An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association.

The U3 small nucleolar ribonucleoprotein (snoRNP) is composed of a small nucleolar RNA (snoRNA) and at least 10 proteins. The U3 snoRNA base pairs with the pre-rRNA to carry out the A0, A1, and A2 processing reactions that lead to the release of the 18S rRNA from the nascent pre-rRNA transcript. The yeast U3 snoRNA can be divided into a short 5' domain (nt 1-39) and a larger 3' domain (73 to the 3' end) separated by a stretch of nucleotides called the hinge region (nt 40-72). The sequences required for pre-rRNA base pairing are found in the 5' domain and hinge region whereas the 3' domain is largely covered with proteins. Mpp10p, one of the protein components unique to the U3 snoRNP, plays a role in processing at the A1 and A2 sites. Because of its critical role in U3 snoRNP function, we determined which sequences in the U3 snoRNA are required for Mpp10p association. Unlike fibrillarin and all the previous U3 snoRNP components studied in this manner, sequences in the 3' domain are not sufficient for Mpp10p association. Instead, a conserved sequence element in the U3 snoRNA hinge region is required, placing Mpp10p near the 5' domain that carries out the pre-rRNA base-pairing interactions in the functional center of the U3 snoRNP.

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.

A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency.

A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo. This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.

A comprehensive database for the small nucleolar RNAs from Saccharomyces cerevisiae.

Small nucleolar RNAs (snoRNAs) are involved in cleavage of rRNA, modification of rRNA nucleotides and, perhaps, other aspects of ribosome biogenesis in eukaryotic cells. Scores of snoRNAs have been discovered in recent years from various eukaryotes, and the total number is predicted to be up to 200 different snoRNA species per individual organism. We have created a comprehensive database for snoRNAs from the yeast Saccharomyces cerevisiae which allows easy access to detailed information about each species known (almost 70 snoRNAs are featured). The database consists of three major parts: (i) a utilities section; (ii) a master table; and (iii) a collection of tables for the individual snoRNAs. The utilities section provides an introduction to the database. The master table lists all known S. cerevisiae snoRNAs and their major properties. Information in the individual tables includes: alternate names, size, family classification, genomic organization, sequences (with major features identified), GenBank accession numbers, occurrence of homologues, gene disruption phenotypes, functional properties and associated RNAs and proteins. All information is accompanied with appropriate literature references. The database is available on the World Wide Web (http://www.bio.umass. edu/biochem/rna-sequence/Yeast_snoRNA_Database/snoRNA_ DataBase.html), and should be useful for a wide range of snoRNA studies.

Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: intron length effects and activity of a precursor snoRNA.

The eukaryotic small nucleolar RNAs (snoRNAs) are involved in processing of pre-rRNA and modification of rRNA nucleotides. Some snoRNAs are derived from mono- or polycistronic transcription units, whereas others are encoded in introns of protein genes. The present study addresses the role of the RNA lariat-debranching enzyme (Dbr1p) in the synthesis and function of intronic snoRNAs in the yeast Saccharomyces cerevisiae. Intronic snoRNA production was determined to depend on Dbr1p. Accumulation of mature intronic snoRNAs is reduced in a dbr1 mutant; instead, intronic snoRNAs are "trapped" within host intron lariats. Interestingly, the extent of intronic snoRNA accumulation in the form of lariats in dbr1 cells varied among different intronic snoRNAs. Intronic snoRNAs encoded within shorter introns, such as U24 and snR38, accumulate more unprocessed lariat precursors than those encoded within longer introns, e.g., U18 and snR39. This correlation was corroborated by experiments conducted with model intron:U24 snoRNA constructs. These results support a splicing-dependent exonucleolytic pathway for the biosynthesis of intronic snoRNAs. Curiously, U24 in a lariat may be functional in directing methylation of ribosomal RNA.

The snoRNA box C/D motif directs nucleolar targeting and also couples snoRNA synthesis and localization.

Most small nucleolar RNAs (snoRNAs) fall into two families, known as the box C/D and box H/ACA snoRNAs. The various box elements are essential for snoRNA production and for snoRNA-directed modification of rRNA nucleotides. In the case of the box C/D snoRNAs, boxes C and D and an adjoining stem form a vital structure, known as the box C/D motif. Here, we examined expression of natural and artificial box C/D snoRNAs in yeast and mammalian cells, to assess the role of the box C/D motif in snoRNA localization. The results demonstrate that the motif is necessary and sufficient for nucleolar targeting, both in yeast and mammals. Moreover, in mammalian cells, RNA is targeted to coiled bodies as well. Thus, the box C/D motif is the first intranuclear RNA trafficking signal identified for an RNA family. Remarkably, it also couples snoRNA localization with synthesis and, most likely, function. The distribution of snoRNA precursors in mammalian cells suggests that this coupling is provided by a specific protein(s) which binds the box C/D motif during or rapidly after snoRNA transcription. The conserved nature of the box C/D motif indicates that its role in coupling production and localization of snoRNAs is of ancient evolutionary origin.

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.

SnoRNAs as tools for RNA cleavage and modification.

Eukaryotic small nucleolar RNAs (snoRNAs) influence rRNA synthesis at two stages: nucleolytic processing and selection of nucleotides to be ribose methylated (Nm) or converted to pseudouridine (psi). The two modification functions and some processing activities involve direct base pairing of snoRNA with rRNA. In addition to rRNA-targeting sequences, snoRNA function depends on the presence of conserved box elements involved in snoRNA synthesis and localization. The present investigation is directed at using snoRNAs as tools for two purposes: 1) introducing nucleotide modifications into novel sites in rRNA and other snoRNAs, and: 2) targeting nucleolar RNAs for destruction using snoRNA:ribozyme chimers ('snorbozymes'). Early results demonstrate that snoRNAs can be used for both applications.

An essential domain in Saccharomyces cerevisiae U14 snoRNA is absent in vertebrates, but conserved in other yeasts.

U14 is a small nucleolar RNA (snoRNA) required for early cleavages of eukaryotic precursor rRNA. The U14 RNA from Saccharomyces cerevisiae is distinguished from its vertebrate homologues by the presence of a stem-loop domain that is essential for function. This element, known as the Y-domain, is located in the U14 sequence between two universal sequences that base pair with 18S rRNA. Sequence data obtained for the U14 homologues from four additional phylogenetically distinct yeasts showed the Y-domain is not unique to S.cerevisiae. Comparison of the five Y-domain sequences revealed a common stem-loop structure with a conserved loop sequence that includes eight invariant nucleotides. Conservation of these features suggests that the Y-domain is a recognition signal for an essential interaction. Several plant U14 RNAs were found to contain similar structures, though with an unrelated consensus sequence in the loop portion. The U14 gene from the most distantly related yeast, Schizosaccharomyces pombe, was found to be active in S.cerevisiae, showing that Y-domain function is conserved and that U14 function can be provided by variants in which the essential elements are embedded in dissimilar flanking sequences. This last result suggests that U14 function may be determined solely by the essential elements.

Characterization of three new snRNAs from Saccharomyces cerevisiae: snR34, snR35 and snR36.

Genes for three novel snRNAs of Saccharomyces cerevisiae have been isolated, sequenced and tested for essentiality. The RNAs encoded by these genes are designated snR34, snR35 and snR36 respectively and contain 203, 204 and 182 nucleotides. Each RNA is derived from a single copy gene and all three RNAs are believed to be nucleolar, i.e. snoRNAs, based on extraction properties and association with fibrillarin. SnR34 and snR35 contain a trimethylguanosine cap, but this feature is absent from snR36. The novel RNAs lack elements conserved among several other snoRNAs, including box C, box D and long sequence complementarities with rRNA. Genetic disruption analyses showed each of the RNAs to be dispensable and a haploid strain lacking all three RNAs and a previously characterized fourth snoRNA (snR33) is also viable. No differences in the levels of precursors or mature rRNAs were apparent in the four gene knock-out strain. Possible roles for the new RNAs in ribosome biogenesis are discussed.