[Truncated abstract] Regulation of mRNA stability is an important posttranscriptional mechanism involved in the control of gene expression. The rate of mRNA decay can differ greatly from one mRNA to another and may be regulated by RNA-protein interactions. A key determinant of mRNA decay are sequence instability (cis) elements often located in the 3' untranslated region (UTR) of many mRNAs. For example, the AU rich elements (AREs), are such well characterized elements, and most commonly involved in promoting mRNA degradation, and specific binding of proteins to these elements leading to the stabilization of some mRNAs. Other cis-elements have been described for mRNA in which mRNA stability is a critical component of gene regulation. This includes the androgen receptor (AR) UC-rich cis element in its 3'UTR. The AR is a key target for therapeutics in human prostate cancer and thus understanding the mechanism involved in regulating its expression is an important goal. The [alpha]CP1 protein, a KH-domain containing RNA-binding protein has been found to bind this UC-rich region of the AR and is thought to play an important role in regulating AR mRNA expression. [alpha]CP1 protein is a triple KH (hnRNP K homology) domain protein with specificity for Crich tracts of RNA and ssDNA (single stranded DNA). Relatively little is known about the structural interaction of [alpha]CP1 with target RNA cis elements, thus the present study aimed to better understand the nature of interaction between 30 nt 3'UTR UC-rich AR mRNA and [alpha]CP1 protein using various biophysical techniques, in an attempt to determine which [alpha]CP1 domain or combination of domains is involved in RNA-binding. These studies could ultimately provide novel targets for drugs aimed to regulate AR mRNA expression in prostate cancer cells. At the commencement of this study little was known about the structure of the [alpha]CP1- KH domains and their basis for poly (C) binding specificity. ... Additional studies addressed the significance of the four core recognition nucleotides (TCCC) using a series of cytosine to thymine mutants. The findings verified some of the results predicted from structural studies, especially the need for maximum KH binding to a core tetranucleotide recognition sequence. Our mutational studies of the four core bases confirmed the importance of cytosine in positions two and three as no binding was observed, while some binding was observed when the fourth base was mutated. In summary, the work presented in this thesis provides new detailed insight into the molecular interactions between the [alpha]CP1-KH domain and AR mRNA. Furthermore, these studies shed light on the nature of protein/mRNA interactions in general, as well as the specific complex that forms on AR mRNA. These studies have provided new understanding into the mode of [alpha]CP1 binding at a target oligonucleotide binding site and, provide a foundation for future studies to define structure of multiprotein/oligonucleotide complexes involved in AR mRNA gene regulation. Understanding the detailed interaction between the AR mRNA and [alpha]CP1 could provide possible targets for drug development at reducing AR expression in prostate cancer cells by interfering with the interaction of [alpha]CP1 and AR-mRNA.
| Identifer | oai:union.ndltd.org:ADTP/194770 |
| Date | January 2008 |
| Creators | Sidiqi, Mahjooba |
| Publisher | University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia. School of Medicine and Pharmacology, Western Australian Institute for Medical Research |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Mahjooba Sidiqi, http://www.itpo.uwa.edu.au/UWA-Computer-And-Software-Use-Regulations.html |
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