The Role of Exoribonucleases in Human Cells using Osteosarcoma as a Model Amy Louise Pashler A thesis submitted in partial fulfilment of the requirements of the University of Brighton and the University of Sussex for the degree of Doctor of Philosophy April 2019 1 Abstract Post-transcriptional control of gene expression is a critical level of regulation in the Central Dogma. One of many layers is control of RNA turnover and metabolism which are vital to maintain cellular homeostasis. In RNA processing, the role of RNA stability is essential in determining how long a species of RNA is able to function within the cell. In addition, RNA stability is central to the process of translation of RNAs, as well as the ability of regulatory RNAs to inhibit or repress their target mRNAs. This thesis focuses on the function of exoribonucleases within human cells and their role in regulating gene expression. Exoribonucleases are key enzymes in RNA degradation, which degrade RNA in either the 5’ – 3’ direction, or in the 3’ – 5’ direction. There is particular focus on the role of the 5’ – 3’ exoribonuclease, XRN1, and how defects in 5’ – 3’ RNA decay can result in defective protein expression, and the onset of disease. This thesis characterises the expression of XRN1 in human bone cancer cell lines, and compares expression to that of a non-cancer cell line control. In doing so, the elucidation of XRN1 as a potential tumour suppressor gene in the progression of the most common bone cancer, osteosarcoma is investigated, alongside characterisation of the expression of the 3’ – 5’ exoribonucleases, DIS3, DIS3L1 and DIS3L2. DIS3 and DIS3L2 have each been previously implicated in the progression of human disease: mutations in DIS3 have been associated with a variety of leukaemias, whereas mutations in DIS3L2 have been associated with congenital overgrowth syndromes and also Wilms’ Tumour of the kidney. This thesis shows that alongside XRN1, DIS3L2 may DIS3L2 also changes in expression in osteosarcoma. This thesis also elucidates phenotypic changes in cells where XRN1 has been knocked down by a lipid-based RNAi transfection system. Using the osteosarcoma cells as a model, XRN1 was knocked down over a period of time, with cellular effects being observed over a time course. With regards to fundamental cellular pathways of proliferation, viability, translation and apoptosis, phenotypic changes in the behaviour of the cell lines were not observed. In contrast, knock down of DIS3L2 in a human embryonic kidney cell line, HEK-293T, resulted in hyper- proliferation, but this effect was not observed in the osteosarcoma cell lines, demonstrating a tissue specific effect for this gene in humans. The existence of synergism between the major exoribonucleases was also studied in this thesis, which found that there may be co-ordinate regulation occurring between XRN1 and DIS3L2, but not with DIS3 and DIS3L1. Finally, this thesis shows the transcriptional changes in SAOS-2 cells when XRN1 is knocked down, in comparison with a control, using RNA sequencing technology to ascertain which pathways this enzyme functions to regulate. RNA sequencing was performed in a global approach to identify transcriptional targets of XRN1 in osteosarcoma cells. In doing so, it was 2 shown that XRN1 is involved in a multitude of cellular processes, such as cellular migration and adhesion, and also may function in the EGF pathway, a known oncogenic pathway. It also became clear that XRN1 targets transcripts with similar motifs in the 3’ UTR, and that transcript targets are conserved across more than one cell line. This thesis marks the stepping stone to elucidating the role of XRN1 in human cells, and serves as an introduction to the function of XRN1 in human disease to pave the way for future research. 3 Table of Contents Abstract .............................................................................................................................. 2 Commonly used abbreviations .......................................................................................... 12 Acknowledgements ........................................................................................................... 15 Candidate’s declaration ..................................................................................................... 16 Chapter 1 .......................................................................................................................... 17 Introduction ...................................................................................................................... 17 1.1 The Central Dogma ........................................................................................................... 17 1.1.2 A Brief Overview of Transcription .............................................................................. 19 1.1.3 A Brief Overview of Translation ................................................................................. 23 1.2 Control of Gene Expression .............................................................................................. 27 1.2.1 Transcriptional control of gene expression ............................................................... 27 1.2.2 Post-transcriptional control of gene expression ........................................................ 28 1.2.3 Translational control of gene expression ................................................................... 29 1.2.4 Post-translational control of gene expression ........................................................... 30 1.3 A description of different RNA species ............................................................................. 31 1.3.1 RNAs involved in protein synthesis ............................................................................ 31 1.3.2 RNAs involved in post-transcriptional modification .................................................. 33 1.3.3 RNAs involved in gene regulation .............................................................................. 33 1.4 The Mechanisms and Roles of RNA Stability .................................................................... 39 1.4.1 RNA stability ............................................................................................................... 39 1.4.2 Regulation of gene expression by 3’ tailing ............................................................... 40 1.5 Mammalian exoribonucleases and how they function..................................................... 40 1.5.1 XRN1: Structure and functions ................................................................................... 41 1.5.2 XRN2: Structure and Functions .................................................................................. 45 1.5.3 DIS3L2: Structure and functions ................................................................................ 47 1.5.4 DIS3 and DIS3L1: Structures and functions ................................................................ 49 1.6 Pathways of RNA Decay .................................................................................................... 53 1.6.1 Summary of 5’ – 3’ RNA decay ................................................................................... 53 1.6.2 Summary of 3’ – 5’ RNA decay ................................................................................... 56 1.6.3 Summary of Nonsense-Mediated Decay ................................................................... 59 1.6.4 Summary of microRNA-mediated decay .................................................................... 62 1.7 RNA stability: implications in human disease ................................................................... 70 1.7.1 Pathologies generally associated with RNA stability and decay ................................ 70 1.7.2 Human diseases associated with XRN1...................................................................... 72 1.7.3 Human pathologies associated with DIS3L2 .............................................................. 77 4 1.8 An overview of Osteosarcoma and Ewing sarcoma .......................................................... 80 1.8.1 Treatments and pathology of osteosarcoma ............................................................. 80 1.8.2 Molecular pathology of the development of osteosarcoma ..................................... 81 1.8.3 A brief overview of Ewing sarcoma............................................................................ 82 1.8.4 Existing RNA studies of OS ......................................................................................... 83 1.9 Preliminary work and previous publications .................................................................... 85 1.10 Project aims and brief results chapter overviews ........................................................... 86 1.10.1 Chapter 3: Characterisation of exoribonuclease expression in OS and EWS........... 86 1.10.2 Chapter 4: The Effects of Changing Exoribonuclease Expression in OS Cells using siRNA ................................................................................................................................... 87 1.10.3 Chapter 5: Identifying Potential Transcript Targets of XRN1 using RNA Sequencing ............................................................................................................................................ 88 Chapter 2 .......................................................................................................................... 89 Materials & Methods