Invertebrates show enhanced immunity and even specific primed immunity in response to repeat infections, analogous to vertebrate adaptive immunity. Little is known of the mechanism for this phenomenon, or which molecules are involved. A candidate gene for the underlying mechanism for a pathogen-specific response in invertebrate immunity is Down syndrome cell adhesion molecule (Dscam). Dscam can produce thousands of different protein isoforms through the mutually exclusive splicing of many exon variants contained within variable regions of the gene. It is an important receptor of the invertebrate nervous system but has been implicated in having a role in immunity. Dscam has been shown to be involved in phagocytosis across members of the Pancrustacea, and it has been reported to respond in a pathogen-specific manner in mosquitoes and crayfish. In this thesis, I have investigated the splicing of Dscam in response to diverse pathogens in different host species. In the Anopheles mosquito, I cloned and sequenced a fragment of Dscam spanning across two of its variable exon regions to enable me to detect mutually exclusively splice variants and their associations in different treatments (Chapter 2). I discovered that the expression diversity of the hypervariable Dscam is higher in parasite-exposed mosquitoes. In Chapter 3, I extended the study to the more experimentally amenable Drosophila fruit fly. A new Illumina-based sequencing assay was developed and implemented to examine more closely Dscam expression in response to diverse pathogens. The new method successfully quantified non-random expression of Dscam variable exons 4 and 6. I also describe a small but detectable effect of pathogen-exposure on the expression of Dscam exon 4 variants. In Chapter 4, I expanded the work of Chapter 3 to study tissue-specific Dscam expression in response to well-characterised immune elicitors of Drosophila. I describe how exon 4 variants were expressed in a tissue-specific manner, but not exon 6 variants. I also found a small exon 4-by-tissue-by-pathogen effect, which although detectable, did not dominate over the tissue effects. Finally, in Chapter 5, I turned to the crustacean, Daphnia, to study Dscam expression in a natural host-parasite interaction and in a clonal organism. I describe the non-random expression of exons 4 and 6, and another small effect of pathogen-exposure on the expression of Dscam exon 4. My work aimed to further investigate the putative pathogen-specific alternative splicing of the hypervariable Dscam receptor. The data presented quantified the constitutive expression of Dscam exons 4 and 6 in different pancrustacean species. The data also suggest that infection-responsive splicing of Dscam may occur but that effects are small, and may be diluted within the background of the highly important Dscam expression of the nervous system if they exist at all. The study supports the high-throughput sequencing method for future studies of alternative splicing and Dscam expression.

Alternative pre-mRNA splicing greatly contributes to the mammalian proteomic diversity. The novel splice variants often emerge through splicing regulation at/nearby the splice sites (SS). A large group of 3′SS in human genes contain REPA (regulatory elements between the Py and 3′AG) G tracts that mostly appear in mammals as splicing silencers. However, the underlying molecular mechanisms and functional consequences remain unknown. We have identified a novel class of REPA G tracts (G)5-8 in a group of human genes including PRMT5 (protein arginine methyl transferase 5) that are significantly enriched in functional clusters of cell growth and proliferation. The PRMT5 G tracts emerged evolutionarily in mammals and repress splicing through recruitment of mainly hnRNP H that interferes with early spliceosome assembly. The splicing repression creates a shorter PRMT5 isoform (PRMT5S) that inhibits cell cycle progression contrary to the role of the full length protein (PRMT5L). Moreover, the expression of a group of genes involved in cell cycle arrest at interphase is preferentially regulated by PRMT5S. We further showed that PRMT5S is differentially expressed among cell and tissue types suggesting tissue-specific regulation. It exhibits distinct subcellular localization pattern from that of PRMT5L and opposite effects on cell cycle-specific structural dynamics of the Golgi apparatus. Moreover, these splice variants are differentially expressed during cell differentiation and PRMT5S promotes the differentiation of dendritic cells whereas PRMT5L shows the opposite effect. The expression of a large number of genes including those involved in crucial cellular processes such as differentiation and apoptosis is regulated by these splice variants of PRMT5. This study provides a direct link between the evolutionary emergence of a novel splicing regulatory G tract element and the generation of a functionally distinct protein isoform. The molecular mechanism underlying the splicing regulation by this G tract is likely common to many mammalian genes and the generation of their protein diversity. / October 2015

Alternative splicing is a common post-transcriptional process in eukaryote organisms by which a single gene can produce more than one distinct transcript. First discovered in the late 1970s, alternative splicing has been the focus of intense attention after the release of the human genome draft revealed a lower than expected gene number. Almost all human protein coding genes are now known to be alternatively spliced. However, how alternative splicing in humans and other well studied model organisms compares to other less characterised taxa such as protists and fungi or what is the functional role of alternative splicing remains poorly understood. Here I analyse alternative splicing in dozens of species using millions of partial transcript sequences ESTs. By applying a transcript normalisation method I showed that alternative splicing in protists and fungi is higher than previously reported and highly variable. I further observed that in representatives of both taxa, associations with translation are overrepresented among alternatively spliced genes. However, no evidence for a relationship between alternative splicing and complex phenotypes was found. Taking human lice as a model I explored the role of alternative splicing in the evolution of phenotypic variants. I found that, despite the fact that the transcriptome profiles of head and body lice are nearly identical, there are markedly differences in alternative splicing patterns. Development related functional associations were found to be enriched among genes with body lice specific alternative splicing events but not in head lice consistent with a scenario of differential patterns of alternative splicing contributing to the phenotypic innovations as human lice adapted to life in human clothing. I further explore the functional relevance of alternative splicing and its possible role in driving genomic innovations even preceding events of gene duplication. Together the work presented show that alternative splicing is widespread among previously understudied fungi and protist species and provide insights on its role in species adaptation to novel environments in using human lice as a model.

Tauopathies refer to a group of neurodegenerative diseases that are characterized by pathological aggregations of the microtubule-associated protein Tau (MAPT). Aberrant alternative splicing of Tau exon 10 leads to the imbalanced expression of Tau isoforms that contain either 3 or 4 microtubule binding repeats (3R-Tau or 4R-Tau) and this is sufficient to cause the formation of Tau inclusions. Nonetheless, the exact molecular mechanisms that regulate aberrant Tau exon 10 splicing regulation and subsequent Tau aggregation in tauopathies remain elusive. In my thesis research, I used RNA Antisense Purification by Mass Spectrometry (RAP-MS) to identify upstream regulators of Tau splicing events. Among the 15 identified novel protein candidates, I validated that hnRNPA2B1 and hnRNPC are required to promote 4R-Tau expression, whereas hnRNPH1 supports 3R-Tau expression. Separately, to elucidate the functional difference between 3R- and 4R-Tau isoforms, I performed proximity-dependent biotin identification (BioID2) for all six human central nervous system Tau isoforms in mouse primary neurons. Followed by tandem mass tag (TMT)-labeling proteomics and data analysis, I observed that 4R-Tau proximal proteins are highly enriched in endocytosis, whereas 3R-Tau proximal proteins show top enrichment in fatty acid metabolism. Through further biochemical validations, I found that MAT2A, a S-adenosylmethionine synthase, has higher binding affinity with 3R-Tau versus 4R-Tau. Overall, using novel proteomics methods, I discovered novel Tau splicing regulators and characterized the neuronal Tau isoform-specific proximity proteome networks. These proteins, once validated through future functional studies in cellular and animal models, can represent therapeutic and diagnostic targets for neurodegenerative tauopathies. / Thesis / Doctor of Philosophy (PhD)

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No description available.

Alternative splicing is a key mechanism by which eukaryotes generate phenotypic complexity without increasing genomic load. In vertebrate evolution, cassette exon alternative splicing is prominent with increasing phenotypic complexity and is specifically enriched in the brain. Apolipoprotein receptor 2 (Apoer2) is a neuronal alternatively spliced transmembrane receptor that binds critical extracellular ligands such as neuroprotective Reelin and Alzheimer’s disease (AD) related risk factor APOE4. Inclusion and exclusion of single exons in Apoer2 regulates isoform specific roles in neuronal processes, such as long-term potentiation (LTP) and neuronal survival. Alternative splicing of APOER2 exon 18, which encodes a functional domain critical for LTP, has been reported as dysregulated in AD. However, the full repertoire and function of APOER2 isoforms in physiological and AD conditions is not well understood. We hypothesize that combinatorial APOER2 alternative splicing events generate a diverse pool of isoforms in the human brain that can become dysregulated in AD and alter receptor function in neurons. Our overall goal is to define the APOER2 transcript pool and understand whether isoform proportions and functions are altered in AD, potentially contributing to synaptic dysfunction. In this work, we observed that Apoer2 has evolved over the course of vertebrate evolution, gaining new exons that alter function at the protein level and increasing the complexity of its alternative splicing events from zebrafish to humans. We generated the first APOER2 specific long-read RNA sequencing dataset in the human cerebral cortex, which identified 48 full-length APOER2 isoforms, some of which are unique compared to full-length murine Apoer2 isoforms and indicate that Apoer2 is spliced in a species specific manner. To determine whether splicing of APOER2 is dysregulated in AD, we generated full-length APOER2 isoform maps in Control and AD parietal cortex and hippocampus. We identified over 200 unique APOER2 isoforms in each brain region with 151 isoforms common between the two brain regions. We also identified region and disease specific APOER2 isoforms suggesting APOER2 splicing is spatially regulated and altered in AD. We found AD and Control-specific APOER2 isoforms exhibited alterations in receptor processing and cleavage patterns, indicating combinatorial splicing across APOER2 dictates protein function and is changed in AD. Sequential cleavage of Apoer2 in response to Reelin generates an intracellular domain (ICD) that translocates to the nucleus and affects transcription; however, whether APOE influences Apoer2 cleavage is unclear. We found Apoer2-ICD is generated in an APOE isoform specific manner and is generated regardless of exon 19 inclusion, which encodes part of the ICD. We generated four novel mouse lines to examine the effects of Apoer2 exon 19 inclusion and APOE isoforms (APOE3 and APOE4) on hippocampal gene expression. We found Apoer2 exon 19 inclusion modulates upregulation of genes such as Serpina3n known to be induced by APOE4 expression, which has strong implications for understanding molecular mechanisms underlying APOE4 as a risk factor in AD. Lastly, since Apoer2 exon 19 confers critical functions at the protein level, including adaptor protein binding and association with the NMDA receptor, as well as potentially modulating APOE4’s transcriptional effects, we were interested in how an RNA binding protein, Srsf1, may influence Apoer2 exon 19 splicing. We and others have found SRSF1 partially represses exon 19 inclusion in primary murine neurons. Because splicing is often modulated by neuronal activity, we examined whether Apoer2 exon 19 and Srsf1 are altered in response to activity stimulation. We found upregulation of exon 19 exclusion and no strong changes in SRSF1 expression or phosphorylation, suggesting modulation of SRSF1 is not a potent regulatory mechanism of activity induced changes in Apoer2 exon 19 splicing. Overall, we have examined the Apoer2 splicing landscape in the brain across multiple vertebrate species. We identified a rich diversity of alternatively spliced APOER2 isoforms in Control and AD brains providing novel APOER2 variants that are significantly changed in AD. These AD related APOER2 isoforms have differential functional impacts on APOER2 biology that may contribute to AD pathogenesis.

The ability of neurons to dynamically regulate their response to changing inputs is essential for the correct development and function of a nervous system capable of learning and memory. The post synaptic compartment of excitatory synapses contains a dense proteinaceous complex of molecules that link excitatory glutamatergic neurotransmission to downstream signalling pathways that ultimately result in modification of the synapse. One of the most abundant of such postsynaptic signalling molecules, synaptic GTPase activation protein, SynGAP, represents a key signalling link between the activation of the NMDA sensitive glutamate receptor to outcomes such as the structural rearrangement of synaptic sites and altered synaptic content of AMPA type glutamate receptors, molecular processes that underly learning and memory. The primary finding of this thesis is that different isoforms of SynGAP, which varies at it N terminus through alternative transcription start sites and at its C terminus through alternative splicing, can differentially affect the function of the synapse. Using primary murine neuronal cultures we show that despite being crucial for the survival of the mouse the absence of SynGAP does not effect mean dendritic spine morphology and density or miniature excitiatory post synaptic currents under a range of experimental conditions (days in vitro 10 – 14, with and without serum, high and low cell plating density). In order to examine the effects of different SynGAP isoforms we cloned two full length transcripts (SynGAP A-alpha-2 and SynGAP Ealpha- 1) which were used to construct a range of isoforms. Whole cell patch clamping of SynGAP transfected neurons revealed that the post synaptic expression of SynGAPs which terminate as an alpha-1 isoform can lead to the elimination of mEPSCs, while isoforms that terminate as an alpha-2 isoform can lead to synaptic strengthening. The magnitude of the effect in both cases is determined by the identity of the N terminus of the protein; SynGAP A-alpha-1 has the largest synaptic weakening effect and SynGAP B-and C alpha-2 strenghten the synapse. The changes in miniature electrophysiological properties are not mirrored by changes in dendritic spine morphology, whole cell AMPA/NMDA currents, or synaptic responsiveness to stimulation suggesting an undefined novel mechanism of action. SynGAPs A, B and C appear to be under the control of different promoters which are differentially regulated by development and synaptic activity, thus the differential function of SynGAP N and C terminal combinations could play a part in the activity dependent regulation of synaptic strength.

RNA-binding proteins play a central role in the post-transcriptional regulation of gene expression; however, little is known about the endogenous transcripts to which they bind. Here, I have used the ultra-violet cross-linking and immuno-precipitation (CLIP) technique to identify RNA targets directly bound to two RNA-binding proteins: Acinus and hnRNP A1. Acinus (apoptotic chromatin condensation inducer in the nucleus) contains a region that is homologous to the RNA binding domain of the Drosophila splicing regulator sex-lethal, and a serine and arginine rich region similar to that seen in the SR family of proteins, which function extensively in splicing. Furthermore it is a component of the multi-protein spliceosome complex, and I have demonstrated it can directly bind polyadenylated RNA. I have shown that Acinus displays a diffuse nuclear localisation pattern, however, overexpression of an epitope-tagged protein results in its accumulation in enlarged nuclear speckles. Together these results suggest a role in pre-mRNA splicing. Acinus is cleaved during apoptosis by caspase-3, resulting in a truncated protein with chromatin condensation inducing activity (Sahara et al., 1999). Accordingly, I have demonstrated that overexpression of epitope-tagged Acinus results in an increased number of cells exhibiting an apoptotic phenotype. The proteolytic fragment contains the RNA binding region, and to determine if the role of Acinus in apoptosis is mediated by RNA interactions I utilised CLIP to identify in vivo RNA targets. I have identified several mRNA targets of Acinus and found that the binding sites in those mRNA targets predominantly map to constitutively expressed exons. This is in agreement with the exon junction complex, of which Acinus is a component, being deposited on mRNAs after splicing. These results may indicate that Acinus is a core RNA binding factor of the exon junction complex. To complement this approach, I also performed CLIP with a known alternative splicing regulator, hnRNP A1. In this manner, the binding site preferences could be compared between the two proteins. As expected, the majority of hnRNP A1 binding sites are located in introns, corresponding with their identified role of antagonizing pre-mRNA splicing by binding intronic splicing elements. Interestingly, a number of the CLIP tags are located in, or adjacent to, alternatively spliced events suggesting a role for hnRNP A1 in the regulation of alternative splicing of these specific pre-mRNAs. In addition to pre-mRNA splicing hnRNP A1 also functions in the cellular stress response. Upon environmental stresses it relocates to the cytoplasm and accumulates in cytoplasmic foci known as stress granules (Guil et al., 2006). Here I show some of the targets identified by CLIP are regulated by hnRNP A1 in times of cellular stress. In summary, I have identified two novel subsets of RNAs, bound by Acinus or hnRNP A1 in vivo. I have shown these proteins exhibit distinct binding preferences, which correspond to their biological function. This work is consistent with hnRNP A1 acting as an alternative splicing regulator, and provides evidence for a dual role of Acinus in mRNA splicing and apoptosis. This study also demonstrates the power of the CLIP technique, as identification of in vivo RNA targets allows greater understanding of the mechanisms by which RNA-binding proteins exert their regulatory control.

Abstract Alternative splicing provides a versatile mechanism by which cells can generate proteins with different or even antagonistic properties. Herein we describe a novel splice variant of the hypoxia-inducible death gene Bnip3. Sequence analysis of the new Bnip3 protein revealed an N-terminus that was identical to Bnip3 but contained an Endoplasmic reticulum (ER) retention motif within the C-terminus, therefore we designated the new Bnip3 isoform NIPLET for (Nip-Like ER Target). While Bnip3 was predominately localized to mitochondria and promoted mitochondrial perturbations and cell death, NIPLET was preferentially localized to the ER and opposed the cytotoxic actions of Bnip3. Interestingly, NIPLET suppressed mitochondrial injury from Bnip3 activation and mitochondrial permeability transition pore opening by a mechanism dependent upon the dynamin motor protein Mitofusin-2 (MFN2). Notably, mutations of NIPLET within the critical ER retention motif rendered NIPLET defective for interacting with MFN2 and suppressed necrosis induced by Bnip3 or hypoxia. Hence, our findings reveal a novel signaling pathway that functionally couples ER and mitochondria for cell survival to a mechanism that is mutually dependent and obligatorily linked to a novel BNIP3 protein in cardiac myocytes. / May 2016