No description available.

Research Doctorate - Doctor of Philosophy (PhD) / Pre-term birth is still the leading cause of perinatal mortality and morbidity. CRH is a hormone that is involved in the timing of labour, therefore investigation of its regulation is of importance in understanding human parturition. The CRE is a central regulatory element on the CRH promoter and in investigating proteins that bind to this element a novel protein was discovered. CREAP or cAMP Regulatory Element Associated Protein, was initially discovered by its ability to bind to the CRE. Its sequence encodes a unique set of modular domains including two zinc fingers, two leucine zippers, two coiled-coils and an RS-rich domain. These domains point to functions in both DNA binding/transcription and RNA splicing, with the leucine zippers being characteristic of bZIP transcription family and the RS domain characteristic of the SR Protein family of splicing factors, to represent a new protein family. In this thesis, molecular reagents were produced for the study of CREAP together with a polyclonal antibody. This antibody was used in western blotting to detect a 58 kDa full-length CREAP protein and a shorter 25-30 kDa truncated splice variant. CREAP was localised to the nucleus and to intranuclear splicing speckles, with co-localisation and co-immunoprecipitation with the splicing factor SC35, strongly suggesting a role in splicing. To test the transcriptional activity of CREAP, specifically if it regulates CRH expression, luciferase reporter studies were conducted. However, CREAP showed negligible effect on CRH or CRE promoter activities suggesting that it is not involved in CRH regulation. CREAP did however react with a large number of transcription factors in an in vitro assay, mostly from the bZIP and zinc finger families. siRNA mediated knockout of CREAP was conducted and the effect on genome-wide expression analysed using a microarray. CREAP knockdown caused an over-representation of genes from the protein transport, metabolism, signal transduction and transcription factor processes. Overall, CREAP appears to be a multifunctional protein that is ubiquitously expressed, and is involved in both splicing and transcriptional processes.

Research Doctorate - Doctor of Philosophy (PhD) / Pre-term birth is still the leading cause of perinatal mortality and morbidity. CRH is a hormone that is involved in the timing of labour, therefore investigation of its regulation is of importance in understanding human parturition. The CRE is a central regulatory element on the CRH promoter and in investigating proteins that bind to this element a novel protein was discovered. CREAP or cAMP Regulatory Element Associated Protein, was initially discovered by its ability to bind to the CRE. Its sequence encodes a unique set of modular domains including two zinc fingers, two leucine zippers, two coiled-coils and an RS-rich domain. These domains point to functions in both DNA binding/transcription and RNA splicing, with the leucine zippers being characteristic of bZIP transcription family and the RS domain characteristic of the SR Protein family of splicing factors, to represent a new protein family. In this thesis, molecular reagents were produced for the study of CREAP together with a polyclonal antibody. This antibody was used in western blotting to detect a 58 kDa full-length CREAP protein and a shorter 25-30 kDa truncated splice variant. CREAP was localised to the nucleus and to intranuclear splicing speckles, with co-localisation and co-immunoprecipitation with the splicing factor SC35, strongly suggesting a role in splicing. To test the transcriptional activity of CREAP, specifically if it regulates CRH expression, luciferase reporter studies were conducted. However, CREAP showed negligible effect on CRH or CRE promoter activities suggesting that it is not involved in CRH regulation. CREAP did however react with a large number of transcription factors in an in vitro assay, mostly from the bZIP and zinc finger families. siRNA mediated knockout of CREAP was conducted and the effect on genome-wide expression analysed using a microarray. CREAP knockdown caused an over-representation of genes from the protein transport, metabolism, signal transduction and transcription factor processes. Overall, CREAP appears to be a multifunctional protein that is ubiquitously expressed, and is involved in both splicing and transcriptional processes.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis supervisor: C. Martin Stoltzfus. Includes bibliographic references (p. 121-143).

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Genetics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

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.