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Nonsense mediated mRNA decay (NMD) is a surveillance mechanism that targets transcripts containing premature stop codons (PTCs) for degradation, and that also regulates up to 10% of the whole transcriptome. During the course of my PhD I set out to identify novel NMD factors by performing a genome-wide RNA interference (RNAi) screen in a transgenic strain of Caenorhabditis elegans carrying an NMD reporter. I identified five novel proteins that are putative NMD factors in worms: NGP-1, NPP-20, AEX-6, PBS-2 and NOAH-2. Knock-down of these proteins led to severe developmental defects: worms were either arrested during various larval stages or died prematurely. The only exception was AEX-6, the knockdown of which led to a milder phenotype. Homology analysis of the novel C. elegans NMD factors showed that these proteins are conserved in human, with the exception of NOAH-2, which only has a homologue in Drosophila melanogaster, NOMPA. By performing an NMD assay in human cells, I demonstrated that GNL2 (NGP-1) and SEC13 (NPP-20) are functionally conserved NMD factors in human. Analysis of the consequences of depletion of GNL2, SEC13, UPF1 or UPF2 on the transcriptome of HeLa cells revealed that these four proteins co-regulate a subset of endogenous NMD targets, whilst also independently regulating the expression of other sets of transcripts. The findings presented in this thesis further our knowledge of the biology of NMD in both nematodes and humans. They demonstrate the existence of further regulators of this surveillance pathway, and add a layer of complexity to this fine-tuned biological process.

Gene expression can be regulated from transcriptional initiation to RNA processing and turnover time and post translational modification of a protein. The majority of studies of gene expression have focussed on transcription. However, it is also important to understand how post-transcriptional pathways are regulated in response to inflammatory stimuli. Chapter 1 introduces background to gene expression during post transcriptional regulation and its regulation related to inflammatory diseases. The innate immune response to LPS is highly dynamic yet tightly regulated. RNA decay pathways include nonsense-mediated decay, the RNA decay exosome, P-body localised deadenylation, decapping and degradation and AU-rich element targeted decay mediated by tristetraprolin. Chapter 2, we examined the regulation of RNA degradation pathways during the lipopolysaccharide response in macrophages and these results have been published. Alternative splicing has been identified as a key process in post-transcriptional regulation of gene expression in higher eukaryotes. In the immune system, alternative splicing provides a major role in regulating gene expression and generating the diverse mRNA transcripts and protein isoforms, therefore giving rise to protein diversity.Intron retention (IR) is a form of alternative splicing where an intron is not removed during transcription coupled splicing and is considered a widely regulated process during gene expression. Chapter 3 we examined its regulation during inflammation. Also, gene expression can be regulated via nonsense-mediated decay, which can precisely control the timing and level of gene expression as well as eliminating unstable or toxic protein production. SMG1 is a member of the PIKK (phosphoinisitide 3-kinase related kinases) family and plays an important role in NMD. For Chapter 4 we investigated the role of SMG1 in the regulating in response to inflammatory stimuli. We generated a novel animal model of total SMG1 loss in macrophages to address this question. For Chapter 5, we showed how to analyse RNA-seq. of BMM from LysM+/CreSmg1fl/fl (Cre) and Smg1fl/fl (wild-type) mice treated with LPS treatment at dedicated time points by gene ontology tools to discover gene enriched clusters during an LPS treatment between LysM+/CreSmg1fl/fl (Cre) and Smg1fl/fl (wild-type) mice. A final discussion and future directions for this field of study are provided in Chapter 6.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the cell bodies of FUS ALS patients motor neurons. The nature of these inclusions has remained elusive, as the proteins localized to them have not been identified. Additionally, the functional consequence of the accumulation of cytoplasmic FUS inclusions has not been established, nor is it understood how they contribute to selective motor neuron death. We carried out two related, but independent studies to characterize the proteins that may be included in FUS-positive inclusions. In this first study, we utilized immunoprecipitation of wild-type and mutant FUS in the presence and absence of RNase, followed by LC MS/MS. The identified proteins represent those that directly or indirectly interact with FUS, with relatively high affinity that can be pulled down with immunoprecipitation. A wide variety of interacting proteins were identified and they are involved in a multitude of pathways including: chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. Their interaction with FUS varied greatly in their requirements for RNA. Most notably, FUS interacted with hnRNPA1 and Matrin-3, proteins also known to cause familial ALS. Immunofluorescent staining of proteins interacting with mutant FUS were localized to cytoplasmic inclusions. We concluded that mis-localization of these proteins potentially lead to their dysregulation or loss of function, thus contributing to FUS pathogenesis. In the second study, we developed a protocol to isolate dynamic FUS inclusions and employed LC MS/MS to identify all proteins associated with FUS inclusions. We identified a cohort of proteins involved in translation, splicing, and RNA export to be associated with the FUS inclusions. Further pathway and disease association analysis suggested that proteins associated with translation and RNA quality control pathways may be the most significant. Protein translation assays using both N2A and ALS patient fibroblasts demonstrated suppression of protein biosynthesis in mutant FUS expressing cells. However, translation initiation was not impaired. To understand how protein synthesis is suppressed by mutant FUS mediated defects in RNA metabolism, we examined changes in a well conserved RNA turnover pathway namely: nonsense mediated decay (NMD). We found that NMD is hyperactivated in cells expressing mutant FUS, likely due to chronic suppression of protein translation shifting the pathways autoregulatory circuit to allow for hyperactivation. We concluded that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation. These defects together likely contribute to motor neuron death.

Using the yeast, Saccharomyces cerevisiae, as a model system, we utilized Affymetrix tiling arrays to globally investigate novel targets of the nonsense mediated decay pathway. The research that has been presented in the first chapter of this dissertation is centered around the investigation of extended transcripts and the degradation pathways that contribute to its rapid turnover in yeast cells. This work was published in the online version of the journal, PLoS Genetics. The work that is presented in the second chapter is based on our findings that intron-retained or unspliced RNAs are markedly stabilized in NMD mutant strains which led us to conclude the widespread impact of the nonsense mediated decay pathway in the degradation of unspliced RNAs. We further expanded our work and showed that introns emanating from NMD-sensitive loci can elicit an NMD-sensitive phenotype in trans, which led us to conclude that the intron (intronic sequences) are necessary for proper NMD-targeting. We further discovered that sequences located within the 5'SS as well as the branchpoint (BP) are responsible in part for the generation of unspliced/inefficiently spliced transcripts. Lastly, we showed that amino acid starvation results in a transient inhibition of nuclear splicing resulting in hyperaccumulation of unspliced transcripts. Under this condition, we elucidated a stress-specific role for the NMD pathway in rapidly targeting and degrading these unspliced transcripts. The results that have been presented in the second chapter of this dissertation have been published in the Molecular Cell journal (August 8th, 2008 issue). The third chapter (part) of this dissertation explores themes that have directly evolved from the first part. Specifically, the second part has shown that there are more complex routes for the degradation for unspliced transcripts besides the NMD pathway. In particular, synergistic modes of degradation involving components of the nuclear exosome and factors involved in NMD function to limit and hence degrade these unspliced transcripts. Decay measurements were undertaken to show that there are unspliced RNAs whose degradation mode depends on a synergistic mode of degradation requiring both the nuclear exosome component, Rrp6p and the NMD factor, Upf1p. Conversely, we found a class of unspliced transcripts whose turnover relies largely on the action of the NMD pathway and not on synergistic degradation. We further expanded our research and showed that inactivation of an essential, conserved, yeast RNA export factor, Mex67p, results in the nuclear sequestration and degradation of normally cytoplasmically degraded unspliced transcripts. This result highlighted the generic features of RNA degradation and has shown that temporal changes (such as malfunction of proper nuclear-to-cytoplasmic RNA trafficking) cause spatial redistribution and degradation of the unspliced RNA. We have also shown that the nuclear periphery factor, Mlp1p, could have a potential role in the retention of nuclear-degraded unspliced RNA. Deletion of this factor led to an increase in the cytoplasmic concentration of the unspliced RNA which was then degraded by the NMD pathway.