Date on Master's Thesis/Doctoral Dissertation
Anatomical Sciences and Neurobiology
Cooper, Nigel G. F.
Polyadenylation; CPEBs; Cytoplasmic polyadenylation; Alternative splicing
Retina; Carrier proteins
The current status of our knowledge of synaptic plasticity comes largely from studies of the hippocampus and the context of learning and memory. We remain largely ignorant of plasticity in other neural systems and contexts. The molecular basis of plasticity has recently been given new impetus due to the discovery of a local control mechanism which can regulate protein synthesis at stimulated synapses. This involves the use of cytoplasmic polyadenylation binding proteins (CPEBs) to regulate translation. The studies presented here attempt to show that these molecular components are present in the retina, a part of the central nervous system that has been seen, historically, as not plastic. Methods used. RT-PCR was used to determine the presence of mRNAs in tissue. In situ hybridization and immunofluorescence microscopy were used for localization of mRNAs and proteins respectively. Real-time PCR and Western blots were used for quantifications of mRNA and proteins during postnatal development. A bioinformatics program "CPE detector" and 3' RACE were used to identify potential mRNA targets for CPEB1 in the UTR databases and in the retina respectively. The PAT assay was used to determine the length of poly(A) tails for some potential mRNA targets. Data mining and sequence alignment were used to identify alternatively spliced isoforms of CPEB3. Major results. Our results demonstrated that CPEB1-4 were all present in the retina. The four CPEBs had similar distributions in the inner retina: predominantly in the retinal ganglion cell layer, and to a less extent, in the inner nuclear layer. However, CPEB1 had a laminar pattern in the inner plexiform layer, whereas CPEB3 was diffuse. The presence of CPEB1 was minimal in the outer plexiform layer in contrast to CPEB3. During postnatal development the levels of CPEB1, 3 and 4 were up-regulated; whereas the level of CPEB2 was constant. Potential mRNAs were identified as targets of CPEB1; some mRNA targets demonstrated elongated poly(A) tails at postnatal day7 or day12, consistent with the up-regulation of CPEB1 at these ages. Multiple isoforms, including a novel one, were identified for CPEB3. The alternative splicing of CPEB3 could occur both in the UTRs and in the coding region. Major conclusions/significance. Our data demonstrated that more than one CPEB paralog is present in mouse retina. Potential mRNA targets for CPEB1 were present in the retina and gained elongated poly(A) tail in accordance with the up-regulation of CPEB1 during development. The increases of CPEB1, 3 and 4 during the development indicate a possible role of such CPEBs in synaptogenesis. Continuing up-regulation of CPEB1, 3 and 4 also indicate a role in the adult retina. Alternative splicing in the UTRs of CPEB3 indicates a complex regulation of CPEB3; multiple isoforms of CPEB3 protein indicate the functional complexity of CPEB3. The presence of CPEBs in the retina indicates the existence of a translational control system in the retina. Future studies. Future studies should focus on the identification of mRNA targets for each CPEB. Such potential targets can be validated using in vitro binding assays to confirm their interaction with CPEB proteins. CPEB can be knocked-down or overexpressed in cultured cells. CPEB knockout mice can be generated for further functional studies.
Wang, Xiangping, "The expression and localization of cytoplasmic polyadenylation element binding proteins in the retina." (2008). Electronic Theses and Dissertations. Paper 1510.