BIOGENESIS AND TRAFFICKING OF U3 SMALL NUCLEOLAR RNPS

University Of Georgia (Uga), Athens GA

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Abstract

DESCRIPTION (adapted from application): The PI proposes to study all posttranscriptional steps involved in biogenesis of the U3 small nucleolar ribonucleoprotein particle (U3 snoRNP). These steps include binding of proteins to the precursor and mature forms of U3 snoRNA; hypermethylation of the 5' cap; nuclear retention of the RNA, RNP intermediates, and the mature snoRNP; and finally, relocalization of the U3 snoRNP from the nucleoplasm to the nucleolus. The experimental system of choice will be the Xenopus oocyte for all the usual reasons. In particular, microinjected U3 snoRNAs are reconstituted into U3 snoRNPs, and the subcellular localization of the resulting RNPs (cytoplasm, nucleoplasm, nucleoli) are easily determined both by biochemical fractionation and by traditional light microscopy. The Xenopus assay system will be used to determine the protein composition of reconstituted U3 snoRNPs, and the role of various U3 snoRNA sequences in protein binding, snoRNP reconstitution, and nucleolar localization. U3 snoRNP proteins will be identified by analytical assays (crosslinking, affinity selection) but cloned by two hybrid screens (for those that might interact with fibrillarin) and three hybrid screens (for those that might interact directly with U3 snoRNA).