- Title
- Characterisation of the multifunctional protein, CREAP
- Creator
- Shipman, Kristy
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2008
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- 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.
- Subject
- transcription; splicing; CRH; gene regulation
- Identifier
- http://hdl.handle.net/1959.13/33061
- Identifier
- uon:3120
- Rights
- Copyright 2008 Kristy Shipman
- Language
- eng
- Full Text
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| View Details Download | ATTACHMENT02 | Thesis | 4 MB | Adobe Acrobat PDF | View Details Download |