ASSEMBLY, LOCALIZATION AND FUNCTION OF THE U3 SNRNP

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The long term objective of our research is to understand the molecular events that occur in pre-ribosomal RNA (rRNA) processing in eukaryotes, focusing particularly of the role of the U3 small nuclear ribonucleoprotein (snRNP) in the initial pre-rRNA cleavage event. The U3 snRNP, one of the abundant snRNPs, has been shown to be essential for the first cleavage event in pre-rRNA processing in multiple organisms, from humans to yeast. In vertebrates, the U3 snRNP consists of an RNA about 200 nucleotides in length bound to several proteins. Although the U3 snRNP has been studied in a wide variety of species and cell types, both in vivo and in vitro, the molecular mechanism underlying its role in pre-rRNA processing remains largely unknown. In particular, it is not clear which of the protein components are critical for its activity or how they contribute to U3 snRNP biogenesis. Fibrillarin, one of the U3 proteins common to other nucleolar snRNPs, is a frequent target of the autoimmune response in patients with systemic sclerosis. Fibrillarin is the only U3 protein that has been previously studied in detail. The role(s) of the remaining five or more proteins in snRNP biogenesis and pre-rRNA processing remain to be elucidated. Five specific aims are proposed to pursue the nature of the assembly, biogenesis and function of the U3 snRNP: 1. Purify the U3 snRNP and its individual protein components from a mammalian source. 2. Clone the genes encoding the U3 proteins using the polymerase chain reaction. 3. Express the U3 proteins in E.coli and use the fusion proteins to derive specific antibodies. 4. Use the clones and antibodies to: a. Study the organization of the U3 proteins on the snRNA, b.Study the cellular biogenesis of the U3 snRNP, including proteins and RNA sequences necessary for nucleolar localization 5. Investigate the U3 snRNP components essential for pre-rRNA processing using an in vitro processing system.