The essential RNT1 (ribonuclease three) gene encodes a double strand-specific endoribonuclease from the yeast Saccharomyces cerevisiae. The RNT1 protein appears similar to bacterial RNase III in structure and function and is required for pre-ribosomal RNA processing events in yeast. Eukaryotic pre-rRNA processing is also dependent on small nucleolar RNAs, and at least one processing event is dependent on both yeast RNase III and U3 snoRNA. In bacteria, RNase III is involved in a wide variety of RNA processing events; thus the identification of the enzyme in yeast offers an unusual opportunity to explore the role of this fundamental enzyme in eukaryotic gene expression and RNA biogenesis, where it may influence mRNA stability or the replication of RNA viruses. An overarching hypothesis to be tested is that yeast RNase III is required for the synthesis or degradation of several unstable nonribosomal RNAs necessary for cell growth. In addition, the mechanism of snoRNA facilitation of RNase III activity deserves investigation.
Three aims are proposed: 1) To characterize the structure, function and substrate specificity of the yeast RNase III enzyme using reverse genetics and biochemistry; 2) to identify and characterize substrates of RNase III whose processing is key to cell growth; and 3) To develop systems to study the mechanism by which snoRNAs function in concert with RNase III in pre-rRNA processing and ribosome assembly. The first specific aim will be addressed by studying the function of RNT1 protein and its mutants to determine which parts of the protein are required for substrate binding, cleavage, metal binding and multimerization. In the second specific aim genetic screens for suppressors and enhancers (synthetic lethal mutations) for two available temperature sensitive mutations in the RNT1 gene will be used to identify key substrates whose cleavage by RNase III is essential for cell growth. The third specific aim will require an effort to reproduce the observed in vivo requirement for both RNase III and the U3 snRNA in the A0 processing event in vitro. Together, the proposed experiments should provide an initial characterization of a conserved and fundamental eukaryotic gene product about which little is known.
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