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

Functional Mapping of the U3 Small Nucleolar RNA from the Yeast Saccharomyces cerevisiae

The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C � and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5 � portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50 % of the molecule, are not required for accumulation or function. The small nucleolar RNAs (snoRNAs) play essential roles in posttranscriptional maturation of rRNAs (reviewed in reference

Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology