The 5’ external transcribed spacer (5’ ETS) of pre-ribosomal RNA, although highly variable in size and sequence, has been shown to be critical for the initiation of rRNA processing. This study further examined the 5’ ETS in Schizosaccharomyces pombe with respect to structural elements that underlie rRNA maturation. Initially, the 5’ ETS/18S rRNA junction region was examined by mutational analyses to detect cis-acting elements critical to known cleavage sites. The results indicated that sequence/structure in the junction region does not direct or strongly influence cleavage at the 5’ end of 18S rRNA. Systematic mutations also were used to examine the significance of previously suggested putative ribosomal protein binding sites or U3 snoRNA binding sites as well as other stem-loop sequences of regions IV, V and VI in the 5’ ETS. The results indicated that the putative U3 snoRNA binding sites were less critical than previously anticipated but have identified elements in regions IV and V with significant influence on the production of mature ribosomal RNA. In vitro studies of interactions between these elements and the U3 snoRNA or cellular protein also were initiated. The results of electrophoretic mobility shift assays indicated a strong interaction between region IV and the U3 snoRNA, suggesting that region IV probably contributes to the function of an important structure in the nucleolar precursor particle, which together with region V and probably other hairpins, may act to organize a stable processing domain. In contrast to the previous studies, which suggested as many as six intermediate cleavage sites in the 5’ ETS of S. pombe, re-examination of termini using hybridization and ligation-mediated RT-PCR indicated only two major cleavage sites. In general the 5’ ETS sequence mutants did not seem to influence the rRNA processing profile significantly but could dramatically affect the quantity of the product, an observation that provided further evidence of quality control, which helps ensure that only functional RNA is incorporated into mature ribosomes. Taken together the results illustrated that various sequence/structural elements in the 5’ ETS could influence or be critical for the maturation of rRNA. The results also support the possibility that the precursor molecule is first organized into one or more processing domains that direct the actual maturation processes. / This study was supported by the Natural Sciences and Engineering Research Council of Canada.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/3886 |
| Date | 29 August 2012 |
| Creators | Nellimarla, Srinivas |
| Contributors | Nazar, Ross |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | http://creativecommons.org/licenses/by-sa/2.5/ca/ |
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