In order to form synaptic connections, an individual neuron needs to coordinate the activities of a large number of proteins. The neuron can modulate the activity of these proteins in a variety of ways. For instance, the neuron can directly target proteins for degradation at the synapse. The neuron can also modulate the activity state of proteins at the synapse through chemical modifications (phosphorylation, glycosylation, etc.). A third way a neuron can modulate synapse structure and connectivity is by regulating transcription. My work has focused on the protein RPM-1, an E3 ubiquitin ligase that modulates protein activity at the synapse in a number of ways (through regulating protein degradation, and indirectly regulating phosphorylation state and transcription). RPM-1 is localized at the synapse directly adjacent to the active zone. Mutations in rpm-1 and its orthologues Highwire/Phr1 disrupt synaptic structure and signaling. Genetic epistasis experiments suggest that RPM-1 controls synaptic development by downregulating MAP Kinase signaling. We have shown that RPM-1 regulates transcription through MAP Kinase. Our data, together with data from Drosophila, suggest that RPM-1/Highwire regulates synaptic morphology by regulating transcription. We have exploited a powerful new technology, mRNA-tagging, to identify the transcriptional targets of RPM-1. A profile of the entire C. elegans nervous system revealed 558 enriched transcripts in an rpm-1 mutant dataset. In vivo GFP imaging confirmed the enrichment of one of these targets, tbb-6. Further work is needed to determine the role these targets play in synaptic development.
C. elegans, with its fully defined neural architecture and powerful genetic and genomic strategies, is an excellent model system for functional and developmental studies of the nervous system. We have exploited the mRNA-tagging technique to generate a gene expression profile of the worm nervous system. We have identified a total of 2149 enriched genes using two different amplification methods. We have confirmed significant enrichment for known neural genes (~90%). These data have also revealed ~30 previously uncharacterized genes that are enriched in the C. elegans nervous system and are conserved in humans. This suggests that these uncharacterized proteins may also function in the human nervous system. In addition to profiling all neurons in the worm, we have utilized the mRNA-tagging technique to generate a gene expression profile of a specific neural class (A-class motor neurons). The use of higher resolution profiles may help direct gene discovery in mammalian systems and may also suggest functions for uncharacterized proteins.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07232007-134517 |
| Date | 30 July 2007 |
| Creators | Watson, Joseph Daniel |
| Contributors | Bruce Appel, Kendal Broadie, Randy Blakely, David Miller |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07232007-134517/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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