The poly(A)-binding proteins (PABPs) are a family of eukaryotic RNA-binding proteins with key roles in mRNA translation and stability. The molecular function of PABPs have been largely revealed through study of the prototypical cytoplasmic poly(A)-binding protein, PABP1. Thus, little is known regarding other PABP family members. PABP5 contains four RNA-recognition motifs characteristic of the cytoplasmic PABPs yet is structurally distinct as it lacks a portion of the C-terminus. This region contains a proline-rich section linked to a globular domain that facilitates a number of protein-protein interactions. To date, little information has been presented regarding the expression of PABP5 and there is no data pertaining to the function of this protein, despite being mapped to a region of the X-chromosome associated with human pathological conditions. In this thesis, I present the first data documenting the expression of PABP5 within mouse tissues, and find it to be expressed at the highest levels within the brain, ovary, and testis. The limited data available suggests that gonads may be the only tissue to contain all PABPs therefore I additionally describe the expression of PABP1 and PABP4 to ascertain their cellular distribution within these tissues. This revealed that PABPs have overlapping yet distinct expression patterns in mouse gonads. The distinct structure of PABP5 suggested that its function may vary from PABP1. Characterisation of its activities in translational regulation was therefore investigated. When tethered to a reporter mRNA PABP5 had limited translational stimulatory activity, and in addition could not be isolated via m7G cap chromatography and failed to interact with translation initiation factors including eIF4G and PAIP-1. These factors interact with PABP1 to positively promote translation, implying that PABP5 function in translational regulation differs from other PABPs investigated. Examining why PABP5 failed to display translational stimulatory activity also revealed an interaction with the negative regulator of translation, PAIP-2. In summary, I present the first description of PABP5 cellular localisation, and have gone some way towards elucidating the molecular function of this uncharacterised protein.

For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways. / QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA. / Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes. / Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.

Post-transcriptional regulation of gene expression sits at the core of proteomic complexity; trans-acting factors that regulate RNA localization and translation capacity are thus indispensible. In this thesis, I present studies of the cytoplasmic polyadenylation element binding protein (CPEB), a sequence specific RNA-binding protein important for cell cycle progression and neural synaptic plasticity. I focus on CPEB because the activity of RNA-binding proteins affects the destiny of their mRNA substrates. As presented in Chapter II, CPEB, though mostly cytoplasmic at steady state, shuttles between the nucleus and the cytoplasm. Surprisingly, the RNA recognition motifs are essential for the nuclear localization. CPEB associates with the polyadenylation machinery in both compartments, suggesting it is involved in both nuclear mRNA processing and cytoplasmic translational regulation. Moreover, the nuclear translocalization is critical to relay a tight translation repression on CPE-containing mRNAs. Chapter III focuses on the regulation of CPEB dimerization. CPEB dimerizes through the RNA-binding domains to inhibit its own RNA binding ability in a cell cycle-dependent manner. By dimerizing, CPEB has enhanced binding to protein destruction factors so that robust active degradation occurs in the later cell cycle. The degradation of CPEB is required for translation activation of a subset of mRNAs and cell cycle progression. In addition, dimerization protects cells from being overloaded with excess CPEB. In sum, the localization and dimerization status of CPEB is dynamic and highly regulated; they in turn regulate the activity of CPEB, which results in responsive translation control. These studies provide a strong foundation to decipher CPEB-mediated gene expression.

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