U14 and U3 are phylogenetically conserved small nucleolar RNAs (snoRNAs). Both RNAs are required for 18S ribosomal RNA synthesis in the yeast Saccharomyces cerevisiae and both have been reported to be associated with rRNA precursors. Loss of either snoRNA disrupts nucleolytic processing of precursor rRNA, suggesting that each has a role in this process. This study addresses three issues related to U14 and U3 biochemistry. The objectives include: (1) development of detailed functional maps of the conserved and essential box C and box D sequence elements in U14; (2) postulation of a secondary folding model for yeast U14 RNA; and, (3) identification of the RNA polymerase responsible for U14 gene expression in S. cerevisiae. The sequence requirements for box C and box D function in U14 have been determined by site-directed mutagenesis. Functional effects were evaluated in a test strain dependent on galactose for expression of wild-type U14; activity of mutant U14 RNAs was assessed in glucose medium. The results show that the first GA bases of the box C sequence UGAUGA and the final GA bases of the box D sequence GUCUGA are essential. Mutations at these positions abolish or severely reduce U14 accumulation. Similar effects were observed for box C mutants transcribed from the heterologous GAL1 promoter. This latter result suggests that box C is not required for expression, but influences turnover. Mutagenesis of the first GA doublet of box C in U3A (CGAUGA) showed that only a G $\to$ C mutation is lethal. The occurrence of another candidate box C element in U3A is discussed. A hypothetical secondary structure model is proposed for U14 based on biochemical and genetic data. In the model the functionally important domains Y2 (specific to yeast), box C element and 18S-A and 18S-B (both complementary to 18S rRNA) are exposed in single-stranded regions. The essential elements box D and Y1 participate in double-stranded regions. Expression of U14 and other small nuclear RNAs has been examined in yeast strains in which RNA polymerase II or III is conditionally defective. The relative abundances of U14, snR190 and snR10 remain surprisingly constant for several generation-equivalents, following a shift to the non-permissive conditions. The significance of these results are discussed in the context of polymerase structure and function and snoRNA turnover rates.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8566 |
| Date | 01 January 1993 |
| Creators | Huang, Guyang Matthew |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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