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Investigating the Role of the Ribonuclease DIS3 In Investigating the role of the ribonuclease DIS3 in haematological cancers Sophie Rebecca Robinson 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 July 2016 Abstract Whole genome sequencing has recently identified DIS3 as a novel tumour suppressor gene in multiple myeloma. DIS3 is a conserved RNA exonuclease and catalytic subunit of the exosome, a protein complex involved in the 3’ to 5’ degradation and processing of messenger RNA and small RNAs. Messenger RNA processing and degradation is important in controlling gene expression and therefore cellular function, however the role DIS3 plays in the pathogenesis of haematological cancer remains unclear. Using RNAi as a means to knock-down DIS3, I have performed various functional assays to investigate the consequences of DIS3 loss-of function on myeloma cells. I have investigated cell viability, drug-sensitivity, mitotic errors, apoptosis and the generation of double-strand breaks in both transiently transfected myeloma cells and stable transfected adherent cells. I have also performed transcript profiling experiments in the form of RNA-sequencing to identify possible targets of DIS3 as well as synthetic lethality screens to identify proteins that may be cooperating with DIS3 mutations in myeloma pathogenesis. Overall, DIS3 knock-down did not appear to affect cellular phenotype in these assays, possibly indicating that DIS3 may be conferring a competitive advantage to cancer cells through a mechanism that only occurs in vivo. Alternatively, DIS3 mutations may not be driving tumourigenesis on their own but may either require another cellular pathway to be disrupted, or, may only be required to maintain the tumour rather than initiate it. In addition to investigating the role of DIS3 in oncogenesis, I have also studied the normal physiological role of DIS3 within the cell. I have confirmed the presence of two alternatively spliced, protein-coding transcripts of DIS3 that differ in the size of their endoribonucleolytic PIN domain. My work has characterised the levels of these two isoforms in cell lines and in tissues from humans with various haematological cancers. Isoform 1 appeared to be the principal transcript in cell lines as well as myeloma and AML patient cells. However, in CMML and healthy controls, the ratios of each isoform are more equal and often isoform 2 is more highly expressed than isoform 1. Activity assays indicated a difference in the ability 1 of the shorter isoform to degrade circular RNAs, suggesting isoform 2 may have reduced endonucleolytic function. Initial work has also identified a link between the higher expression of isoform 2 in CMML patients and common mutations in the splicing gene SRSF2. This suggests the expression of the endonucleolytically- reduced DIS3 isoform 2, may contribute towards a CMML phenotype. Although this project was unable to identify the role of DIS3 in myeloma development, there is strong evidence that mutations in this gene are being positively selected and confer an advantage to cancer cells. More sophisticated experiments may need to be conducted whereby the in vivo environment is mimicked more effectively, through the generation of a mutant mouse model. Only once we understand the picture more fully, can we begin to design targeted molecular therapies for affected patients. 2 Contents Chapter 1: Introduction ............................................................................................. 21 1.1 The expansive role of RNA within the cell ........................................................ 21 1.1.1 Messenger RNA .......................................................................................... 21 1.1.2 Non-protein coding RNAs .......................................................................... 22 1.1.2.1 Non-coding RNAs in translation .......................................................... 24 Transfer RNAs .............................................................................................. 24 Ribosomal RNA ............................................................................................ 24 1.1.2.2 Non-coding RNAs in gene regulation .................................................. 26 MicroRNAs ................................................................................................... 26 Short-interfering RNAs ................................................................................ 28 Piwi-interacting RNAs .................................................................................. 28 Long non-coding RNAs ................................................................................ 30 1.1.2.3 Non-coding RNAs in RNA processing .................................................. 31 Small nuclear RNAs ...................................................................................... 31 Self-splicing introns ..................................................................................... 32 Small nucleolar RNAs ................................................................................... 32 1.2 RNA Degradation and Maturation ................................................................... 33 1.2.1 mRNA decay ............................................................................................... 33 1.2.1.1 mRNA quality control pathways ......................................................... 36 1.2.1.2 Regulated mRNA decay pathways ...................................................... 37 1.2.2 Small-RNA processing and decay ............................................................... 39 1.3 An overview of the exoribonuclease DIS3........................................................ 40 1.3.1 Conservation, structure, mechanistic functions and sub-cellular localisation of DIS3.............................................................................................. 40 1.3.2 Molecular functions of DIS3 ...................................................................... 47 3 1.3.2.1 Role of DIS3 in mRNA decay................................................................ 47 1.3.2.2 Role of DIS3 in small non-coding RNA processing and decay ............. 48 1.3.3 Biological functions of DIS3 ....................................................................... 51 1.3.3.1 Role of DIS3 in cell-cycle regulation .................................................... 51 1.3.3.2 Role of DIS3 in generating antibody diversity ..................................... 55 1.3.4 DIS3 and Disease ........................................................................................ 56 1.3.4.1 DIS3 and cancer ................................................................................... 56 1.3.4.2 DIS3 and multiple myeloma ................................................................ 58 1.4 Multiple Myeloma ....................................................................................... 62 1.4.1 General overview and pathophysiology .................................................... 62 1.4.2 Genetics ..................................................................................................... 68 1.4.3 Epidemiology ............................................................................................. 72 1.4.4 Aetiology .................................................................................................... 72 1.4.5 Treatment .................................................................................................. 73 1.5 Aims of this project........................................................................................... 75 1.5.1 Chapter 3 ................................................................................................... 75 1.5.2 Chapter 4 ................................................................................................... 76 1.5.3 Chapter 5 ................................................................................................... 76 1.5.4 Chapter 6 ................................................................................................... 76 Chapter 2: Materials and Methods ........................................................................... 77 2.1 Cell Culture ....................................................................................................... 77 2.1.1 Cell Lines .................................................................................................... 77 2.1.2 Primary Cells .............................................................................................. 77 2.1.3 Cell Passage ................................................................................................ 79 2.1.4 Cryopreservation of cells ........................................................................... 79 2.2 Cell Transfection ............................................................................................... 80 4 2.2.1 Transient transfection of siRNA ................................................................. 80 2.2.2 Stable transfection of shRNA vectors ........................................................ 80 2. 3 Synthetic lethality screens ............................................................................... 84 2.4 Growth assays................................................................................................... 85 2.5 Cell Viability assays ........................................................................................... 85 2.6
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