proposal): In the proposed investigation two small RNAs will be investigated as model systems for furthering our understanding of RNA traffic and localization. These studies are a logical extension of recent work from this laboratory in which the intracellular traffic of various RNAs in living cells was investigated by microinjection of fluorescent RNAs, coupled with parallel localization of the endogenous RNAs by in situ hybridization and immunocytochemistry.
In Aim 1 the intranuclear traffic of the RNA subunit of the ribonucleoprotein enzyme RNase P will be investigated, with emphasis on the nucleolar phase of its biosynthesis, RNP assembly and conceivably function. Mutants of RNase P RNA will be investigated by injection of fluorescent RNA and by expression of marked genes to define nucleolar targeting signals. These studies will be complemented by analysis of the sequences involved in RNP assembly, using recently developed antibodies. The possibility that RNase P functions in pre-ribosomal RNA processing or other nucleolar RNA processing will be investigated in Xenopus oocytes, and if warranted in mammalian cells.
In Aim 2 the applicant's recent discovery that signal recognition particle (SRP) RNA is localized (at least transiently) in the nucleolus will be the basis for a search for sequences required for this localization. The same studies should provide information on sequences required for nuclear export. These two inter-related projects test the hypothesis that the nucleolus is a more pluri-functional organelle than previously thought.
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