The 80 S cytoplasmic ribosome is the largest of three populations of ribosomes responsible for protein synthesis in plants. It is comprised of two RNA/protein subunits of unequal size: the small (40 S) subunit selects messages to be translated and performs proofreading, while the large (60 S) subunit has peptidyl transferase acitivity, adding new amino acids to the growing polypeptide. In the model flowering plant <i>Arabidopsis thaliana</i> (hereafter <i>Arabidopsis</i>), four ribosomal RNAs and 81 ribosomal proteins (r-proteins) assemble to form the 80S ribosome. Although the <i>Arabidopsis</i> ribosome contains only a single copy of each of the 81 r-proteins (with the exception of small number of acidic phophoproteins), all r-proteins are encoded from multi-gene families containing two or more expressed members. Herein, I investigated r-protein paralogy in Arabidopsis via specific examination of a two member gene family, RPL23a. By analyzing patterns of reporter gene expression driven by full-length and truncated regulatory regions, I was able to identify a core promoter that is largely conserved between paralogs. Regulation was found to be complex, involving transcriptional, post-transcriptional and translational components. The effects of knocking-out a single RPL23a paralog (RPL23aB) were determined. Results indicated that this paralog is broadly dispensable, and Arabidopsis does not compensate for its loss at the transcriptional level. Subcellular localization was investigated by tagging RPL23aA/B with fluorescent proteins, demonstrating that RPL23aA is targeted to nucleolus more efficiently than RPL23aB, possibly due to a stronger nucleolar localization signal. RNA-interference was used to individually silence RPL23a paralogs to characterize functional overlap. Results showed that RPL23aA, and not RPL23aB, is required for normal development. Mutants with reduced levels of RPL23aA develop a pointed first leaf phenotype that I postulate may be due to disruption of miRNA-mediated degradation of specific auxin response genes. Lastly, the 26 S proteasome was inhibited to determine the importance of protein turnover in regulating RPL23a levels. Findings suggest that proteasome-mediated degradation of RPL23a is essential for preventing accumulation of unincorporated r-proteins. Overall, results indicate that the Arabidopsis RPL23a paralogs have diverged from each other: RPL23aA has become the predominant paralog, while RPL23aB functions in an anciliary capacity and/or is undergoing neofunctionalization.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-07142008-061230 |
| Date | 15 July 2008 |
| Creators | Degenhardt, Rory Frank |
| Contributors | Bonham-Smith, Peta C., Berleth, Thomas, Fowke, Larry C., Gray, Gordon R., Wilson, Ken E. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-07142008-061230/ |
| 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 University of Saskatchewan 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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