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Most triggers of programmed cell death or apoptosis promote cytochrome c release from the mitochondria into the cytosol which initiates the activation of a panel of pro-apoptotic caspases. Activation of caspase-8 in this pathway is not well defined but may be important because it activates Bid which promotes further cytochrome c release. A range of experimental approaches have suggested different mechanisms for caspase-8 activation in this pathway. In order to identify the proteases directly responsible for caspase-8 activation, caspase-8 cleaving activity was purified from cytochrome c activated cytosolic extracts. Caspase-6 was found to be the only soluble protease in cytochrome c activated cytosolic extracts that has significant caspase-8 cleaving activity. Furthermore, caspase-6 was sufficient to induce Bid-dependent cytochrome c releasing activity in cell extracts. Inhibition of caspase-6 activity in cells significantly inhibited caspase-8 cleavage and apoptosis, therefore establishing caspase-6 as a major activator of caspase-8 in vivo and confirming that this pathway can have a critical role in promotion of apoptosis. The intrinsic activity of caspase-6 was found to be dependent on removal of the short prodomain. The requirement for two distinct cleavage steps to activate an effector caspase may represent an effective mechanism for restriction of spontaneous caspase activation and aberrant entry into apoptosis. The caspase activites which cleave the two cleavage sites of caspase-6 were purified from cytochrome c activated cell extracts. Caspase-3 and caspase- 7 were found to have different specificities for the caspase-6 cleavage sites, despite having the same preferred peptide substrates.

The genomes of eukaryotes exist as chromatin. The organisation of chromatin provides a means of regulating access to the underlying genetic information. One way in which eukaryotes regulate chromatin is through the action of chromatin remodelling motor proteins. Human forms of the SWI/SNF remodelling complex are a series of chromatin remodelling ATPases comprised of some 15 partially overlapping subunits. These complexes function at enhancers and other regulatory elements. They have been observed to be mutated at high rates in a range of human cancers. Characterisation of the structure and biochemical activities of these complexes is limited. We aimed to address this by developing a system for the expression of human SWI/SNF complex subunits in insect cells as recombinant proteins. A complex of the core proteins was successfully expressed and purified. This complex was found to have robust biochemical activity, and enabled the investigation of the effects on remodelling resulting from small molecule inhibitors targeting SWI/SNF domains, and the effects of introduced subunit mutations. Complexes bearing a tumour-associated mutation in the SMARCA4 ATPase were purified and their enzymatic activity on nucleosomes was characterised. The presence of mutant complexes was found to reduce the activity of wild-type complexes, consistent with their dominant negative phenotype in vivo. A sub-complex of human PBAF was purified by the incorporation of PBAF-specific subunits PBRM1 and BRD7 into the core complex. The biochemical activity of both complexes on nucleosomes was tested. Both complexes were then investigated for their remodelling activity on nucleosomes bearing acetylated H3 histones, which are known to stimulate binding to and remodelling of nucleosomes by yeast SWI/SNF complexes. The PBRM1 sub-complex was found to remodel acetylated nucleosomes at a reduced rate compared to the core complex. Structural analysis by cryo-EM was undertaken for both of the complexes, however only low resolution structures were obtained.

The mRNA cap consists of a series of co-transcriptional modifications on nascent transcripts that confer protection from degradation and mediate RNA processing and translation. The addition of an inverted guanosine cap to the 5'-end of the transcript and its subsequent methylation along with the methylation of the first and second transcribed nucleotides are catalysed by several enzymes to form a complete cap structure. Growing evidence suggests that the mRNA cap and the mRNA capping enzymes can be regulated in a context-dependent manner by different molecules and signalling pathways either through co-factors or posttranslational modifications.

C-MYC is a transcription factor and a potent driver of many human cancers. In addition to regulating transcription, C-MYC promotes formation of the mRNA cap which is important for transcript maturation and translation. However, the mechanistic details of C-MYC-dependent mRNA capping are not fully understood. Since anti-cancer strategies to directly target the C-MYC protein have had limited success, enzymatic co-factors or effectors of C-MYC present attractive alternatives for therapeutic intervention of C-MYC-driven cancers. mRNA capping enzyme (CE) initiates mRNA cap formation by catalysing the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide. The involvement of CE in C-MYC-dependent mRNA capping and C-MYC function has not yet been explored. Therefore, I sought to determine whether C-MYC regulates CE, and whether CE is required for C-MYC function. I found that C-MYC promotes CE recruitment to RNA polymerase II (RNA pol II) transcription complexes and to regions proximal to transcription start sites on chromatin. Consistently, C-MYC increases RNA pol II-associated CE activity. Interestingly, cells driven by C-MYC are highly dependent on CE for C-MYC-induced target gene expression and cell transformation, but only when C-MYC is overexpressed; C-MYC-independent cells or cells retaining normal control of C-MYC expression are insensitive to CE inhibition. C-MYC expression is also dependent on CE. Taken together, I present a bidirectional regulatory relationship between C-MYC and CE which is potentially therapeutically relevant. Studies here strongly suggest that inhibiting CE is an attractive strategy to selectively target cancer cells which have acquired deregulated C-MYC.

The mRNA cap added to the 5’ end of nascent transcripts is required for the efficient gene expression in eukaryotes. In vertebrates, the guanosine cap is methylated at N7 position by RNMT, which is in complex with its activating subunit RAM. Additionally, the first and second transcribed nucleotides can be methylated at ribose O2 position by CMTR1 and CMTR2 respectively. The mRNA cap protects transcripts from degradation and recruits cap-binding factors to promote pre-mRNA processing, nuclear export and translation initiation. In mouse embryonic stem cells (mESCs), high levels of RAM maintain expression of pluripotency factors. Differentiation of mESCs to neural progenitors is accompanied by a suppression of RAM, resulting in downregulation of pluripotency factors and efficient formation of neural cells. Here, I demonstrated that the suppression of RAM during neural differentiation is promoted via ubiquitination and proteasomal degradation. Concurrently, neural differentiation is associated with an increase in CMTR1 expression, creating a developmental cap methyltransferase switch. Moreover, differentiation into endodermal and mesodermal lineages exhibited distinct changes in the mRNA capping enzymes expression. In mESCs, RAM promotes expression of translation-associated proteins and promotes global loading of mRNA on ribosomes. RAM contributes to the ESC-specific gene expression program, by maintaining optimal expression of pluripotency-associated transcripts and inhibiting expression of neural genes. Chromatin immunoprecipitation revealed that RAM, RNMT and CMTR1 promote binding of RNA polymerase II at gene loci. In RAM-repressed cells, RNA polymerase II binding was reduced at pluripotency-associated genes, but relatively increased at neural genes. Moreover, knock-down of RNMT or CMTR1 induced increased or decreased accumulation of RNA polymerase II at promoter proximal regions respectively. In naïve T cells, Rnmt or Cmtr1 conditional knock-outs caused downregulation of translation-related transcripts and upregulation of cell cycle transcripts. Furthermore, many transcripts were specifically dependent on RNMT or CMTR1 for expression, demonstrating distinct roles of these cap methyltransferases. Thus, the mRNA cap methylation emerges as an important regulator of pluripotency and differentiation, modulating gene expression at transcriptional and post-transcriptional levels.

The synthesis of the N7-methylguanosine cap at the 5’ end of pre-mRNA occurs co-transcriptionally and is catalysed by a series of enzymes including the N7 RNA methyltransferase (RNMT), which along with its recently discovered activating subunit RAM, methylates the cap. RAM, which contains an RNA binding domain, is required to promote RNMT activity both in vitro and in vivo. Although the biochemical function of RAM has been characterized, its biological relevance remains elusive to date. The addition of the cap moiety is a crucial event in gene expression as it affects several processes within the mRNAs life cycle including mRNA processing, stability and translation. In stem cells, every step of mRNA metabolism is tightly regulated to maintain the undifferentiated state, allowing the expression of pluripotency genes and the concomitant repression of the lineage-specific ones. Here, I describe a critical role for the mRNA cap methylation in the maintenance of pluripotency. RNMT and RAM are highly expressed in mESCs compared to differentiated cells. The reprogramming of MEFs to iPS totally restores the elevated expression levels of RNMT and RAM suggesting that high levels of the two proteins are a feature of pluripotent cells. Even more exciting, the same expression is conserved amongst species as also hESCs and hiPSCs exhibit high levels of RNMT and RAM compared to fibroblasts. So far, RNMT and RAM were described as a complex in all cells lines examined and it was assumed that are similarly regulated, instead surprisingly, during in vitro neural differentiation a specific reduction in RAM protein levels is observed. Gain- and loss-of-function studies have been employed to demonstrate that specifically high RAM levels are required for the maintenance of pluripotency. In fact, RAM depletion causes a major reduction in the methyl cap levels of important pluripotency factors, ultimately resulting in a decreased of the protein levels. Therefore, RAM is found to function as modulator of RNMT activity, whereby it promotes cap methylation of fundamental transcripts required for the maintenance of ESCs pluripotency. I have also found that during differentiation RAM is down regulated post-transcriptionally, and therefore current studies are focused on investigating the role of RAM phosphorylation at Serine-36, which correlates with proteosomal degradation. Together the data corroborate previous findings about the methyl cap formation being a critical and regulated process within gene expression and propose a novel implication of this modification in the maintenance of pluripotency.

Contrasting physiological requirements for T. brucei survival between procyclic (vector) and bloodstream (mammal) forms necessitate different molecular processes and therefore changes in protein expression. Transcriptional regulation is unusual in T. brucei because the arrangement of genes is polycistronic; however, genes which are transcribed together are subsequently cleaved into separate mRNAs by trans-splicing and are individually regulated. During the process of trans-splicing, a 39-nucleotide splice-leader RNA is added to the 5´ end of mRNA. In this study, gene regulation in trypanosomes will be examined in the context of the 7-methylguanosine cap attached to the 5´ end of the splice-leader. Interestingly, in addition to the capping enzymes identified in other eukaryotes, trypanosomatids have an additional guanylyltransferase and methyltransferase in the form of a bifunctional enzyme (TbCGM1). TbCGM1 was found to be essential in bloodstream form T. brucei, although the purpose of this bifunctional capping enzyme remains unclear. Null mutants of a related enzyme, monomeric methyltransferase TbCMT1, did not show an effect on cell viability in culture, however, the enzyme proved to be important for virulence in vivo. Complementary to the study of T. brucei capping enzymes, we worked to develop a method to allow structural analysis of the 5´mRNA cap by mass spectrometry. Following pre-mRNA processing, regulation of the mature mRNAs is a tightly controlled cellular process. While multiple stage-specific transcripts have been identified, previous studies using RNA-seq found that the changes in overall transcript level do not necessarily reflect the abundance of the corresponding proteins. We hypothesized that in addition to mRNA stability, mRNA recruitment to ribosomes may play a significant role in the regulation of gene expression in T. brucei. To approach this question, we performed RNA-seq of total, subpolysomal, and polysomal mRNA. This transcriptomic data was then correlated with published proteomic studies to obtain a global picture of the relative translation efficiencies and their relationship to steady-state protein levels between bloodstream and procyclic form T. brucei.

Breast cancer is the leading cause of cancer death in women worldwide. Although significant advances have been made in the treatment of breast cancer, new therapeutic approaches are required. Protein synthesis is often found to be deregulated in cancer and there has been significant effort into developing strategies to inhibit protein synthesis for anti-cancer therapeutics. The addition of the 7-methylguanosine cap (methyl cap) at the 5’ end of mRNA is essential for efficient protein synthesis and cell viability. In humans, RNA guanine-7 methyltransferase (RNMT) methylates the guanosine cap. RNMT expression is essential for cell viability and efficient gene expression. This thesis demonstrates that a subset of breast cancer cells exhibit an enhanced dependence on RNMT for survival, in comparison to immortalised mammary epithelial cells. Moreover, RNMT depletion induces apoptosis in a subset of breast cancer cell lines. Further analyses reveal that this cellular sensitivity to RNMT depletion does not correlate with cellular RNMT expression, RNMT enzymatic activity, global protein synthesis levels, basal cell proliferation rate or c-Myc protein expression. It was found that activating mutations in the gene PIK3CA, which encodes the p110α catalytic subunit of PI3K, contributes to cellular RNMT dependency. Moreover, inhibition of PI3K signalling in a RNMT depletion sensitive breast cancer cell line reverses the sensitivity. These findings provide the first evidence that RNMT dependency in breast cancer is related to PI3K activity/ signalling. Since an estimated 18-40% of breast tumours have activating mutations in PIK3CA, my findings could potentially have significant therapeutic implications.

The N-7 methylguanosine cap structure is conserved from yeast to man. It is essential for cell proliferation as it influences several steps in eukaryotic gene expression including transcription, pre-mRNA processing, RNA export and translation. The N-7 methylguanosine cap is added co-transcriptionally to RNA pol II transcripts. In mammals, two enzymes catalyse the synthesis of the N7-methylguanosince cap. RNGTT adds an inverted guanosine group to the first transcribed nucleotide and RNMT methylates the guanosine cap at the N7-position. RNMT consists of a catalytic domain and an N-terminal domain that is absent in lower eukaryotes. Experiments presented in this thesis revealed that the N-terminus mediates RNMT recruitment to transcription start sites. Furthermore, it was found that the RNMT N-terminal domain is phosphorylated at Threonine-77 (T77) by CDK1/Cyclin B in a cell cycle-dependent manner during G2/M-phase. RNMT T77 phosphorylation activates cap methyltransferase activity in vitro. Furthermore, it negatively regulates the interaction of RNMT with KPNA2 (Importin-a), which was found to inhibit RNMT activity in vitro. RNMT T77 phosphorylation is required for normal cell proliferation suggesting an important biological function. Initial experiments indicated that RNMT T77 phosphorylation functions to regulate gene expression in a gene-specific manner. Future work is focused on establishing an experimental system to perform a genome-wide study in order to elucidate which transcripts are affected by RNMT T77 phosphorylation. To summarise, this study for the first time revealed that the RNA cap methyltransferase activity is regulated in a cell-cycle dependent manner.

Gene expression in eukaryotes is dependent on the N-7 methylguanosine cap, located at the 5’ end of RNA pol II transcripts, which marks pre-mRNA for processing, stabilisation and translation initiation. The enzymes that catalyse the formation of the N-7 methylguanosine cap are recruited to RNA pol II at the initial stages of transcription. The final step in this process, N-7 methylation of the guanosine cap, is catalysed by the RNA guanine-7 methyltransferase, RNMT. RNA guanine-7 methylation is an essential process for cell viability and its up-regulation has been associated with cell transformation. However, the mechanistic details of RNMT function in mammalian cells remain elusive. In order to gain better understanding of the molecular mechanisms associated with RNA guanine-7 methylation, cellular RNMT complexes were purified from human cells and constituent proteins were identified using mass spectrometry. A novel component of the RNA guanine-7 methyltransferase complex was identified and designated as RAM (RNMT activating mini-protein). The vast majority of RNMT is found in a complex with RAM and vice versa.RAM is an RNA-binding protein, promoting recruitment of RNA to RNMT. RAM increases recombinant and cellular RNMT cap methyltransferase activity and is required for cap methylation in vivo. We therefore, describe RAM as an “obligate activator” of the human cap methyltransferase. As expected of a protein essential for cap methylation, RAM is required for gene expression, and RAM depletion results in loss of cell viability. Current studies are being focused on determining RAM/RNMT crystal structure as well as determining how the RNA guanine-7 methyltransferase complex is regulated within cells.