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Box C/D small nucleolar RNAs: Biogenesis, structure and utilization for in vivo ribozyme studies
Eukaryotic cells contain scores of small nucleolar RNAs (snoRNAs), which are required for maturation of pre-rRNA. Two large snoRNA families exist defined by vital box C/D and box H/ACA motifs. The goal of the present study was to gain new insights into the structure and biogenesis of the box C/D snoRNAs; the knowledge developed from this effort was then recruited for practical applications. The investigation was conducted with the phylogenetically conserved U14 and U3 box C/D snoRNAs, from the yeast Saccharomyces cerevisiae. The specific aims included: (1) identification of cis-elements sufficient for biogenesis of the U14 snoRNA; (2) development of a functional map for the U3 snoRNA, and; (3) development of a U3-based model ribozyme system for in vivo studies. Conclusions derived from the U14 biogenesis studies are: (1) production of U14 involves ordered folding of the precursor RNA, and this step is required for formation of the vital box C/D structure motif, and; (2) the active box C/D motif, which is now predicted to consist solely of the box C and D elements, is necessary and sufficient for both accumulation and targeting RNA to the nucleolus. A general model for box C/D snoRNA biogenesis is proposed. Functional mapping of U3 revealed that: (1) boxes C$\sp\prime$ and D and flanking helices are critical for U3 accumulation; (2) boxes B and C are not essential for U3 production, but are important for function, due most likely to binding of a trans-acting factor(s); (3) the 5$\sp\prime$ portion of U3 is required for function, but not stability, and; (4) the non-conserved hairpins, which account for 50% of the molecule, are not required for accumulation or function. Based on the knowledge obtained with U14 and U3, a model ribozyme system featuring chimeric U3:ribozyme RNAs, or "snorbozymes", was developed and tested in vivo. Remarkably, the cleavage efficiency by a hammerhead ribozyme, both in cis- and in trans-configurations, appears quantitative! Other advantages of the system are: (1) a final product is stable, and; (2) authentic in vivo cleavage can be easily distinguished from artifactual cleavages. Snorbozymes are predicted to be useful for targeting natural transcripts in any eukaryotes, for fundamental research or practical applications.
Molecular biology|Cellular biology|Genetics
Samarsky, Dmitry A, "Box C/D small nucleolar RNAs: Biogenesis, structure and utilization for in vivo ribozyme studies" (1998). Doctoral Dissertations Available from Proquest. AAI9841920.