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.

No description available.

<p>Alternative RNA splicing is one of the leading mechanisms contributing towards transcript and protein diversity. Several alternative splicing surveys have confirmed the frequent occurrence of exon skipping in human genes. However, the occurrence of exon skipping in mouse genes has not yet been extensively examined. Recent improvements in mouse genome sequencing have permitted the current study to explore the occurrence of exon skipping in mouse genes orthologous to human genes on chromosome 22. A low number (5/72 multi-exon genes) of mouse exon-skipped genes were captured through alignments of mouse ESTs to mouse genomic contigs. Exon-skipping events in two mouse exon-skipped genes (GNB1L, SMARCB1) appear to affect biological processes such as electron and protein transport. All mouse, skipped exons were observed to have ubiquitous tissue expression. Comparison of our mouse exon-skipping events to previously detected human exon-skipping events on chromosome 22 by Hide et al.2001, has revealed that mouse and human exon-skipping events were never observed together within an orthologous gene-pair. Although the transcript identity between mouse and human orthologous transcripts were high (greater than 80% sequence identity), the exon order in these gene-pairs may be different between mouse and human orthologous genes.<br /> <br /> Main factors contributing towards the low detection of mouse exon-skipping events include the lack of mouse transcripts matching to mouse genomic sequences and the under-prediction of mouse exons. These factors resulted in a large number (112/269) of mouse transcripts lacking matches to mouse genomic contigs and nearly half (12/25) of the mouse multi-exon genes, which have matching Ensembl transcript identifiers, have under-predicted exons. The low frequency of mouse exon skipping on chromosome 22 cannot be extrapolated to represent a genome-wide estimate due to the small number of observed mouse exon-skipping events. However, when compared to a higher estimate (52/347) of exon skipping in human genes for chromosome 22 produced under similar conditions by Hide et al.2001, it is possible that our mouse exon-skipping frequency may be lower than the human frequency. Our hypothesis contradicts with a previous study by Brett et al.2002, in which the authors claim that mouse and human alternative splicing is comparable. Our conclusion that the mouse exon-skipping frequency may be lower than the human estimate remains to be tested with a larger mouse multi-exon gene set. However, the mouse exon-skipping frequency may represent the highest estimate that can be obtained given that the current number (87) of mouse genes orthologous to chromosome 22 in Ensembl (v1 30th Jan. 2002) does not deviate significantly from our total number (72) of mouse multi-exon genes. The quality of the current mouse genomic data is higher than the one utilized in this study. The capture of mouse exon-skipping events may increase as the quality and quantity of mouse genomic and transcript sequences improves.</p>

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

No description available.

Alternative splicing (AS) is a common post-transcriptional process in eukaryotic organisms, by which multiple distinct functional transcripts are produced from a single gene. Because of its potential role in expanding transcript diversity, interest in alternative splicing has been increasing over the last decade, ever since the release of the human genome draft showed it contained little more than the number of genes of a worm. Although recent studies have shown that 94% human multi-exon genes undergo AS while aberrant AS may cause disease or cancer, evolution of AS in eukaryotic genomes remains largely unexplored mainly due to the lack of comparable AS estimates. In this thesis I built a Eukaryote Comprehensive & Comparable Alternative Splicing Events Database (ECCASED) based on the analyses of over 30 million Expressed Sequence Tag (ESTs) for 114 eukaryotic genomes, including protists (22), plants (20), fungi (23), metazoan (non-vertebrates, 29) and vertebrates (20). Using this database, I addressed two main questions: 1) How does alternative splicing relate to gene duplication (GD) as an alternative mechanism to increase transcript diversity? and 2) What is the contribution of alternative splicing to eukaryote transcript diversity? I found that the previous “interchangeable model” of AS and gene duplication is a by-product of an existing relation between gene expression breadth, AS and gene family size. I also show that alternative splicing has played a key role in the expansion of transcript diversity and that this expansion is the best predictor reported to date of organisms complexity assayed as number of cell types. In addition, by comparing alternative splicing patterns in cancer and normal transcript libraries I found that cancer derived transcript libraries have increased levels of “noisy splicing”.

Regulation of pre-mRNA splicing is a key process for most if not all eukaryotes. The process can, in the abstract, be considered as a series of trans-acting factors that interact with cis-motifs in the RNA to enable the removal of introns and joining of exons. As the cis factors need not only be the splice sites themselves, but also motifs in the exons, the splicing process has the potential to impose selective constraint on exonic sequence in addition to the normal selection on the amino acid content of the protein. To understand this more clearly, in this thesis, I mainly focus on a type of important and widely investigated cis-motifs, exonic splicing enhancers (ESEs), which bind with SR proteins to re-enforce the splice sites and so ensure splicing correctly. First, I explore splice-related cis-motif usage of the Ectocarpus genome, which is a species phylogenetically very distant from vertebrates but, like vertebrates in having abundant large introns. A deep phylogenetic conservation of exonic splice-related constraints is observed (Chapter II). Then I extend the analysis across taxa in a phylogenetically explicit framework. In this section stronger selection on exon end synonymous sites can be detected within humans when the exons are flanked by larger introns. Additionally I report evidence that reduced Ne might lead to larger introns and weakened splice sites. Thus I suggest an unusual circumstance in which selection (for cis-motifs to control error-prone splicing) might be stronger when population sizes are smaller; this is unexpected and would be a necessary complement to nearly-neutral theory (Chapter III). Third, I ask whether what we know about biases in the usage of ESEs and splicing control elements allows us to understand where in human genes pathogenic mutations tend to occur (Chapter IV). By examining the relationship between determinants of the usage of splice-associated cis-motifs and the distribution of human pathogenic SNPs, I found certain exons are vulnerable to splice disruption owing to low ESE density and a “fragile” exon model we proposed could describe and explain this phenomenon (Chapter IV). Finally I perform preliminary analysis, with a view to biotechnological optimization of transgenes, to address whether there might be such a thing as a tissue specific ESE. To this end I examine ESE usage in tissue specific genes. I find some preliminary evidence for tissue specific biased usage of certain ESEs.

Les dysferlinopathies sont des maladies musculaires dues à une déficience en protéine dysferline, codée par le gène DYSF. Dans ce travail de thèse, trois approches thérapeutiques ont été évaluées pour ces pathologies, sur des modèles cellulaires et murins. Un variant transcriptionnel court de la dysferline a été vectorisé dans un AAV8r et injecté dans le modèle murin Bla/J, déficient en dysferline. L’analyse des muscles des animaux traités montre une augmentation de la résistance des fibres musculaires au stress mécanique, mais n’apporte pas de correction histologique. Cette étude souligne également la toxicité de cette miniprotéine. L’anoctamine 5, impliquée dans des pathologies et des activités similaires à la dysferline, a été testée en tant que protéine compensatrice. L’anoctamine 5 surexprimée dans le modèle Bla/J ne permet pas la restauration d’un phénotype normal. La compensation de DYSF par ANO5 n’est donc pas une voie thérapeutique à exploiter pour les dysferlinopathies. Enfin, une thérapie génique par chirurgie de l’ARN dysferline a été évaluée en utilisant le trans-épissage médié par le splicéosome (SMaRT). La preuve de principe de la reprogrammation d’un minigène dysferline a été faite (ARN et protéine trans-épissée obtenus in vitro). L’efficacité du SMaRT dans un contexte endogène s’est en revanche révélée faible, et n’a pas permis la restauration d’une protéine dysferline fonctionnelle dans des myoblastes humains. De plus, l’observation de l’expression de protéines directement à partir du RTM (RNA-trans-splicing molecule) a fait apparaître des limites à l’utilisation du SMaRT pour la thérapie génique, et en particulier pour les dysferlinopathies. / Dysferlinopathies are muscular diseases due to mutations in DYSF gene, inducing dysferlin protein deficiency. In this thesis, three therapeutic approaches have been investigated for these pathologies, on cell or mice models. A short transcriptional dysferlin variant has been injected into Bla/J dysferlin deficient mouse model, using AAV8r vector. Muscle fibers of treated animals displayed an increased resistance to mechanical stress without therapeutic benefit. These experiments also pointed out the toxicity of this strategy. A protein compensation approach has been tested using anoctamin 5, known to be involved in pathologies and activities similar to dysferlin’s ones. AAVr mediated Anoctamin 5 overexpression in Bla/J model does not rescue their muscle phenotype. Overexpression of ANO5 does not seem to be a valuable therapeutic strategy for dysferlin deficiency. Dysferlin RNA surgery was evaluated as a possible genetic therapy using Spliceosome-Mediated RNA Trans-splicing (SMaRT). On a Minigene target, SMaRT is able to induce RNA reprogramming by trans-splicing, and produce the corresponding protein. But efficiency is by far decreased in endogenous context and not good enough to restore functional dysferlin in human myoblasts. Moreover, we described proteins resulting from RNA-trans-splicing molecule (RTM) self-expression, limiting the value of SMaRT as therapeutic strategy, especially for dysferlinopathies.

Alternative splicing of pre-mRNA is a critical mechanism for enabling genetic diversity, and is a carefully regulated process in neuronal differentiation. RNA binding proteins (RBPs) are developmentally expressed and physically interact with RNA to drive specific splicing changes. This work tests the hypothesis that RBP-RNA interactions are critical for regulating timed and coordinated alternative splicing changes during neurodevelopment and that these splicing changes are in turn part of major regulatory mechanisms that underlie morphological and functional maturation of neurons. I describe our efforts to identify functional RBP-RNA interactions, including the identification of previously unobserved splicing events, and explore the combinatorial roles of multiple brain-specific RBPs during development. Using integrative modeling that combines multiple sources of data, we find hundreds of regulated splicing events for each of RBFOX, NOVA, PTBP, and MBNL. In the neurodevelopmental context, we find that the proteins control different sets of exons, with RBFOX, NOVA, and PTBP regulating early splicing changes and MBNL largely regulating later splicing changes. These findings additionally led to the observation that CNS and sensory neurons express a variety of different RBP programs, with many sensory neurons expressing a less mature splicing pattern than CNS neurons. We also establish a foundation for further exploration of neurodevelopmental splicing, by investigating the regulation of previously unobserved splicing events.