generation of CNBr Sepharose 4B Activated Columns

• 2.2.5 Purification of Antibody by CNBr activated Sepharose

• 2.2.6 Generation of HiTrap NHS-activated HP column

• 2.2.7 Purification of Antibody by HiTrap NHS activated HP column

• 2.2.8 Cross linking Nt-GST to Glutathione Sepharose

• 2.2.9 Purification of Antibody using Cross linked Glutathione Beads

• 2.2.11 Cell Lysis

• 2.2.12 BCA Protein Estimation

• 2.2.13 SDS PAGE

• 2.2.14 Staining of SDS PAGE

• 2.2.15 Western Blotting

• 2.2.16 Development of Blots

• 2.3.1 Mammalian Cell Culture

• 2.3.2 Transfection of Mammalian Cell Lines

• 2.3.3 Immunofluorescence

49 Chapter Three Properties of Nrap and its domains

• 2.3.4 Cell Treatment with Proteasomal Inhibitor MG132

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3.3 RESULTS

3.3.1 Production of anti N-terminal Nrap Antibody

A polyclonal antibody against Nrap was raised in rabbits using the N-terminal region of Nrap. The polypeptide antigen consisted of amino acids 8-114, fused to GST (Nt-GST), giving it a calculated molecular mass of 38 752 Da. Expression and purification of the Nt-GST fusion protein for injection into the rabbits was performed as outlined in Materials and Methods (section 2.2.1). This procedure was successful in purifying large quantities of protein but resulted in significant amounts of degradation of the protein. SDS-PAGE analysis showed that the Nt-GST was stable for only a short period before it was rapidly degraded (when stored at 4°C) (figure 3.1). The purification procedure of Nt-GST was optimised to reduce the rate of degradation (achieved by slowing the growth of the bacteria induced to express the fusion protein) and the protein was freshly prepared and analysed by SDS PAGE before each injection.

Figure 3.1 SDS-PAGE of Nt-GST. Panel A shows Nt-GST freshly prepared. Panel B shows Nt-GST that had been stored for 1 week and a significant degradation band running below it. M lanes contain protein markers with weights indicated.

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Purified Nt-GST suspended in MPL + TDM + CWS adjuvant was injected into rabbits. This adjuvant was used as an alternative to the classical Freund emulsion and is a powerful immuno-stimulant containing little or no toxic allergenic properties. Progression of the antibody response was monitored, by probing western blots loaded with purified Nt-GST, GST and NIH3T3 cell lysate with the crude rabbit serum. These results (figure 3.2) showed that the serum was able to strongly detect the recombinant Nrap protein (figure 3.2, lane 2) but did not detect endogenous protein from the cell lysate (figure 3.2, lane 1). The blots also showed that a high response had been raised against GST in the rabbits with the serum strongly detecting purified GST protein (figure 3.2, lane 3).

Figure 3.2. Western analysis using serum from Nt-GST immunized rabbits. Western blot analysis was used to evaluate the ability of the crude serum to recognize Nt-GST, GST and endogenous Nrap from a cell lysate. Lane 1 shows the serum detecting no endogenous Nrap from a NIH3T3 cell lysate. Lane 2 shows the serum strongly detecting the Nt-GST protein along with a large amount of non-specific binding. Lane 3 shows the serum recognizing GST. M lane contains protein markers with weights indicated to the left.

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GST cleanups were performed on the serum to remove the unwanted anti-GST antibodies raised. It was discovered that multiple cleanups (approximately four times per 5ml of serum) were required to eliminate all of the unwanted antibodies. As the crude serum was unable to detect endogenous Nrap from a cell lysate, it was decided to concentrate the antibodies. Affinity chromatography using CNBr activated sepharose beads conjugated to Nt-GST was used in an attempt to purify the anti-Nrap antibody. Coomassie stained SDS-PAGE analysis indicated that no antibody was located in the eluted solution but was instead found in the column wash solution (figure 3.3), indicating that the anti-Nrap antibody was not binding to the fusion protein bound to the column.

Alternatively, a HiTrap NHS activated column was used to purify Nrap antibodies. The efficiency of Nt-GST to couple to the column was determined to be 56%, yet again no antibody could be found in the eluate. It was suspected that the Nt-GST was degrading on the column and therefore had a reduced capacity to bind the antibody.

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Figure 3.3. Coomassie stained SDS-PAGE of serum and eluant from CNBr column. Lane 1 shows the bound fraction and as can be seen indicates low yield. Lane 2 shows almost all the antibody was contained in the unbound fraction. This indicates that the Nrap antibody was not binding to the Nt-GST on the column. The arrow indicates the position of the heavy chain of the antibody at approximately 50kDa. M lane contains protein markers with weights indicated to the right.

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A third method to purify the antibody involved cross-linking Nt-GST to glutathione beads to help reduce the rate of degradation of the recombinant protein. Imidoester Dimethyl adipimidate dihydrochloride (DMP) was used to cross link the primary amine groups from the GST portion of the fusion protein to the glutathione sepharose. It was found that cross-linked Nt-GST to glutathione sepharose was able to purify the Nrap antibody (figure 3.4). Interestingly, cross-linked Nt-GST was found to still adequately purified Nrap antibody several weeks after its expression and cross-linking. This result indicated that cross-linking with DMP substantially increased the stability of Nt-GST bound to glutathione sepharose.

The purified antibodies were evaluated using western blot analysis. Results showed that the antibody recognized the original protein used to raise the antibody, Nt-GST, and its degradation products (figure 3.4A). The western blots also demonstrated the antibody recognizing endogenous Nrap (figure 3.4B) shown by the clear band running at approximately 128 kDa. Interestingly, a second band was observed with the anti-Nrap antibody when the NIH3T3 cell lysate was not prepared fresh and immediately analysed by western blot (figure 3.4C). All lysates included optimised concentrations of protease inhibitors to limit degradation and those that were not immediately analysed were stored at 4°C. However, lysates older than one day (figure 3.4C shows lysate 2 days old) always contained the second band running at approximately 120 kDa. This delayed appearance of the second band suggested that it might be a degradation or cleavage product. An alternative explanation for the appearance of the second band was also considered. The western blot (figure 3.4C) showed that the top band corresponded in size to the larger Nrap-α isoform and the lower corresponded to the predicted molecular weight of the smaller Nrap-β isoform. It could therefore also be speculated that the anti- Nrap antibody detects both Nrap isoforms in NIH3T3 cells rather than degradation products. However, it was unable to be resolved why fresh lysate produced a single Nrap band while older lysate produced two.

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Figure 3.4. Western blot analysis of the purified anti-Nrap antibody. Blot A, lane 1, shows the antibody detecting Nt-GST. Blot B shows the antibody recognizing Nrap (128kDa) from a freshly prepared cell lysate of NIH3T3 cells (lane 1). Blot C is of NIH3T3 cell lysate after 2 days storage (lane 1). The Nrap antibody is detecting the same Nrap (128kDa) along with a second band (approximately 120kDa). Lanes labelled M contain protein markers with weight indicated.

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Despite the eventual identification of an adequate antibody purification procedure, extensive antibody concentrating was required to detect the endogenous Nrap on a western blot. Ultimately, a low yield of anti-Nrap antibody was generated which needed to be used at a low dilution of 1/500 on a western blot. As a result, the quantity of Nrap antibody was too low to use in all of the intended experiments. Therefore, some results in the following chapters use an alternative to the anti-Nrap antibody despite the fact that using the Nrap antibody would have been the more ideal choice.

3.3.2 Cloning of mouse full length Nrap

Due to the limited amount of Nrap antibody purified, an alternative tool, a mammalian expression vector containing the full length Nrap sequence, was developed. The complete mouse Nrap-β isoform had previously been cloned into the pBluescript vector (provided by Budi Utama, Baylor College of Medicine) and was used as a template for subcloning the Nrap-β sequence into the mammalian expression vector pFLAG-CMV- 4. Using gateway cloning (outlined in materials and methods section 2.1.8), a construct containing the β isoform was successfully generated with a N-terminal Flag tag.. Extensive attempts were also made to clone the Nrap-α isoform. For reasons unknown, all efforts to clone the α isoform remained unsuccessful. As there was no functional data on either isoform to suggest if one was predominant over the other, it was decided to investigate only the smaller Nrap-β isoform. For simplicity, the Nrap-β flag tagged construct was referred to as Nrap-flag.

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3.3.3 Nrap is an unstable protein that may undergo proteasomal degradation.

Expression of the Nrap-flag construct in NIH3T3 was analysed by western blot. When probed with the anti-flag antibody, over expression of Nrap-flag displayed what appeared to be extensive degradation. A time course of Nrap transfection as well as differing DNA concentrations was conducted to find a procedure that would limit the degradation. Figure 3.5A shows the result of the western blot. It was observed that even after as little as 3 hr, considerable degradation had already occurred. More importantly, expression of the Nrap-flag construct at the predicted size was not observed.

As the degradation bands were so intense, it was thought that it might be possible that the non-degraded Nrap band was too weak to be seen in comparison. Hence, the degradation bands were removed by cutting the blot just below the 100 kDa band. When the blot was exposed again, a single band running at approximately 120 kDa was observed (figure 3.5B, bottom panel). The blot was also probed with the anti-Nrap antibody, shown in figure 3.5B top panel, and the endogenous Nrap running at a similar molecular weight was observed. Levels detected with the anti-Nrap antibody were shown increased with increasing levels of Nrap-flag transfection. This data suggested that recombinant Nrap is being expressed as expected and is detected by both anti-Nrap and anti-flag antibodies, but undergoes rapid degradation soon after. It was speculated that protein levels of Nrap were under tight expressional control, with over expressed levels regulated at the cellular level by rapid degradation to compensate for the overall increase in total Nrap (endogenous and exogenous).

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Figure 3.5 Western blot analysis of full length Nrap expression. (A) Western blot of Nrap- flag transfections in NIH3T3 cells over differing time course and DNA concentrations. Blot was probed with anti-flag antibody that detected degradation of expressed recombinant protein. (B) Lysates of Nrap-flag transfected NIH3T3 cells were subjected to western blotting. The blot was cut just below the 100 kDa marker to remove degradation products. The top panel shows a western blot of endogenous Nrap detected by the anti-Nrap antibody and bottom panel shows recombinant Nrap detected by the anti-flag antibody.

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It was established that Nrap is an unstable protein but the mechanism behind the degradation remained unknown. Many nucleolar proteins have been found to undergo degradation by the 26S proteasome (Itahana et al., 2003; Kuo et al., 1996). To ascertain if this may also apply to Nrap, the proteasome inhibitor, MG132, was administered to NS47 cells transfected with recombinant Nrap. The turnover of the protein p53, known to be proteasome dependent, was also examined to show that the experiment was performing as intended. Western blot analysis of p53 levels showed an increase after MG132 treatment (figure 3.6, middle panel). Actin levels were also analysed to demonstrate equal loading (figure 3.6, bottom panel). Interestingly, the levels of Nrap- flag (figure 3.6, top panel) were found to increase slightly in cells treated with MG132 compared to those without treatment. This data suggests that degradation by the proteasome might be responsible for the rapid turnover of Nrap.

Figure 3.6 Western blot analysis of MG132 treatment on Nrap-flag transfected cells. NS47 cells transfected with Nrap-flag were treated with and without 5 μg/mL MG132 for 16 hr. Total cell lysates were analysed by western blot with the anti-flag pAb (detects Nrap-flag), anti-p53 mAb, and anti-actin pAb to detect changes in the indicated protein levels.

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3.3.4 Recombinant Nrap localises to sub-nuclear components

The ability of the anti-Nrap antibody to recognize endogenous Nrap in immunofluorescence studies was examined. NIH3T3 cells grown on cover slips were subjected to various fixation, permeabilisation and blocking procedures including 4% paraformaldehyde fixation, acetone/chloroform fixation, with altered levels of Triton-X and BSA (blocking agent). Different concentrations of anti-Nrap antibody ranging from 1/50 dilution through to 1/1500 dilution were also examined. Immunofluorescence results showed that endogenous Nrap was not specifically recognized under the assessed conditions (data not shown). This result was confirmed by comparison to the anti-B23 mAb, which showed pronounced staining of B23 in the nucleolus. Hence it was concluded that the anti-Nrap polyclonal antibody was not effective for immunofluorescent studies under these conditions.

To overcome this, the subcellular localisation of Nrap-flag was investigated. Cos-7 cells grown on cover slips were transfected with Nrap-flag and subjected to immunofluorescence after 24 hr. In approximately 30% of transfected cells, Nrap-flag was observed to localise to multiple sub-nuclear components (figure 3.7, panel A). This was confirmed by DAPI staining of the nucleus (panel B) and merging the images (panel C). The cell integrity was observed by DIC (panel D). Interestingly, a higher proportion of transfected cells (approximately 70%) displayed Nrap-flag in the cytoplasm (panels E-H). It is probable, that this large subset of Nrap-flag was in the process of degradation. Proteins sequestered for proteasomal degradation are exported to the cytoplasm for their degradation. This may explain the presence of Nrap-flag in the cytoplasm after over expression.

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Figure 3.7 Sub-cellular localisation of Nrap-flag. COS-7 cells were transfected with Nrap- flag for 24 hr. (A) A subset of transfected cells displayed Nrap-flag staining in sub-nuclear components (E) while a greater fraction were localised to the cytoplasm. (B, F) Corresponding DAPI stain of the nucleus from images A and E respectively. (C, G) Images of Nrap-flag staining were merged with images of nuclear staining to confirm sub-nuclear and cytoplasm localisation respectively. (D, H) Integrity of cells observed by DIC.

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To determine if the sub-nuclear regions that Nrap-flag localised to were nucleolar, the experiment was repeated but cells were also labelled with anti-B23 mAb, an established nucleolar marker (Olson et al., 2002). The results showed that the sub-nuclear regions of Nrap-flag staining did not correspond to the nucleolus (figure 3.8), indicated by the lack of overlap with the B23 staining (panel D). In fact, Nrap-flag staining was seen to accumulate near the periphery of the nucleolus. This data indicated that recombinant Nrap behaved different to endogenous Nrap and only endogenous Nrap co-localised with B23 in the nucleolus (Utama et al., 2002).

Fig 3.8 Nrap-flag localises to sub-nuclear structures. COS-7 cells were transfected with Nrap-flag for 24 hr. (A) Nrap-flag staining (B) Nucleolar B23 staining (C) DAPI staining of the nucleus (D) Images A-C were merged to show that only a small portion of Nrap-flag co- localised with B23 in the periphery of the nucleolus. (E) Cell integrity observed by DIC.

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3.3.5 Multiple domains are required for nucleolar localisation of Nrap

To determine the region of Nrap responsible for nuclear and subsequently nucleolar localisation, different sub-regions of Nrap were sub-cloned into the mammalian expression vector pFLAG-CMV-4 using gateway cloning (outlined in materials and methods section 2.1.8). These were designed around putative domains previously identified by database searches (outlined in introduction section 1.11).

Two NLSs had been identified in the mouse Nrap sequence. Previously, a GFP construct containing the first 933 base pairs of mouse Nrap had been transfected into NIH3T3 cells and by fluorescent microscopy, shown to localise to the nucleus. Although this data suggested that the NLSs were functional, it did not exclude the possibility that the construct contained another region responsible for directing it to the nucleus. Therefore, the region containing only the two identified NLSs and the small region between the two (base pairs 75-345) was sub-cloned (NLS-flag) (figure 3.9).

As mentioned previously (Introduction section 1.11), Nrap showed weak homology to the PAP/25A core (PAP) domain. It was suspected that this domain would be responsible for nucleolar localisation. This was because the PAP domain in other proteins was involved in interactions with nucleotides. From this information, it was inferred that the PAP domain might interact with rRNA, directing Nrap to the nucleolus to achieve this. Therefore base pairs 907-1039 were sub-cloned to generate the PAP- flag construct (figure 3.9). A third construct was generated, N-terminal-flag (Nt-flag), comprising base pairs 75-1039 to include the NLSs (figure 3.9). This construct was envisaged to target the nucleolus more efficiently because it contained the NLS and PAP domains.

The remainder of the Nrap sequence was sub-cloned to generate the C-terminal-flag (Ct-flag) construct (base pairs 1040-3196) (figure 3.9). No functional domains had been identified in this region and therefore it was of interest to see if it displayed any unique properties. In addition, another clone was constructed, CtPAP-flag, which encompassed the PAP region through to the end of the C-terminal (base pairs 907-3196) (figure 3.9).

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It must be noted that all the constructs were sub-cloned from the Nrap-β construct. The additional C-terminal region of the Nrap-α isoform was not studied.

Figure 3.9 Schematic of the regions of Nrap sequence sub-cloned into pFLAG-CMV-4. The NLS, PAP, N-terminal, C-terminal and PAP/C-terminal regions of Nrap were sub-cloned into the pFLAG-CMV-4 vector to generate the NLS-flag, PAP-flag, Nt-flag, Ct-flag and CtPAP-flag constructs respectively. The Nrap-flag construct was used as a template.

Expression of each construct was confirmed by western blotting. 293T cells were transfected with the five different construct and cells lysates were analysed on a western blot to determine that each construct migrated at the predicted molecular weight. The western blot (figure 3.10) showed that each of the constructs did run at the correct molecular weight. Each construct also showed good expression levels except for PAP- flag that showed lower expression levels.

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Figure 3.10 Western blot analysis of Nrap deletion construct expression. NLS-flag (lane 1), PAP-flag (lane 2), Nt-flag (lane 3), Ct-flag (lane4), CtPAP-flag (lane 5), flag vector (lane 6) and no transfection (lane 7) were transfected into 293T cells and their expression was analysed by western blot using the anti-flag mAb.

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The subcellular localisation of each construct in COS-7 was examined by fluorescent microscopy to determine the region responsible for nuclear and nucleolar localisation. It was observed that the NLS-flag construct localised specifically to the nucleus but not the nucleolus as expected (figure 3.11). PAP-flag on the other hand was excluded from the nucleus and localised to the cytoplasm (figure 3.11). Interestingly, Nt-flag was also shown to localise to the nucleus but not the nucleolus (figure 3.11). Finally, expression of Ct-flag and CtPAP-flag was found to be cytoplasmic only. Localisation of the empty flag vector was also observed as a control (figure 3.11). This data suggested that the NLS is required for nuclear localisation but not sufficient for nucleolar localisation. The region of Nrap responsible for nucleolar localisation was not identified. It is likely that multiple domains working together are necessary for nucleolar localisation, explaining why a single domain could not be identified in the above study.

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3.4 DISCUSSION

Nrap is a novel nucleolar protein with a previously unidentified function in mammalian cells. A polyclonal antibody as well as expression vectors of Nrap were developed to further study its function. These tools provided a powerful method to analyse Nrap and in this chapter were able to reveal important information about the properties of full- length Nrap as well as its putative domains.

Investigation into Nrap found it to be an unstable protein under certain conditions. This was observed in a number of the experiments conducted. Firstly, the Nt-GST fusion protein was unstable, making it difficult to purify the anti-Nrap antibody. The most pronounced degradation was demonstrated when recombinant Nrap was over expressed in mouse fibroblast cells, with degradation being detected as early as three hours after transfection. Treatment of cells with the proteasome inhibitor, MG132, indicated that the mechanism of degradation is likely to involve the 26S proteasome. Interestingly, significant degradation of endogenous Nrap was not observed in fresh NIH3T3 cell lysates. Together, this suggested that Nrap levels are tightly regulated. In normal cells, Nrap levels might be maintained within a very tight threshold. When levels are elevated as a result of transfection with a recombinant Nrap protein, it is expected that regulatory processes would be activated to immediately restore nominal Nrap levels. The reason why Nrap levels would be so tightly regulated is unknown but it suggests an important biological role for Nrap.

The anti-Nrap antibody was observed to detect two bands in some NIH3T3 cells lysates. The bands were suspected to correspond to the two Nrap isoforms, α and β, although this was not confirmed. If the assumption that these bands are indeed the alternative isoforms is correct, then it is interesting that their expression in NIH3T3 cells was relatively equal. This is because it was reported that in mouse tissue, the Nrap-α isoform was predominately expressed over Nrap-β (Utama et al., 2002). Spleen was the only mouse tissue to display higher expression levels of the β isoform, although it was still lower than Nrap-α. This indicated that the putative isoforms in NIH3T3 cells behave differently in regards to Nrap expression in comparison to mouse tissue. It is possible that the rate of proliferation is the controlling factor in expression of the two 69 Chapter Three Properties of Nrap and its domains

Nrap isoforms, as both spleen tissue and transformed cell lines both have relatively high cell divisions.

Using fluorescent microscopy, the subcellular localisation of the Nrap-flag construct was observed to be in unidentified sub-nuclear compartments in a fraction of cells expressing the recombinant protein. This data was unlike that of endogenous Nrap, observed to be strictly nucleolar in all mouse fibroblast cells (Utama et al., 2002). It must be noted that although the antibody from this study and that from Utama and colleagues were raised from the same peptide, for reasons unknown, each were substantially different in their ability to detect Nrap on western blots and in immunofluorescence. In this study it was demonstrated that the two Nrap isoforms are relatively equally expressed in mouse fibroblast cells. The report by Utama et al., (2002) showing endogenous Nrap localised to the nucleolus was also performed in NIH3T3 cells, indicating that both isoforms are nucleolar. Interestingly, the higher proportional of Nrap-flag expressing cells displayed the recombinant protein localised in the cytoplasm. It was found that over expression of Nrap-flag resulted in its rapid degradation, possibly through the proteasome. Therefore, it was assumed that the cytoplasmic pool of Nrap-flag represented the proteasomal degradation induced by the excessive levels of Nrap. This was based on reports that indicate proteins such as p53 and Smad3 are degraded by cytoplasmic 26S proteasomes (Shcherbik and Haines, 2004). Combined, this data supported the preliminary degradation studies of Nrap. A possible scenario may be that both endogenous Nrap isoforms may nominally reside in the nucleolus, but upon induction of excessive levels through over expression of the Nrap-flag construct, the tightly regulated threshold is compromised. This may result in the rapid degradation of the recombinant Nrap, observed by its export from the nuclear region to the cytoplasm where it would undergo proteasomal degradation.

Immunofluorescent studies of the sub-cellular localisation of the constructs containing the different regions of Nrap revealed that the NLS is required for import of Nrap into the nucleus. Both Nt-flag and NLS-flag contained the NLS and were the only constructs found to localise to the nucleus. The NLS-flag construct consisted only of the two identified NLS as well as the small region in between, and for the first time, demonstrated that it was specifically that region required for nuclear import. It would be

70 Chapter Three Properties of Nrap and its domains interesting to ascertain if both or just one of the NLS’s are required, but at this stage that remains unknown. The motif recognised in these studies to direct Nrap to the nucleus most closely resembles a bipartite NLS. However, point mutations within the identified NLS would be required to confirm this.

It was hypothesised that the PAP region would be responsible for nucleolar localisation of Nrap. The sub-cellular localisation of this domain (PAP-flag) was found to be cytoplasmic and apparently excluded from the nucleus and nucleolus. When the NLS and the PAP region of Nrap were included in the one construct (Nt-flag), again the sub cellular localisation was found to be nuclear, but not nucleolar. This suggested that the PAP region on its own is not responsible for directing Nrap to the nucleolus. Although it is speculated that this domain is involved in interactions with RNA, it may be that another unidentified domain involved in protein-protein interactions is accountable for nucleolar localisation of Nrap. It must be noted that the putative PAP domain identified within Nrap was found to have weak homology when compared with the other established PAP domain containing proteins (Utama et al., 2002). Therefore it is also possible that this region of Nrap is not a functional PAP domain at all, and it was purely coincidental that some similarity was identified. This would explain the lack of nucleolar localisation of PAP containing constructs.

The results of the localisation studies of the different Nrap regions revealed that more than one domain is necessary for nucleolar import. The NLS is most likely an essential component, initially required for nuclear import with other domains facilitating translocation of Nrap specifically to the nucleolus. Hence it was not surprising to find the Ct-flag and CtPAP-flag constructs were cytoplasmic. It could be deduced that the C- terminal region is involved in nucleolar accumulation of Nrap, simply because the N- terminal region was observed to be only nuclear. To verify this, ideally the NLS would need to be incorporated into these constructs and their sub-cellular localisation re- examined. To date, no functional domains have been identified in the C-terminal region of Nrap. Considering the size of this region (approximately two thirds of Nrap) it is logical to assume that it accounts for a number of Nrap’s functions. It cannot be dismissed that the PAP domain, in conjunction with a section of the C-terminal, are both required for nucleolar import.

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CHAPTER FOUR

NRAP ASSOCIATES WITH NUCLEOLAR PROTEINS B23 AND p19ARF TUMOUR SUPPRESSOR

72 Chapter Four Nrap associates with B23 and p19ARF

4.1 INTRODUCTION

Many proteins are implicated in the process of ribosome synthesis including ribonucleases, rRNA processing proteins, helicases, molecular chaperones, snoRNPs and RNA modifying enzymes, to name a few. Consequently, a crowded environment is created in the nucleolus, perfect for the myriad of interactions taking place. Despite over a century of research into the nucleolus, many of the proteins and subsequently their corresponding interactions remain uncharacterised. In this chapter, possible interactions between Nrap and other nucleolar proteins were investigated. Two potential binding partners, B23 and p19ARF, were identified and the significance of this relationship was examined.

Recently, the interaction between B23 and p19ARF tumour suppressor received a great deal of attention in relation to nucleolar function. B23 is a multifunctional protein predominantly involved in ribosome biogenesis (Okuwaki et al., 2002; Wang et al., 2002). B23 also possesses molecular chaperone activity, and shuttles between the nucleolus and cytoplasm (Li, 1997; Szebeni and Olson, 1999). On the other hand, p19ARF is widely regarded as an upstream activator of p53-dependent growth arrest, but it also plays an active role in rRNA processing and cellular growth (Bertwistle et al., 2004; Itahana et al., 2003; Sugimoto et al., 2003). It is well established that B23 and p19ARF interact (Bertwistle et al., 2004; Itahana et al., 2003). It was identified that the acidic and the domain responsible for oligomerisation of B23 were essential for an interaction with the extreme N-terminal region of p19ARF (Bertwistle et al., 2004). Subsequently, p19ARF has been found to induce the polyubiquitination and proteasomal degradation of B23 (Itahana et al., 2003). In return, B23 appeared to stabilise p19ARF despite it also being vulnerable to its own polyubiquitination (Kuo et al., 2004). In addition, p19ARF appeared to impede nucleoplasmic shuttling of B23. Interestingly, Mdm2 was found to antagonise these effects (Brady et al., 2004).

Different assumptions have been made concerning the functional significance of the B23-p19ARF relationship. Ithana et al., (2003) proposed that p19ARF regulates ribosome biogenesis in response to oncogenic signals by inhibiting B23 function. Interestingly, B23 has been reported to inhibit p19ARF function by targeting it to the nucleolus

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(Korgaonkar et al., 2005). Korgaonkar et al., (2005) proposed that B23 and p19ARF exist in a negative regulatory feedback loop. More specifically they state that their data suggests that B23 retains p19ARF in the nucleoli, consequently rendering it unable to interact with nucleoplasmic Mdm2, and ultimately inhibiting p53 activation leading to inhibition of cell growth.

Recent data on the B23-p19ARF interaction has suggested that it may play an intimate role in assisting in regulation of cell proliferation (Itahana et al., 2003; Korgaonkar et al., 2005). This was not entirely unexpected, as B23 has already been shown to affect cell proliferation in a number of ways. Its expression has been shown to be higher in actively proliferating cells and indeed in cancerous cells (Nozawa et al., 1996; Subong et al., 1999). Also, studies into its down regulation showed cells had delayed entry into mitosis (Jiang and Yung, 1999). Important to its involvement with p19ARF, B23 was shown to regulate the stability of p53 (Colombo et al., 2002). Although much remains to be elucidated about this functional interaction, the evidence thus far has emphasised the importance this interactions plays in key cellular processes.

74 Chapter Four Nrap associates with B23 and p19ARF

4.2 MATERIALS AND METHODS

The materials and methods performed in this chapter have been described in detail in Chapter Two sections:

• 2.1.1 Transformation of DNA into Competent Cells

• 2.1.2 Plasmid DNA Preparation

• 2.1.3 Determination of DNA/RNA Concentration

• 2.1.4 Restriction Endonuclease Digest

• 2.1.5 Agarose Gel Electrophoresis

• 2.1.7 Polymerase Chain Reaction

• 2.1.8 Gateway Cloning ARF • 2.1.11 Sub-cloning p19 into pFLAG-CMV-4

• 2.1.12 PCR Screening of Recombinant Plasmids

• 2.1.13 Sequencing of DNA

• 2.1.14 Isolation of Total RNA

• 2.1.15 Reverse Transcription PCR

• 2.1.16 Real-Time Quantitative PCR

• 2.2.10 Purchased Antibodies

• 2.2.11 Cell Lysis

• 2.2.12 BCA Protein Estimation

• 2.2.13 SDS PAGE

• 2.2.15 Western Blotting

• 2.2.16 Development of Blots

• 2.2.17 Co-immunoprecipitation

• 2.3.1 Mammalian Cell Culture

• 2.3.2 Transfections of Mammalian Cell Lines

• 2.3.3 Immunofluorescence

• 2.3.5 Nrap siRNA Knockdown

• 2.3.6 Cell Proliferation Assay

75 Chapter Four Nrap associates with B23 and p19ARF

4.3 RESULTS

4.3.1 Nrap associates with the nucleolar protein B23

It had previously been shown using immunofluorescence analysis that endogenous Nrap co-localised with B23 in the nucleolus (Utama et al., 2002). To explore a potential association between Nrap and B23, immunoprecipitations were performed. Asynchronous NIH3T3 total cell lysate were initially pre-cleared with protein A sepharose to remove non-specific binding. All proteins co-immunoprecipitated by anti- Nrap were subjected to SDS PAGE and subsequently western blotted. Blots were then probed for B23. Figure 4.1A shows the result of the co-immunoprecipitation of Nrap and B23. The top panel showed the western blot probed with the anti-Nrap antibody and demonstrated that endogenous Nrap is binding to and is immunoprecipitated by the antibody used in the pull down experiment. The results also showed that the antibody detected two Nrap bands in the pre-cleared cell lysate (top panel, lysate lane), presumably corresponding to the hypothesized isoforms Nrap-α (top band) and Nrap-β (bottom band) (must be noted that existence of isoforms not confirmed and may instead be degradation). Surprisingly, the co-immunoprecipitation and supernatant from co- immunoprecipitated beads did not display both Nrap bands at the same intensity. Instead the supernatant displayed predominately Nrap-α (top panel, supernatant lane) while the co-immunoprecipitation showed predominately Nrap-β (top panel, Co-IP lane). This result suggests that the majority of Nrap binding the anti-Nrap antibody used in the co-immunoprecipitation was Nrap-β.

Figure 4.1A, bottom panel, shows the same blot but was subsequently probed with the anti-B23 antibody. B23 (36kDa) was shown to be located in the pre-cleared lysate (bottom panel, lysate lane) but was not found to be non-specifically binding to the protein A sepharose (bottom panel, beads lane). The supernatant from the beads used in the co-immunoprecipitation was also found to contain B23 (bottom panel, supernatant lane). Finally, B23 was found to be co-immunoprecipitated by the anti-Nrap antibody (bottom panel, Co-IP lane). This result demonstrated an association between Nrap and B23. More specifically, this result indicated that B23 was pulled down by the Nrap-β isoform predominately bound to the antibody. Alternatively, the small amount of Nrap- 76 Chapter Four Nrap associates with B23 and p19ARF

α bound to the anti-Nrap antibody may have been responsible for the co- immunoprecipitation of B23.

Due to the limited quantities of the anti-Nrap antibody, the immunoprecipitation was repeated using over expression of the Nrap-flag construct in NS47 cells (mouse skin fibroblast cell line with low but detectable levels of endogenous p19ARF, an important feature for latter experiments) (figure. 4.1B). Any interaction with this construct was of interest because it was not shown to localise to the nucleolus. In this experiment, lysates were pre-cleared with mouse and rabbit IgG bound to the sepharose beads (IgG lane). This step removed non-specific binding against the beads as well as the antibodies. Using the anti-Flag pAb to bind to recombinant Nrap, B23 was again immunoprecipitated. In the reciprocal, anti-B23 mAb precipitated recombinant Nrap, confirming their association. Lysates were also treated with and without RNase A to determine if the interactions were dependent on RNA. Results showed that the amount of protein immunoprecipitated was reduced when treated with RNase A suggesting some involvement of RNA in the interaction.

77 Chapter Four Nrap associates with B23 and p19ARF

Figure 4.1 Nrap associates with B23. (A) Western blot analysis of NIH3T3 cell lysates immunoprecipitated with anti-Nrap and probed with anti-Nrap (top) or anti-B23 (bottom) (Co- IP lane). Whole cell lysate (lysate) and the supernatant (supernatant) from the immunoprecipitation were loaded to show total and unbound protein levels respectively. Beads used to pre-clear lysate (beads) were used as a control for non-specific binding. (B) NS47 cells that over express Nrap-Flag were immunoprecipitated with anti-flag and anti-B23 antibodies and probed with antibodies as indicated. Normal IgG was used as a control for non-specific binding. 10% of the total lysate volume was loaded to show whole cell lysate protein levels. Lysates were treated with and without 100 μg/ml RNase A as indicated.

78 Chapter Four Nrap associates with B23 and p19ARF

4.3.2 Nrap is in complex with B23 and p19ARF Tumour Suppressor

The interaction between B23 and p19ARF has recently received a great deal of attention in relation to its involvement in ribosome biogenesis and cell proliferation (Andrique et al., 2005; Ayrault et al., 2004; Itahana et al., 2003; Korgaonkar et al., 2002). This raised the question of whether Nrap also associates with p19ARF, possibly through its association with B23. Therefore, p19ARF was investigated as a potential binding partner of Nrap.

Endogenous levels of p19ARF in most cells lines is very low or non existent as its expression leads to p53 stabilisation and often cell death. The mouse skin fibroblast cell line NS47 showed detectable levels of endogenous p19ARF, although still relatively low, and was therefore used to examine endogenous p19ARF. NIH3T3 cells are also mouse fibroblasts but do not express p19ARF, making them a suitable control. To aid in the investigation, the p19ARF sequence was sub-cloned into the mammalian expression vector p-FLAG-CMV-4. Over expression of the p19ARF-flag construct for short periods would overcome problems with low endogenous p19ARF levels.

Fluorescent microscopy was utilised to confirm previous reports that B23 co-localised to the nucleolus with p19ARF. COS-7 cells were transfected with p19ARF-flag for 24 hr and subjected to immunofluorescence. Results showed that the recombinant p19ARF-flag did co-localise with endogenous B23 in this cell line (figure 4.2B), seen by the merged image in panel D. The anti-Nrap pAb was not effective in immunofluorescence, but previous reports (Utama et al., 2002) demonstrated that endogenous Nrap localised with B23 to the nucleolus. From this data, it was inferred that it was likely that endogenous Nrap would also co-localise with p19ARF even though the experiment could not be performed at this stage.

79 Chapter Four Nrap associates with B23 and p19ARF

Figure 4.2 B23 and p19ARF are located in the nucleolus. Indirect immunofluorescence microscopy of COS-7 cells over expressing the p19ARF recombinant protein. (A) Staining of p19ARF detected with anti-flag pAb and visualised by the texas red secondary Ab. (B) Staining of B23 detected by anti-B23 mAb, visualised with the Alexa Fluor 488 secondary Ab. (C) The nucleus was visualised by DAPI staining. (D) Panels A-C were merged to show co-localisation of B23 and p19ARF in the nucleolus. (E) Same field of cells observed with DIC.

80 Chapter Four Nrap associates with B23 and p19ARF

The potential interaction between Nrap and p19ARF was investigated by co- immunoprecipitation. Although Nrap-flag had been shown to predominately localise to unidentified sub-nuclear components, a small portion did co-localise with B23. In addition, recombinant Nrap was still able to precipitate B23. Therefore Nrap-flag was over expressed and immunoprecipitated using anti-p19ARF pAb (figure 4.3A). Western blot analysis of the immunoprecipitates revealed extremely low levels of p19ARF binding to the anti-p19ARF antibody (middle panel). Despite this, reasonable levels of recombinant Nrap were immunoprecipitated, detected by the anti-flag antibody (top panel). This suggests that Nrap is indeed a new binding partner of endogenous p19ARF tumour suppressor in NS47 cells. The presence of B23 in the immunoprecipitate complex was also examined. The western blot probed with anti-B23 (bottom panel) detected B23 indicating that the three proteins exist in the same protein complex.

To overcome low p19ARF expression levels in NS47 cells, the co-immunoprecipitation was repeated using over expressed Nrap-flag and p19ARF-flag (figure 4.3B). 293T cells were used to increase the transfection efficiency and expressed levels of the recombinant proteins. However, relatively low levels of p19ARF were still observed despite the over expression of p19ARF-flag (bottom panel), possibly a result of the antibody having a low affinity for p19ARF. Using the anti-flag antibody instead in the pull down was not an option because both constructs contained the flag tag. Regardless of the complications, anti-p19ARF antibody immunoprecipitated Nrap-flag, again confirming the association (top panel). RNase A treatment of lysates suggested that the Nrap - p19ARF association is also somewhat dependent on RNA. However verification will need to wait for better anti-p19ARF and anti-Nrap antibodies to be made available.

81 Chapter Four Nrap associates with B23 and p19ARF

Figure 4.3 Nrap associates with p19ARF. (A) NS47 cells that over express Nrap-flag were immunoprecipitated with anti-p19ARF and probed with anti-flag (top), anti-p19ARF (middle), and anti-B23 (bottom) (B) 293T cells over expressing Nrap-flag and p19ARF-flag were immunoprecipitated with anti-p19ARF and probed with anti-flag (top) and anti-p19ARF (bottom). Normal IgG was used as a control for non-specific binding and a 10% volume of the total lysate was loaded to show whole cell lysate protein levels. Lysates were treated with and without 100 μg/ml RNase A as indicated.

Reports indicate that p19ARF interacts with p53 and Mdm2 in response to hyperproliferative signal. Therefore, the potential interactions of Nrap with Mdm2 and p53 were also investigated. Using the approach described above, Nrap was not immunoprecipitated using either anti-p53 polyclonal antibodies or anti-Mdm2 monoclonal antibodies in either NIH3T3 or NS47 asynchronized cells (data not shown). This evidence suggests a lack of interaction between Nrap with either Mdm2 or p53 in both cell lines. This implied that the interactions reported here between Nrap, B23 and p19ARF are not a non-specific consequence of the protocols used to assess protein- protein interactions. This data is also in agreement with a recent report that the nucleolar function of p19ARF is independent of its interactions with Mdm2 and p53 (Itahana et al., 2003).

82 Chapter Four Nrap associates with B23 and p19ARF

4.3.3 Knockdown of Nrap using siRNA.

Although it was observed that Nrap associates with B23 and p19ARF in the nucleolus, the functional significance of this complex remained unknown. To further evaluate the association between these proteins, the consequence of reducing Nrap levels to a minimum was sought in the hope it would provide further insight into the role of this complex.

To knockdown Nrap levels, small interfering RNA (siRNA) was designed to target exon-2 of mouse Nrap. Evaluation of the knockdown procedure was performed by real time quantitative PCR to measure the changes in the mRNA levels of Nrap. As a control for specificity of the Nrap siRNA, it was decided to also examine the mRNA levels of B23 and p19ARF due to their close association with Nrap. The efficiency of each primer in the real time quantitative PCR were determined by melting curve analysis (figure 4.4).

Nrap siRNA was transfected into NS47 and NIH3T3 cells. As a control, parallel transfection with unrelated control siRNA, and no transfection, all under the same conditions were used to evaluate non-specific effects of the siRNA transfection. Total RNA was isolated after 24 hr from cells and the levels of mRNA specific to the genes of interest were measured by real-time quantitative PCR and normalized to the corresponding levels of actin mRNA. The Nrap siRNA was found to effectively knockdown mRNA levels of Nrap in both cells lines (figure 4.5A and B). As expected, there was no significant change in the mRNA levels of B23 and p19ARF after treatment with Nrap siRNA compared to the non-related siRNA and non-transfected NS47 cells (figure. 4.5A), and again in the levels of B23 in NIH3T3 cells (figure. 4.5B), indicating the specificity of the Nrap siRNA. This evidence also suggested that the reduction in the Nrap mRNA levels by siRNA silencing does not affect the transcriptional expression or stability of B23 and p19ARF mRNA despite there being an established association between these proteins.

83 Chapter Four Nrap associates with B23 and p19ARF

Figure 4.4 Melting curve analysis of primers in real time quantitative RT-PCR. The efficiency of Nrap, Actin, B23 and p19ARF primers in the real time quantitative RT- PCR was determined by examining the melting curve for a specific product seen as a clear single peak.

84 Chapter Four Nrap associates with B23 and p19ARF

Figure 4.5 siRNA mediated knockdown of Nrap does not affect the levels of B23 and p19ARF mRNA. (A) Real Time quantitative PCR was used to analyse the effective knockdown of Nrap and the relative levels of B23 and p19ARF mRNA in NS47 cells and (B) the relative level of B23 in NIH3T3 cells (no p19ARF expression). Cells were transfected with 100nM Nrap siRNA (Nrap-siRNA) for 24 hr. Transfection with unrelated control siRNA (ctr-siRNA) and untransfected cells (NS47-ctr and 3T3-ctr) were used as controls. All values were normalised against corresponding actin mRNA expression levels, and represent mean (± SEM) of triplicate measurement.

85 Chapter Four Nrap associates with B23 and p19ARF

4.3.4 Knock-down of Nrap affects the protein levels of B23 and p19ARF

To determine if Nrap knockdown was effective at the protein level, Nrap siRNA was transfected into NS47 cells at concentrations ranging from 0-100 nM. After 24 hr, total cell lysates were separated by SDS-PAGE, transferred to membrane, and probed with the anti-Nrap antibody to determine the changes in protein levels. Nrap knockdown displayed a quick response to treatment with more than 75% of its protein level diminished in a dose dependent manner (figure 4.6A). The experiment was not repeated in the NIH3T3 cell line as the anti-Nrap antibody had been fully expended at this stage.

The protein levels of B23 and p19ARF after Nrap knockdown were also examined in NS47 and NIH3T3 cell lines (Nrap knockdown in NIH3T3 cells was confirmed by real time quantitative PCR). In addition, western blots were probed for actin levels to show equal loading (figure 4.6A). B23 was observed to increase significantly as Nrap levels decreased (figure 4.6A). This result was repeated in NIH3T3 cells, indicating that the relationship between Nrap and B23 is irrespective of p19ARF presence. This evidence suggested that Nrap might play a role in maintaining the basal level of B23 in these cell lines.

Interestingly the protein levels of p19ARF in NS47 cells were also found to increase, albeit modestly, when treated with Nrap siRNA at 20-60 nM concentrations (figure 4.6A). However, a significant decrease was observed when higher concentrations of siRNA (80 nM or more) were administered to the cells. As described above, it has been confirmed that the Nrap siRNA targeting sequence was specific for this gene without any cross-reaction with any other associated genes which suggested the increase in B23 and the silencing of p19ARF in NS47 cells treated with high concentrations of Nrap siRNA was a direct consequence of the low levels of Nrap rather than a non-specific interaction of the Nrap siRNA with p19ARF mRNA.

86 Chapter Four Nrap associates with B23 and p19ARF

Figure 4.6 siRNA mediated knockdown of Nrap affects the protein levels of B23 and p19ARF. (A) Western blot of Nrap, p19ARF, B23, and actin expression levels. NS47 cells were transfected with a titrated range of Nrap siRNA (0-100 nM). After 24 hr, 20 μg of total protein lysate was loaded per lane (actin levels confirms equal loading) and changes in protein level were analysed (upper panel). The procedure was repeated in NIH3T3 cells showing expression levels of p19ARF, B23, and actin levels in lower panel. (B) Protein levels from NS47 cells were quantified using Image-J software analysis, and the values were normalized against its corresponding actin levels. (C) B23 protein levels were removed from the graph to highlight the changes in p19ARF protein levels.

87 Chapter Four Nrap associates with B23 and p19ARF

Observed protein levels from NS47 cells were quantitated using Image-J analysis software (NIH), normalized against the corresponding actin level, and graphed to emphasise the changes seen (figure 4.6B, C). Surprisingly the level of B23 dramatically increased up to eleven fold as the level of Nrap decreased (figure 4.6B). In the second graph (figure 4.6C), values for B23 were removed so that the changes in Nrap and p19ARF levels could be observed more clearly. This graph showed that the dramatic reduction in p19ARF levels unmistakably coincided with low levels of Nrap. It is unlikely that the decreased p19ARF levels were a result of a global reduction in cellular translation as B23 proteins levels were shown to increase in parallel experiments. These findings indicated that the effect of Nrap knockdown on these proteins is occurring by altering the rate of translation or (more likely) turn-over/degradation of these associated proteins.

4.3.5 Nrap may play a role in cell proliferation through its association with B23 and p19ARF

The effect of Nrap knockdown on cell proliferation was examined by BrdU incorporation. NS47 and NIH3T3 cells were transfected with 100 nM Nrap siRNA along with parallel control transfections of non-related control siRNA and no transfection. The BrdU label was administered to the cells 24 hr after transfection and allowed to incorporate for a further 24 hr, permitting 48 hr for any changes in cell proliferation to occur. After this time, changes were measured with the BrdU Cell Proliferation Assay Kit. The results were graphed (figure 4.7A) and it was observed that there were no changes between Nrap knockdown (Nrap-siRNA) and the controls (ctr- siRNA, 3T3-ctr, NS37-ctr). This indicated that a reduction in Nrap levels to both of these cells lines had no effect on cell proliferation over this time period.

Next, the effect of Nrap over expression in cell proliferation was investigated. This was performed by transfecting Nrap-flag into cells for 24 hr and then following the same procedure as outlined above. Over expressions of B23 and p19ARF were also included in the experiment for two reasons. Firstly, B23 and p19ARF over expressions have been shown to alter cell proliferation (Bertwistle et al., 2004; Itahana et al., 2003;

88 Chapter Four Nrap associates with B23 and p19ARF

Korgaonkar et al., 2005). Therefore their inclusion would provide an ideal control to show that manipulations to cell proliferation could be induced, and that the assay could successfully measure the changes. Secondly, it has been shown that when p19ARF is over expressed in conjunction with B23 or Mdm2, its anti prolific effects upon cells are inhibited (Brady et al., 2004). Hence, it was examined if Nrap-flag expressed together with p19ARF-flag could also rescue cells. This experiment was conducted in NIH3T3 cells due to their lack of endogenous p19ARF expression, allowing the effects of recombinant ARF-flag to be observed more clearly.

The results of the experiment (figure 4.7B) showed that over expression of Nrap-flag alone did not show any changes to cell proliferation when compared with expression of the empty vector. Taken alone, this would have suggested this is because exogenous Nrap is rapidly degraded when over expressed.

In the same experiment, ARF and B23 were over expressed. As stated in the literature, p19ARF resulted in an expected decrease in cell proliferation and B23 caused an increase, validating the ability of the assay to detect changes in cell proliferation. However, it must be noted that the observed changes were not as significant as shown in other studies (Brady et al., 2004). The reason for this is that this experiment employed transient transfections as the method to increase recombinant expression of the proteins of interest. Therefore the efficiency of the transfection must be taken into account. Using immunofluorescence, the transfection efficiency of the Flag construct in NIH3T3 cells was determined to be approximately 10%. This indicated that the majority of cells do not over express the proteins of interest thereby explaining why much smaller changes were observed in this experiment compared to those in the literature.

Nrap-flag was then over expressed in conjunction p19ARF-flag at two different concentrations. Interesting, both concentrations showed that Nrap antagonized the ability of p19ARF to reduce cell proliferation levels, observed by no change in the cell proliferation level when compared with the empty vector. Although rapid degradation of Nrap after over expression has been shown in the previous chapter, these results indicated the functional activity of Nrap-flag before its degradation. These preliminary cell proliferation studies indicated that much more research is needed to fully

89 Chapter Four Nrap associates with B23 and p19ARF understand the role of Nrap in cell proliferation, but highlight the importance Nrap plays. Moreover, this data reconfirmed that Nrap exhibits a close functional relationship with nucleolar proteins B23 and p19ARF.

90 Chapter Four Nrap associates with B23 and p19ARF

Figure 4.7 The effect of Nrap in cell proliferation. BrdU incorporation assays were used to measure changes in cell proliferation when (A) NS47 and NIH3T3 cells were transfected with 100 nm Nrap siRNA (Nrap siRNA), non-related control siRNA (control siRNA), and no transfection (No transf.) for 24hr and then cell proliferation was assessed after a further 24 hr. (B) Cells were transfected with empty vector (vector), Nrap, p19ARF, Nrap and p19ARF combined at a concentration of 50 ng and 100 ng each, or B23 for 24 hr and then cell proliferation assessed after a further 24 hr. All values were normalised against the empty vector and represent mean (±SEM) of four replicate measurements.

91 Chapter Four Nrap associates with B23 and p19ARF

4.4 DISCUSSION

In this chapter, it was demonstrated that Nrap associates with B23 and p19ARF tumour suppressor using immunoprecipitation analyses in mouse fibroblast cells. The association came as little surprise as endogenous Nrap shared the same subcellular location and a similar pattern of nucleolar translocation to the nucleoplasm after treatment with actinomycin D as B23 and p19ARF (Bertwistle et al., 2004; David-Pfeuty and Nouvian-Dooghe, 2002; Utama et al., 2002). Interestingly, the endogenous Nrap responsible for precipitating B23 (figure 4.1A) was probably the β isoform. Again this isoform was found to associate with B23 with p19ARF when the experiment was repeated with recombinant Nrap. However, in contrast to endogenous Nrap, recombinant Nrap was observed to localise more intensely to other sub-nuclear compartments. Small portions of recombinant Nrap, not visualised by fluorescent microscopy, are likely to be nucleolar, possibly for a short time, in order to explain the association with B23 and p19ARF. It is therefore likely that the Nrap-β isoform was involved in the association with B23 and p19ARF but at this stage, it cannot be dismissed that this band is simply a degradation product. In the previous chapter, the Nrap deletion constructs were shown to localise in the cytoplasm. Unfortunately, analysis of the region of Nrap binding to B23 in the nucleolus therefore could not be performed with the cytoplasmic deletion constructs.

The interaction between Nrap and B23 was shown to occur in cells lines with and without endogenous p19ARF expression (ie, NS47 versus NIH3T3 cells), indicating that this interaction occurred independently of the presence of p19ARF. A likely explanation for this is that all three proteins are members of a multi-component nucleolar complex. B23 and p19ARF are found in a functional high molecular mass complex (2-5 MDa) in the nucleolus with its formation thought to be dependent on rRNA (Bertwistle et al., 2004). This complex also consists of many other proteins (Bertwistle et al., 2004), with Nrap possibly being one of these (Leung et al., 2003). Therefore, it cannot be determined from this data if the interactions are direct or indirect.

To explore the dynamics of the interaction among the three proteins of interest, Nrap levels were knocked down using siRNA at different concentrations. Under these

92 Chapter Four Nrap associates with B23 and p19ARF conditions, it was found that the levels of B23 were up regulated in a dose-dependent manner. This result was found in two cell lines, one of which did not express p19ARF, suggesting the observed changes in B23 after Nrap knockdown were irrespective of p19ARF presence. Interestingly, the level of p19ARF was shown to increase modestly when treated with Nrap siRNA at 20-60 nM concentrations, although a significant decrease in p19ARF was observed at higher concentrations of siRNA (80 nM or more). Stable expression of B23 and p19ARF mRNA levels when Nrap was suppressed suggested that the effects are post-transcriptional, either at translational efficiency or more likely at the level of protein stability.

Previous reports indicated that B23 stabilizes p19ARF (Korgaonkar et al., 2005; Kuo et al., 2004). Therefore it is reasonable to assume that the modest increase in p19ARF levels is due to its increased stability, an action mediated by increased levels of B23 resulting from Nrap knockdown. It is interesting to note that although B23 enhances p19ARF levels, it also simultaneously inhibits p19ARF function by targeting the protein to the nucleolus (Korgaonkar et al., 2005). When Nrap was further suppressed to approximately 20% of its original level by siRNA treatment, p19ARF rapidly decreased, which was accompanied by a continuous increase in B23 levels. The trigger for this change in p19ARF behaviour is currently unknown. Korgaonkar et al, (2005) suggested that the magnitude of B23 expression impacts p19ARF regulation. It is plausible then, that B23 may reach a threshold in which it not only inhibits p19ARF function but also plays a role in mediating its degradation. Currently it is known that p19ARF is polyubiquitinated at its N-terminus which results in its subsequent degradation, although the mechanism remains to be elucidated (Kuo et al., 2004).

In addition, p19ARF promotes the polyubiquitination and degradation of B23 (Itahana et al., 2003) and has also been shown to prevent nucleoplasmic shuttling of B23 (Brady et al., 2004). Korgaonkar et al., (2005) suggested that p19ARF and B23 exist in a negative regulatory feedback loop. B23 might stabilise p19ARF, which in turn promotes the degradation of B23, maintaining levels possibly in response to hyperproliferative signals. Interestingly, the data from this study does not support this hypothesis, with the opposite being shown by B23 levels continuing to increase, and p19ARF levels suddenly plummeting when Nrap levels were diminished to 20% of its original. There was no

93 Chapter Four Nrap associates with B23 and p19ARF indication in the experiment that the increased stability of p19ARF was promoting the degradation of B23. It was probable that reducing levels of Nrap to 20% of its original level was the contributing factor to the unexpected decrease in p19ARF levels. This may explain the observation that p19ARF can decrease while B23 continues to increase has not been made in previous reports. It was speculated that Nrap regulated the interaction between p19ARF and B23, although it was undetermined if the signal passed through a number of unidentified regulators first.

Nrap knockdown was found to have no effect on cell proliferation. This was unexpected, as it had been previously reported that in yeast cells, Nrap knockout was lethal (Utama et al., 2002). In addition, it was shown that Nrap knockdown increased B23 levels. Previous studies showed that high levels of B23 induced cell growth (Colombo et al., 2002), yet this was not shown after Nrap knockdown. It is noteworthy to mention that Nrap knockdown over 48 hr or more did not demonstrate any change in Nrap levels (data not shown), unlike the data produced after 24 hr. This observation suggested that the siRNA became unstable after 24 hr of transfection, allowing the translation of Nrap to return to normal levels. Although Nrap knockdown induced a rapid change in B23 and p19ARF levels (within 24 hr), it was thought that variations in cell proliferation might require another 24 hr to respond. The results indicated that levels of Nrap, B23 and p19ARF had returned to normal by this stage, explaining why using this method of experimentation, no changes in cell proliferation was observed after Nrap knockdown.

On the other hand, when Nrap was over expressed with p19ARF, changes were seen in cell proliferation. More specifically, Nrap was found to rescue the cells from the anti- prolific effects of p19ARF. This data confirmed that the Nrap-β isoform (represented by recombinant Nrap) is functional before it is rapidly degraded (section 3.3.3). In the same manner as Nrap, proteins B23 and Mdm2 have also been reported to antagonise the activity of p19ARF when over expressed in unison (Brady et al., 2004). Simply, it was suggested p19ARF binding partners were triggered to switch and that the ability of p19ARF to dictate growth arrest through the p53 pathway was determined by the stoichiometry of its binding partners (Brady et al., 2004). In addition, reports indicated that B23 is distributed throughout a number of discrete functional pools within cells and

94 Chapter Four Nrap associates with B23 and p19ARF only a small fraction of the total B23 is associated with p19ARF (Bertwistle et al., 2004). It was also discovered that under certain condition, in this case over expression of a deletion mutant construct, caused p19ARF to redistribute from the 2-5 MDa high molecular weight complex to smaller 500 kDa complexes. Curiously, this inhibited p19ARF to retard rRNA processing, suggesting that a change in binding partners and hence translocation is a mechanism employed to regulated at the very least the activity of p19ARF. It is possible Nrap activity is also regulated by the complex it associates with. Moreover, it appeared increased levels of Nrap, or more specifically the Nrap-β isoform, like B23 and Mdm2, causes p19ARF to switch binding partners or possibly induce competition for the binding of p19ARF. Ultimately this enabled Nrap to inhibit the most well characterised function of p19ARF, the induction of growth arrest or apoptosis through the p53 pathway.

95 Chapter Five Nrap is Involved in rRNA Processing

CHAPTER FIVE

NRAP IS INVOLVED IN rRNA PROCESSING

96 Chapter Five Nrap is Involved in rRNA Processing

5.1 INTRODUCTION

The results presented in the previous chapters demonstrate that Nrap is a nucleolar protein that exists in a complex with other nucleolar proteins B23 and p19ARF. It was established that knocking down Nrap levels dramatically affected the levels of B23 and more modestly p19ARF, both in a dose dependent manner. These results indicate that Nrap has a specific functional role in the nucleolus. Considering that the major role of the nucleolus is ribosome biogenesis, it is reasonable to assume that Nrap may participate in this process. In addition, previous results using cytotoxic drugs that affect different stages of ribosome biogenesis in mammalian cells suggest that Nrap may be involved in early processing of rRNA (Utama et al., 2002). Moreover, both B23 and p19ARF have been shown to participate in rRNA processing, lending to the hypothesis that Nrap may associate with these proteins in regulating rRNA processing.

There is further evidence that suggests a role for Nrap in ribosome biogenesis. In Saccharomyces cerevisiae, the Nrap homolog YGR090w has been implicated in early processing and assembly of pre-rRNA. In a number of different studies it was shown to fractionate with the 90S pre-ribosome (Bernstein et al., 2004; Grandi et al., 2002; Schafer et al., 2003). Proteomic studies have revealed that many components of this 90S pre-ribosome are also mutual to a large RNP, the small-subunit (SSU) processome (Krogan et al., 2004). The SSU processome is required for processing of the small 40S ribosomal subunit rRNA and is known to contain the U3 snoRNA and over 30 proteins (Bernstein et al., 2004; Dragon et al., 2002; Grandi et al., 2002). YGR090w was identified as a component of the SSU processome by coimmunoprecipitation with U3 snoRNA and Mpp10, a protein specific to the SSU processome (Bernstein et al., 2004; Grandi et al., 2002). YGR090w was later renamed Utp22 to reflect its function in biogenesis of the small ribosomal subunit (Bernstein et al., 2004). In the same study, Utp22 depletion resulted in defects in rRNA processing (Bernstein et al., 2004).

When investigating rRNA processing, it must be noted that significant differences exists between species. This is partly due to considerable variation in the rDNA sequence. Interestingly, a high level of conservation is found in the regions encoding the 18S, 5.8S and 28S rRNA genes, unlike the rest of the rDNA sequence. Consistent with previous

97 Chapter Five Nrap is Involved in rRNA Processing chapters, Nrap was to be examined in the context of mammalian cells, specifically mouse NS47 cells (chosen because known to express detectable levels of p19ARF). The mouse rDNA repeat unit consists of 45309 base pairs with base pairs 1 through to 13404 being transcribed as the primary transcript or the 47S rRNA. This transcript is then processed to the mature rRNA transcripts (outlined in detail in Chapter One, figure 1.2).

Northern blot analysis was used to study rRNA processing defects after manipulations to Nrap levels. This technique provided a direct relative comparison of the abundance between the RNA samples. Even though there has recently been an influx of more powerful technologies to quantitate RNA, such as RT-PCR, northern blotting is a straightforward technique still widely performed. Moreover, the northern blotting procedure remains the preferred method for determining the size of transcripts. This made it the ideal technique to study processing of rRNA, as the transcripts are abundant and easily distinguished by size.

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5.2 MATERIALS AND METHODS

The materials and methods performed in this chapter have been described in detail in Chapter Two sections:

• 2.1.1 Transformation of DNA into Competent Cells

• 2.1.2 Plasmid DNA Preparation

• 2.1.3 Determination of DNA/RNA Concentration

• 2.1.5 Agarose Gel Electrophoresis

• 2.1.7 Polymerase Chain Reaction

• 2.1.14 Isolation of Total RNA

• 2.1.17 Generation of DIG Labeled DNA Probes

• 2.1.18 Northern Blot

• 2.1.19 Probe Detection

• 2.3.1 Mammalian Cell Culture

• 2.3.2 Transfections of Mammalian Cell Lines

• 2.3.5 Nrap siRNA Knockdown

• 2.3.7 Cycloheximide and DRB Treatment of Cells

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5.3 RESULTS

5.3.1 Generation of probes for northern blot analysis

To determine if Nrap participates in rRNA biogenesis, changes in the processing of rRNA after different Nrap treatments was analysed by northern blot analysis. DNA probes, to detect rRNA on the northern blot, were designed so that each transcript in the rRNA processing pathway could be detected. To achieve this, five probes were generated using primers listed in Table 2.3. The region complementary to each rRNA transcript is outlined in Table 5.2. Probes were designed to bind the mature rRNA transcripts 18S, 5.8S and 28S (figure 5.1). These probes would allow all intermediate and mature transcripts to be visualized by northern blot. The rRNA transcripts 47S, 46S and 45S differ very little in size and subsequently run at the same weight on a northern blot unlike each of the other transcripts. If changes were seen in this band, the precise transcript affected could not be determined. Therefore, the 5′ETS probe was designed to detect the 47S transcript only and the 3′ETS probe to detect the 47S-46S (figure 5.1).

Figure 5.1 Schematic of the region of mouse rDNA that is transcribed as the primary transcript. The underscores represent the relative position of the 5′ETS, 18S, 5.8S, 28S and 3′ETS probes in the rDNA sequence (not drawn to scale).

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Table 5.1: DNA probes for northern blot analysis. Name Complementary Region Transcript detected 5′ETS Base pairs 115-300 of 5′ETS 47S 18S Base pairs 4375-4706 of 18S 47-45S, 41S, 18S 5.8S Base pairs 6882-7035 of 5.8S 47-45S, 41S, 36S, 32S, 12S, 5.8S 28S Base pairs 9682-9962 of 28S 47-45S, 41S, 36S, 32S, 28S 3′ETS Base pairs 12841-13191 of 3′ETS 47S-46S

In order to visualize the transcripts on the northern blot, probes were labelled with DIG- 11-dUTP (DIG) (Materials and Methods section 2.1.19). This was achieved by PCR incorporation during synthesis of the probes in which DIG was incorporated approximately every 20-25 base pairs into the probe. To examine successful DIG incorporation, labelled probe was compared to unlabelled probe by agarose gel. The labelled probe migrated slower due to the increased mass caused by labelling with DIG (figure 5.2).

Figure 5.2 Evaluation of PCR labelled probes by agarose gel. Efficient labelling of probes was examined by comparison to unlabeled probe. DIG incorporation resulted in the same probe migrating slower.

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To confirm each probe detected the predicted rRNA transcripts, total RNA isolated from NIH3T3 and NS47 cells was separated on formaldehyde agarose gels and transferred to nylon membrane for northern analysis (figure 5.3). The 5′ETS and 3′ETS probes displayed a single band corresponding to the 47S primary transcript and 46S transcript respectively, as expected. The 5.8S probe showed the expected 47-45S, 41S, 36S, 32S, 12S and the 5.8S transcripts. The 47-45S, 41S, 36S, 32S and 28S transcripts were also visualised by the 28S probe. In both probes, visualisation of the 41S and 36S were not as clear as the other transcripts detected. The mature 18S transcript was detected by the 18S probe only, although it was anticipated that it would also display the 47-45S and 41S transcripts. However, the 47-45S and 41S rRNA was detected by the 5.8S and 28S probes, and was used in place of the 18S probe. Therefore, further examination into why the 18S probe did not perform as predicted was not pursued. Used in combination, these probes detected all the rRNA transcripts providing a powerful tool to study changes in the rRNA processing pathway.

Figure 5.3 Northern blot analysis of rRNA transcripts by DIG labelled probes. Northern blots of total RNA isolated from NIH3T3 (lane 1) and NS47 (lane 2) were hybridised with the 5′ETS, 18S, 5.8S, 28S and 3′ETS probes to visualize the rRNA transcripts.

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5.3.2 Northern blot analysis detects cytotoxic drug induced changes in rRNA processing

Although it had been established that northern blotting using each of the probes described earlier could detect all the rRNA processing transcripts, it was unclear if this technique would be sensitive enough to detect changes in the rRNA processing pathway. Cytotoxic drugs known to affect ribosomal biogenesis without interfering with transcription of the rDNA were employed to induce changes in the rRNA processing pathway. Total RNA was analysed by northern blot using the probes designed earlier. The first drug utilised, DRB, causes significant modifications to nucleolar function (Louvet et al., 2005) and more specifically to ribosome biogenesis induced a defect in the production of the 60S ribosomal subunit (Granick, 1975; Louvet et al., 2005). A second drug was also examined to ensure the reliability of the northern blotting procedure for this appliciation. Cycloheximide is well known for it ability to inhibit translation but also affects rRNA processing by causing the rapid degradation of the newly processed 32S and 18S rRNA transcripts (Soeiro et al., 1968; Willems et al., 1969).

To induce changes to rRNA processing DRB (25 μg/mL) was administered to NS47 cells for 4 hr and the total RNA was isolated, along with untreated cells. The RNA was analysed by northern blot using each of the probes. In addition, to control for equal loading, all northern blots were stained with methylene blue prior to hybridisation with the probes to show total levels of RNA loaded, seen by the abundant 28S and 18S transcripts. The northern blots showed (figure 5.4) that nearly all the transcripts were reduced after DRB treatment compared with no treatment. This was especially apparent for the 5.8S probe when analysing the 32S band. The levels of the 28S, 18S and 5.8S transcripts remained at levels closer to that of the RNA without treatment. This may be caused by the stable abundance of the fully processed rRNA, a product not normally influenced by changes in early rRNA processing, such as those induced by DRB treatment. Overall, the results indicated that DRB causes a general down regulation in rRNA processing. This was successfully detected by the northern blotting procedure for the following analysis of Nrap on rRNA processing.

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Figure 5.4 Northern blot analysis of rRNA processing from DRB treated cells. (A) Northern blots of total RNA isolated from NS47 cells treated with 25 μg/mL DRB for 4 hr (+) were compared to cells with no treatment (-). (B) Northern blots were stained with methylene blue to show loading.

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The experiment was repeated again in NS47 cells, using 25 μg/mL cycloheximide incubated for 24 hr. Northern blot analysis of the total RNA showed a reduction in the levels of the 47S-36S transcripts as well as the 32S transcript as expected after cycloheximide treatment when compared with no treatment (figure 5.5). No changes were observed in downstream transcripts. Interestingly, the 18S probe detected no change in levels of the 18S transcript. This may be a result of the probe detecting large quantities of mature 18S transcript. Inhibition of newly processed 18S transcripts may be difficult to observe over the existing levels of abundant mature 18S using this technique over the 4 hr period of treatment. On the other hand, the 32S is very short lived before it is rapidly processed, enabling degradation of this transcript to be easily observed. The results from treatment with DRB and cycloheximide showed that changes in rRNA processing could be successfully observed. This indicated that the northern blotting procedure using the current battery of transcript specific probes is an effective procedure to study changes in the rRNA processing pathway.

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Figure 5.5 Northern blot analysis of rRNA processing from cycloheximide treated cells. (A) Northern blots of total RNA isolated from NS47 cells treated with 25 μg/mL cycloheximide for 24 hr (+) were compared to cells with no treatment (-). (B) Northern blots were stained with methylene blue to show loading.

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5.3.3 Over expression of Nrap affects rRNA processing

The effect of over expression of the Nrap-flag construct on rRNA processing was compared against transfection with the empty flag vector by northern blot analysis (figure 5.6). NS47 cells were transfected and after 24 hr the total RNA was isolated. After being hybridised with each probe, the northern blots displayed a general decrease in all transcripts after over expression of Nrap-flag compared to the empty flag vector. This was once again visualised by examining the 32S band when the 5.8S probe was used. The intensity of each band was quantitated using Image J software and the values normalised against the corresponding quantitated levels of the 18S rRNA from the methylene blue stain (figure 5.6B). This enabled the observed changes on the northern blot to be analysed in relation to the exact amount of RNA loaded. The Image J analysis indicated there was a small reduction in rRNA processing of approximately 10% for each transcript. Although a clear down regulation of rRNA transcripts was observed on the northern blot after Nrap over expression in comparison to flag, the analysis suggested that when taking the amount of total RNA loaded into the blot in account, the reduction in processing was very minor.

The transient transfection efficiency of the flag constructs in NS47 cells was calculated to be approximately 25%. Therefore only one quarter of all cells assessed over expressed Nrap-flag, possibly indicating why only a small reduction in processing was observed. In addition, the rapid degradation of Nrap-flag may inhibit any significant changes being observed when over expressed.

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Figure 5.6 Northern blot analysis of rRNA processing after Nrap over expression. (A) Northern blots of total RNA isolated from NS47 cells transfected with Nrap-Flag for 24 hr (Nrap) were compared with transfection of the empty flag vector (Control). (B) Northern blots were stained with methylene blue to show loading.

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5.3.4 Knockdown of Nrap inhibits rRNA processing

To examine the effects of Nrap knockdown on rRNA processing, total RNA was isolated from NS47 cells transfected with Nrap siRNA, control siRNA, and without transfection respectively, and analysed by northern blot (figure 5.7). The 5.8S probe showed a reduction in the levels of the 32S transcript in the Nrap siRNA lane compared to the control lanes. Consistent with the results from the 5.8S data, the 28S probe also detected a marked reduction in the levels of 32S through to 28S rRNA. A slight decrease was also detectable in the 47S-45S bands although this was not detectable by the 5.8S probe. The 5′ETS and 3′ETS probes showed no changes between lanes in the corresponding 47S and 46S transcripts, indicating that down regulation of Nrap affects expression levels or the stability of the 45S rRNA transcript as well.

As mentioned, a reduction in the 28S transcript was observed after Nrap knockdown. This was consistent with the slight decrease seen in the Nrap siRNA lane on the methylene blue stain. However, the equal amounts of the 47S and 46S transcript detected by 5′ETS and 3′ETS probes respectively indicated the same amount of starting material was loaded into each lane, validating the equal loading. Image J software was then utilised to analyse the changes seen in relation to the loading observed on the methylene blue stain. Band intensities of the 45S, 32S and 28S transcripts detected with the 28S, 5.8S and 28S probes respectively were quantified and the values normalised against the levels of the 18S rRNA from the methylene blue stain (figure 5.8). The 28S band from the methylene blue stain was not used to normalise value because significant changes were seen in processing of the 28S transcript after Nrap knockdown. The image J analysis indicated that the 45S, 32S and 28S transcripts are each approximately half as intense as the no transfection and control siRNA lanes. As this analysis also takes into consideration relative loading levels, it was apparent that Nrap knockdown resulted in significant changes to rRNA processing.

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Figure 5.7 Northern blot analysis of rRNA processing after Nrap siRNA treatment. (A) Northern blots of total RNA isolated from NS47 cells transfected with Nrap siRNA for 24 hr, compared to cells transfected with control siRNA and without transfection. (B) Northern blots were stained with methylene blue to show loading.

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Figure 5.8 Image J analysis of rRNA transcripts affected by Nrap knockdown. (A) Transcripts affected by Nrap siRNA from northern blots in figure 5.9 were analysed by Image J software. (B) Band intensities from the 47-45S band detected by the 28S probe, 32S band detected by the 5.8S probe, and the 28S band detected by the 28S probe were quantified and the values normalised against the 18S levels from the methylene blue stain (18S was used as a control for loading instead of the 28S as significant changes were seen in the 28S processing) and presented as a percentage relative to no transfection.

The reduction in the levels of the 45S, 32S and 28S rRNA after Nrap knockdown might be a consequence of the concurrent perturbation in the levels of p19ARF and B23. It was also possible and likely that Nrap itself participates in processing of rRNA.

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5.4 DISCUSSION

In this chapter, northern blot analysis was employed to observe changes in the abundance of each transcript in the mouse rRNA processing pathway. The probes generated to visualise rRNA transcripts proved to be an effective tool. This was shown by induced changes to the rRNA processing pathway (by DRB and cycloheximide treatment) being successfully observed by the developed northern blotting procedure.

A very minor down regulation in processing of all the rRNA transcripts was observed after over expression of the Nrap-flag construct. Although this construct had been shown to be functional in the previous chapter (section 4.3.2), it was not unexpected when over expression did not cause a significant change in rRNA processing in comparison to the empty flag vector. It was probable that the instability of the Nrap-flag construct when over expressed inhibited its potential to function in the rRNA processing pathway, explaining the lack of significant changes observed.

On the other hand, knockdown of Nrap levels did result in changes to rRNA processing when compared to the control knockdown and no knockdown. Silencing Nrap levels in NS47 cells was found to be an effective method to study Nrap function in rRNA processing, unlike over expression of Nrap-flag, where instability complicated its functional activity. More specifically, Nrap knockdown was found to cause significant reductions in the observed levels of the 47-45S transcripts, detected by the 28S probe, and the 32S and 28S bands detected by the 5.8S and 28S probes respectively.

No differences in the 47S and 46S rRNA levels were observed between the Nrap knockdown and control lanes (verified by the 5′ETS and 3′ETS probes respectively). A slight decrease was detected in the 47-45S rRNA band with the 28S probe (figure 5.7) though. As the 5′ETS and 3′ETS probes specifically detect the 47S and 46S rRNA transcript, the reduction was presumed to be in the processing of the 45S rRNA. This result was not confirmed by the results of the 28S probe, with the 47-45S bands displaying little difference in abundance between lanes. The reason for this discrepancy was unknown but it may be a result of particular probes displaying a higher affinity to certain transcripts than others.

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In the previous chapter, Nrap knockdown was found to have effects on the regulation of B23 and p19ARF, both of which have been reported to also participate in rRNA processing. One of the functions of B23 is to preferentially cleave the ITS2 region of the 32S pre-rRNA transcript to yield the mature 28S rRNA (Savkur and Olson, 1998). The reduction in the level of the 32S rRNA is likely to be the result of Nrap knockdown enabling B23 levels to significantly increase. The excess B23 results in accelerated processing of the 32S rRNA to 28S rRNA compared with other processing steps, visualised by a reduction in the levels of this transcript. Interestingly, p19ARF has also been reported to inhibit rRNA processing, not only through its interaction with B23, but also through a broader spectrum by targeting the 47/45S pre-rRNA (Sugimoto et al., 2003). It is possible that the reduction in the 45S rRNA is a result of Nrap suppression changing p19ARF levels or activity.

As these results were not consistent with previous reports, it is likely that Nrap has a broader range of functions than displayed thus far which may account for the changes in rRNA processing. In particular, the reduction in the 28S levels at this stage can only be contributed to Nrap loss of function. It is therefore plausible to speculate that Nrap has a functional role in rRNA processing although further study is needed to clarify the precise mechanism. As Nrap is a component of a large nucleolar complex, its loss of function is likely to influence a number of other nucleolar factors, possibly explaining why a number of different rRNA transcripts were affected after reduced Nrap levels. In addition, it is unknown if Nrap directly participates with rRNA, or if it functions through a number of unidentified pathways to regulate factors that do interact with the rRNA. The observed processing defects in multiple locations of the rRNA processing pathway after Nrap knockdown would suggest that Nrap is more to likely act as an auxiliary regulatory protein. Either way, these results indicate that Nrap is an essential component in proper processing of rRNA.

At this stage the functional role of mammalian Nrap has not correlated with its yeast homolog YGR090w/Utp22. It is possible that mammalian Nrap may have different or even additional roles to its yeast homolog. It was identified that Nrap is part of a large nucleolar complex in mammalian cells lines (section 4.3.5), a feature also common to YGR090w/Utp22, with it being identified as a component of the SSU processome, also

113 Chapter Five Nrap is Involved in rRNA Processing a large multi-factored complex (Krogan et al., 2004). rRNA processing in mammalian cells has not been elucidated as extensively as in Saccharomyces cerevisiae, mainly due to its increased complexity. It is anticipated that further study into the components of the mammalian nucleolus and Nrap, will provide insight into the differences in biological activity between species.

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CHAPTER SIX

GENERAL DISCUSSION AND FUTURE DIRECTIONS

115 Chapter Six General Discussion and Future Directions

6.1 GENERAL DISCUSSION

The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. The functional role of the novel nucleolar protein, Nrap, was examined in mammalian cells. Nrap was found to associate with other established nucleolar proteins B23 and p19ARF, both with functional roles in ribosome biogenesis. More importantly, Nrap was shown to regulate protein expression levels of these two nucleolar proteins. Upon further investigation, Nrap was demonstrated to participate in the predominant nucleolar function of ribosome biogenesis, possibly through its association with B23 and p19ARF.

The sub-cellular localisation of endogenous Nrap had previously been confirmed as nucleolar (Utama et al., 2002). For that reason, it was not expected that recombinant Nrap (β isoform) would accumulate in unidentified sub-nuclear structures in a substantial number of transfected cells (section 3.3.4). A variety of nuclear bodies have been revealed in previous reports including cajal bodies, perinucleolar regions, and additional nuclear bodies (Zimber et al., 2004). Apart from the semi characterised cajal bodies, the structure and function of these sub-nuclear components remain relatively undefined. Remarkably, a number of well established nucleolar proteins have also been found present in nuclear bodies. Fibrillarin is one example where it has been identified as a component of the DFC of the nucleolus as well as being found in cajal bodies (Biggiogera et al., 2001; Bohmann et al., 1995). In addition, nucleolin is redirected from the nucleolus to the nucleoplasm under certain conditions of stress (Daniely et al., 2002). Finally, the major nucleolar protein B23, found to associate with Nrap in this study (section 4.3.1), has been described in unidentified nuclear bodies (Zatsepina et al., 1997). Although, it was not anticipated that recombinant Nrap would not be nucleolar, the literature would suggest that it is not uncommon for predominately nucleolar proteins to be present in other nuclear components under certain conditions. The excess quantities of the recombinant Nrap from over expression is the likely reason for the observed difference in the sub-cellular localisation compared to endogenous Nrap that was nucleolar. It is probable that the surplus recombinant Nrap was rapidly translocated from the nucleolus out to the nucleoplasm and eventually the cytoplasm to undergo

116 Chapter Six General Discussion and Future Directions proteasomal degradation, a control mechanism to keep Nrap protein levels in check (further discussed in section 6.2.1).

Mammalian expression vectors of full-length mouse Nrap and selected deletion constructs were generated to further study the role of Nrap (section 3.3.5 figure 3.8). Fluorescent microscopy was employed to observe the sub-cellular localisation of the different Nrap regions sub-cloned into the flag plasmid. From this study, the region responsible for directing Nrap to the nucleolus was unable to be identified. However, it was revealed that multiple domains are required, with the NLS being found as one of these. This assessment is not unique to Nrap. In fact, a number of domains are required for the nucleolar localisation of ribosomal L7a protein and nucleolin (Messmer and Dreyer, 1993; Russo et al., 1997; Schmidt-Zachmann and Nigg, 1993), just to mention a few. Like these two nucleolar proteins, it was presumed that Nrap requires the cooperative action of multiple domains in protein-protein or protein-nucleic acid interactions for nucleolar accumulation.

A study into the nucleolar localisation of the p120 nucleolar protein has demonstrated that the domain involved in directing this protein to the nucleolus binds to the nucleolar protein B23 (Valdez et al., 1994). B23 has a range of functions including shuttling proteins between the cytoplasm and the nucleolus (Li, 1997; Valdez et al., 1994). It was established in this study that Nrap associates with B23. Therefore, a possible scenario could involve B23 acting as a shuttle to facilitate Nrap nucleolar localization, by interacting with the currently unidentified region/s of Nrap needed for nucleolar accumulation.

The poly (A) polymerase-1 protein has been discovered to interact with B23 through its identified nucleolar localisation sequence (Meder et al., 2005). As mentioned in Chapter One (section 1.11), both the poly (A) polymerase proteins and Nrap share homology to the domain referred to as the PAP/25A core domain in this thesis (Rogozin et al., 2003). In the study performed by (Meder et al., 2005), a construct containing the N-terminal region of poly (A) polymerase-1, inclusive of the homologous region to Nrap, was found responsible for its association with B23. This evidence would suggest that B23 might interact with the putative PAP region of Nrap. However, it was observed that the

117 Chapter Six General Discussion and Future Directions

N-terminal region of Nrap, inclusive of the PAP domain and the NLS, did not direct Nrap to the nucleolus (section 3.3.5). Therefore, the speculated B23 interaction with the PAP region of Nrap would not assist in the translocation of Nrap to the nucleolus. Alternatively, B23 might interact with the PAP region of Nrap in another context that is currently unknown and that another region is necessary for Nrap nucleolar localisation.

In this study, it was found that over expression of recombinant Nrap resulted in its rapid degradation. Cells treated with the proteasome inhibitor MG132, showed reduced degradation indicating the mechanism of degradation is likely to involve the 26S proteasome. The dependency on the proteasome pathway also suggested Nrap may be polyubiquitinated. Noteworthy was the large percentage of cells over expressed with Nrap-flag displaying expression in the cytoplasm (section 3.3.4). It was speculated that cytoplasmic localisation of Nrap-flag may represent the proteasomal degradation of the excess recombinant protein. Ubiquitination promotes the nuclear export and subsequent degradation by cytoplasmic proteasomes of a range of nuclear proteins (Shcherbik and Haines, 2004). p53 is a well studied example. It is widely accepted that Mdm2 acts as an E3 ubiquitin ligase, promoting the ubiquitination and accelerating the nuclear export of p53 for degradation in cytoplasmic proteasomes (Haupt et al., 1997; Momand et al., 1992). Curiously, p53 was recently reported to undergo degradation by nuclear proteasomes as well (Xirodimas et al., 2001). Hence, it cannot be excluded that Nrap also undergoes degradation in the nucleoplasm and the cytoplasm.

B23 and p19ARF, nucleolar proteins found to associate with Nrap (section 4.3.2), have also been reported to be polyubiquitinated and subsequently degraded by the 26S proteasome (Itahana et al., 2003; Kuo et al., 2004). Interestingly, p19ARF was found to promote the polyubiquitination and proteasomal degradation of B23 (Itahana et al., 2003). It has been established that B23 and p19ARF exist in a complex relationship (Brady et al., 2004; Korgaonkar et al., 2005; Lee et al., 2005), and in this study it was found that Nrap is also intimately involved. In fact, Nrap knockdown induced a dramatic elevation in B23 protein levels, and levels of p19ARF to increase more modestly initially and then plummet at higher concentrations of Nrap knockdown (section 4.3.4). This data indicated that a loss of Nrap was able to regulate the protein levels of B23 and p19ARF. It was interesting to speculate then that Nrap may be involved

118 Chapter Six General Discussion and Future Directions in mediating their polyubiquitination and proteasomal degradation. In addition, the results of this study indicated that Nrap levels itself were maintained within a tight threshold (section 3.3.3). Therefore, it was plausible to suggest that Nrap regulated p19ARF and B23 protein levels, possibly through mediation of their polyubiquitination and proteasomal degradation, through its own tightly regulated stability. It would also be interesting to investigate the potential role B23 and p19ARF may play in turn mediating the degradation of Nrap (discussed further in section 6.2.2).

A recent report indicated that p19ARF not only promotes polyubiquitination of B23 but also induces sumoylation of B23 (Tago et al., 2005). SUMO (small ubiquitin-related modifier) resembles ubiquitin in structure and in the mechanism of ligation but does not mark proteins for degradation (Melchior, 2000). Instead, sumoylation has been reported to affect localisation and transport, gene expression, DNA repair, stress response, and cell cycle progression (Johnson, 2004).

The mouse Nrap amino acid sequence was analysed for a putative sumoylation consensus sequence. A previous study identified 2683 potential SUMO substrates by searching for proteins containing firstly the motif, a-K-x-E (where a is a hydrophobic amino acid and x is any amino acid), and secondly a NLS (Zhou et al., 2005). Reports indicated a NLS suffices for SUMO conjugation in vivo (Rodriguez et al., 2001). Based on this data, it was discovered that Nrap contained a short segment of amino acids at its N-terminal, G-K-D-E, closely resembling the above SUMO consensus sequence. In addition, in the present study, the NLS of Nrap was shown to be functional. One derivation from the expected consensus in the putative Nrap sequence was the presense of a glycine residue in the position of the predicted hydrophobic amino acid. Most SUMO consensus sequences contain a proline or glycine residue between 2-5 amino acids upstream or downstream of the essential lysine residue. Further examination of the Nrap sequence revealed that it also complied with this observation with a proline situated 4 amino acids upstream of the lysine. Together, this data suggested that Nrap might be sumoylated. It would also be interesting to investigate if p19ARF induces sumoylation of Nrap like it does with B23. One could surmise that Nrap could be potentially sumoylated, in a similar manner as the nucleolar proteins it associates with,

119 Chapter Six General Discussion and Future Directions possibly to control important cellular processes such as cell proliferation (further described in section 6.2.2).

B23 and p19ARF are found together in high molecular weight complexes (2-5 MDa) (Bertwistle et al., 2004). A study by Bertwistle and colleagues were able to purify 28 proteins in the recombinant p19ARF containing complex in NIH3T3 cells. Of these, 13 were identified as large ribosomal subunit proteins, 1 small ribosomal subunit protein, as well as the well documented nucleolar proteins nucleolin and B23. Immunoprecipitations revealed Nrap exists in complex with these two proteins (section 4.3.1) suggesting Nrap is also a previously unidentified component of this large nucleolar complex. In conjunction with other reports, the characteristics (Pinol-Roma, 1999) and RNA dependent formation (Bertwistle et al., 2004) of this complex suggested that it represents precursor ribosomal subunits.

By northern blot analysis, Nrap was found to participate in rRNA processing. Other data presented here suggested that this newly identified function of Nrap might be modulated in conjunction with B23 and p19ARF. B23 and p19ARF are known to localize in the granular region of the nucleolus (Brady et al., 2004), a location common to most proteins that are involved in processing and maturation of rRNA species. The fact that Nrap associates with these proteins suggests its involvement in processing or maturation of rRNA. Indeed, after 24 hr treatment of 100 nM Nrap siRNA in NS47 cells, changes were identified in the 45S, 32S and 28S rRNA levels. The accumulative data presented in the current study demonstrating a functional role for Nrap in the nucleolus supports the notion that Nrap is involved in the most prominent function of the nucleolus, ribosome biogenesis. Further analysis of Nrap’s function in rRNA processing (see section 6.2.3) is necessary to elucidate its unique properties as well as how it regulates the functions of B23 and p19ARF in this essential cellular process.

The results from this study implicated Nrap in sub-nuclear structures as well as in important processes such as cell proliferation and ribosome biogenesis. Combined, this suggested that Nrap is an essential protein with a number of different functions. Regulation of these functions may be contolled by a number of mechanisms. Firstly, the two Nrap isoforms may possess individual properties, allowing each to serve a unique

120 Chapter Six General Discussion and Future Directions function. Therefore, controlling the expression of each isoform could be a mechanism to induce or inhibit specific Nrap activities. Secondly, the data presented suggested that the protein levels of Nrap were sensitive to fluctuations, best seen by the over expression of recombinant Nrap (section 3.3.3). Hence, it is likely that the proteasomal degradation of Nrap is another mechanism to contol the activity of Nrap. Finally, the putative sumoylation consensus senquence within Nrap indicated that Nrap function might also be controlled by sumoylation, although in what context remains to be solved. In addition to the mechanisms controlling Nrap, it was apparent that Nrap regulates B23 and p19ARF through an unknown mechanism, to control ribosome biogenesis and cell proliferation.

Hence this study has achieved its goal to increase the knowledge of Nrap function in mammalian cells. Moreover, the characterisation of Nrap has contributed to the general understanding of nucleolar function and behaviour in a number of ways. The ability of Nrap to associate with other nucleolar proteins B23 and p19ARF and affect cell proliferation supported existing evidence of a plurifunctional nucleolus. Most importantly, the data presented in this thesis supports recent suggestions that specific nucleolar proteins may mediate cross talk between ribosome biogenesis and cell cycle progression (Itahana et al., 2003), although the molecular mechanism is not understood and requires investigation. It was proposed by (Korgaonkar et al., 2005) that B23, in response to hyperproliferative signals, might serve to impair the ability of p19ARF to interact with Mdm2 and stimulate p53. Nrap was also found to affect the levels of B23 and p19ARF although in what context is unknown. However, with further investigation of Nrap function, it may not be too tantalising to speculate that Nrap may represent a novel target in the battle against cancer.

121 Chapter Six General Discussion and Future Directions

6.2 FUTURE DIRECTIONS

It was evident from the work performed in this study that Nrap has a functional role in the nucleolus. Not only was it found to participate in the predominant nucleolar function of rRNA processing, it also was implicated in the more recently discovered nucleolar function of regulating cell proliferation. Despite the advancements made from this study, it was apparent that further investigation is required to fully elucidate the functional role of Nrap in mammalian cells. Outlined below are several important experiments that would further characterize Nrap function.

6.2.1 Functional differences between Nrap-α and Nrap-β.

To date, little is known of the regulation and subsequent differences in function between the two mouse Nrap isoforms, α and β. Several lines of evidence in the present study supported the existence of two Nrap isoforms, particularly the ability of the anti-Nrap antibody to detect two bands corresponding to the predicted molecular weight of the two isoforms in NIH3T3 cells. However, the functional differences between the two remain unresolved. For reasons unknown, all efforts to clone Nrap-α remained unsuccessful, unlike Nrap-β. To further investigate the biological significance of each isoform, it would be wise to encompass techniques other than cloning. One option would be to raise antibodies specific to each isoform in addition to the anti-Nrap antibody created that theoretically detects both isoforms. Polypeptides to raise the antibodies against in rabbits, would need to consist of a short segment of amino acids unique to each of the extreme C-terminal regions of both isoforms (Nrap is alternatively spliced at the extreme C-terminal region to generate Nrap-α and Nrap-β).

Upon successful purification of specific antibodies against each isoform, a number of experiments could be performed to study the differences between the two isoforms. Firstly, any variation in their sub-cellular localisation could be observed by fluorescent microscopy. It would also be interesting to ascertain in this potential study if

122 Chapter Six General Discussion and Future Directions endogenous Nrap-β localised in the same manner as the Nrap-flag construct (recombinant Nrap-β).

In conjuction with the generation of antibodies against the specific Nrap isoforms, siRNA could be designed to target either Nrap-α or Nrap-β, to study the role of each isoform. This would allow a specific Nrap isoform to be reduced to minimal levels, enabling the function of the other Nrap isoform, still at nominal levels, to be analysed. Effective knockdown would be examined by Real-Time PCR at the mRNA level, and by western blot with the newly generated Nrap isoform specific antibodies for the protein levels. This technique would greatly assist in confirming any results obtained using the Nrap isoform specific antibodies. It would also allow changes in rRNA processing and cellular proliferation to be monitered in cells defective in one Nrap isoform.

Immunoprecipitations using the specific Nrap isoform antibodies could be performed to assess differences in binding partners. To overcome problems with low protein levels, purification of nuclear or nucleoli fractions would be used in place of whole cell lysates. An appealing direction to investigate would be if one isoform displayed a higher affinity to associate with B23 or p19ARF. In the present study, Nrap was found to regulate protein levels of B23 or p19ARF. The specific Nrap isoform antibodies could also be employed to evaluate if either or both isoforms direct these regulatory processes.

In addition to functional differences between the isoforms, the control of their expression would be interesting to evaluate. Again the specific Nrap isoform antibodies could be utilised in analysis of the protein expression levels. In addition, real time PCR using specific primers to each isoform could be incorporated to examine the regulation of the mRNA levels of each Nrap isoform. Analysis of the mRNA and protein expression levels after perturbations to ribosome biogenesis (eg cycloheximide, DRB, and Actinomycin D treatment of cells) and cell cycling (serum starvation of cells) would be particularly informative to perform. Detailed analysis of the differences between the Nrap isoforms would lead to a greater understanding of the general role of Nrap in the mammalian nucleolus.

123 Chapter Six General Discussion and Future Directions

6.2.2 Further analysis of Nrap degradation

The dependency of recombinant Nrap stability on the 26S proteasome, shown by MG132 treatment of cells over expressing Nrap-flag (section 3.3.3), suggested Nrap might be polyubiquitinated. To answer this, in vivo ubiquitination assays would be performed. As described by (Itahana et al., 2003; Kuo et al., 2004) mouse fibroblast cells would be co- transfected with the Nrap-flag construct as well as a plasmid expressing HA tagged ubiquitin. The Nrap complex would then be immunoprecipitated with the anti-flag antibody and analysed by western blot. The presence of ubiquitin would be detected by probing the western blot with anti-HA antibody. If Nrap were polyubiquitinated, it would be expected that a ladder of HA-marked molecules would be observed on the western blot. These would then be confirmed as being conjugated to Nrap by probing parallel western blots with anti-flag antibodies.

Reports indicated p19ARF promotes the polyubiquitination of B23. Nrap too, is intimately involved in the B23- p19ARF interaction. It would therefore be logical to next examine the effects of B23 and p19ARF levels on Nrap polyubiquitination and degradation. The in vivo ubiquitination assay would be repeated in the presence of over expressed B23 and p19ARF to determine if either induced the polyubiquitination of recombinant Nrap, observed by an increase intensity of HA detected molecules on the western blot. In addition, the sub-cellular localisation of the Nrap-flag construct could be analysed by microinjection into cells, followed by a time course analysis of its expression. This would allow Nrap-flag to be observed at its lowest possible expression levels and the relative degradation levels in comparison to the standard transfection of the Nrap-flag construct to be examined.

The identification of a putative SUMO consensus sequence in Nrap suggests that Nrap may be sumoylated as well as ubquitinated. Examination of this hypothesis would be undertaken in a similar manner as for ubiquitin, using a modified procedure described by (Tago et al., 2005). Mouse fibroblast cells would be co-transfected with Nrap-flag and a His tagged SUMO expression vector. Using nickel beads, complexes associated with the expressed SUMO would be precipitated from the cell lysate and analysed by western blot. The blot would then be probed for the presence of Nrap in the SUMO

124 Chapter Six General Discussion and Future Directions complex. The reciprocal immunoprecipitation could also be performed using the anti- flag antibody. Unlike ubiquitin, the observation of a ladder of sumoylated molecules indicates monosumoylation of multiple lysine residues (Tago et al., 2005). Investigation of the role of B23 and p19ARF in Nrap sumoylation would also be performed as described above.

6.2.3 RNA targets of Nrap

Northern blot analysis revealed Nrap participates in rRNA processing. Knockdown of Nrap levels resulted in perturbations to processing of the 45S, 32S and 28S transcripts. This data demonstrated for the first time a functional role for Nrap in ribosome biogenesis. One draw back of the method used to examine the role of Nrap in rRNA processing was that the observed changes could not be distinguished between direct and indirect interactions with the rRNA. Therefore, it would be interesting it ascertain if Nrap interacts directly with one or more rRNA transcripts or if it dictates changes through an unidentified pathway.

Using sucrose density gradients, the pre-rRNP particle that Nrap co-sediments with could be identified. The specific fractions containing Nrap could be analysed to determine not only the proteins associated with Nrap but also the rRNA transcripts included with the pre-rRNP. Briefly, this could be performed by preparing nuclear extracts from mouse fibroblast cell lines as described by (Strezoska et al., 2000) and analysing them on 10-30% sucrose density gradients. Each gradient fraction would be resolved by SDS PAGE (8% gels) and analysed by western blot to determine the Nrap containing fraction. In addition, western blots could be probed for other known nucleolar proteins to determine other co-sediments.

To determine the rRNA transcripts fractionating with Nrap, total RNA would be isolated from the same gradient fractions confirmed to incorporate Nrap by western blot analysis and separated on a 1% formaldehyde agarose gel. Total RNA would then be transferred to membrane and analysed by northern blot. The DIG labelled probes

125 Chapter Six General Discussion and Future Directions created in the present study could be hybridised to the total RNA to determine which rRNA transcripts co-sedimented with Nrap.

Confirmation of the identified RNA transcripts associating with Nrap would be essential. Immunoprecipitation of the associated RNA and examination of the sequence would confirm any putative interaction. A protocol modified from (Chu et al., 1999) would involve the immunoprecipitation of the complex with an anti-Nrap antibody from mouse fibroblast cells. The complex containing the RNA would then undergo a phenol/chloroform extraction to isolate the associated RNA. The amount of RNA immunoprecipitated would likely be at submicromolar levels. For this reason, RT-PCR with the use of universal primers would be employed to amplify the RNA to large quantities of cDNA. The amplified cDNA could be cloned into a plasmid and the sequenced identified.

126 Chapter Six General Discussion and Future Directions

6.3 POTENTIAL MODEL OF NRAP ACTIVITY

From the data presented in this thesis and reports from other research groups, a model that explains how Nrap regulated the interaction between B23-p19ARF was hypothesised (figure 6.1). It is proposed that Nrap negatively regulates the interaction between B23 and p19ARF, through an unidentified mechanism, to ultimately control processing of rRNA as well as cell proliferation.

B23 and p19ARF exist in a negative feedback loop (Korgaonkar et al., 2005). B23 stabilises levels of p19ARF by targeting it to the nucleolus. This probably rescues p19ARF from proteasomal degradation, but also inhibits its ability to interact with Mdm2 in the nucleoplasm and stabilise p53. In return, the nucleolar levels of p19ARF, promote the polyubiquitination of B23. Through this cycle, levels of both B23 and p19ARF are maintained. It is speculated that Nrap negatively regulates the above interaction. In response to cellullar signals, levels of Nrap may be altered, resulting in changes to the levels of B23 and p19ARF through an unidentified pathway, in order to adjust processing of rRNA and or cell proliferation. It appears that Nrap negatively controls the B23- p19ARF interaction by being maintained within a tightly regulated threshold. Therefore, small changes in Nrap levels, probably through proteasomal degradation, quickly induce the appropriate changes to the levels of B23 and p19ARF. As revealed by Bertwistle et al., (2004), in normal cells, the bulk of B23 resides in 500 kDa complexes within the nucleolus. In conjunction with Nrap and low levels of p19ARF, a small subset of B23 is situated in a large 2-5 MDa nucleolar complex, representing pre-ribosome particles. It is likely, that Nrap negatively regulates the B23 - p19ARF by maintaining the appropriate levels of B23 and p19ARF in the high molecular weight nucleolar complex.

127 Chapter Six General Discussion and Future Directions

Figure 6.1 Potential Model of Nrap activity. Protein levels of Nrap are maintained within a highly regulated threshold, probably through proteasomal degradation. This allows Nrap to negatively regulate the interaction between B23 and p19ARF through an unidentified mechanism. As a result, the negative feedback loop in which B23 and p19ARF exist can be altered in response to cellular signals to regulate rRNA processing and cell proliferation.

128 Chapter Six General Discussion and Future Directions

Using the proposed model, the data presented in this thesis can be explained. When siRNA at concentrations between 20-60 nM reduced Nrap levels to approximately 50%, both B23 and p19ARF levels were found to increase. Nrap might potentially modulate the polyubiquitination of B23 and p19ARF. According to the model, the drop in Nrap would no longer inhibit an interaction between B23 and p19ARF. This would allow levels of B23 to increase and stabilise p19ARF by targeting it to the nucleolus, seen by increased levels. One of the normal functions of p19ARF is to promote the degradation of B23. This was not observed because the lack of Nrap modulating the B23- p19ARF interaction caused B23 and p19ARF to accumulate in the nucleolus.

It is likely that only very small changes in Nrap levels are required to negatively regulate the B23 - p19ARF interaction. Therefore, the sudden and unexpected decrease in Nrap levels to less than 25% of their original by high concentrations of Nrap siRNA, resulted in the levels of B23 to continue to increase while levels of p19ARF suddenly plummeted. It is hypothesised that the cell sensed critical levels of Nrap and other cellular responses were activated to rectify the regulation between B23 and p19ARF levels. As p19ARF is crucial in maintaining normal cell proliferation, it is not unlikely that unidentified pathways were probably able to compensate for the loss of Nrap function in extreme situations, and able to induce degradation of the excess p19ARF levels.

This model suggests an important role for Nrap in regulating rRNA processing and cell proliferation through B23 and p19ARF. Although much remains unknown on the mechanisms controlling these interactions, it is apparent that nucleolar proteins play a more critical role than first anticipated in important cellular processes. It is likely that ribosome biogenesis and other nucleolar functions are tightly linked with maintaining healthy levels of cell growth and survival. With further study, this area of research has potential to revolutionise the field of cancer research.

129 References

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