Biogenesis of Eukaryotic Ribosomes

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DESCRIPTION(applicant's abstract): The production of mature ribosomal RNA (rRNA) species from the- rRNA precursor requires an ordered sequence of cleavage events. The main subject of this grant is to understand the roles of small nucleolar RNAs (snoRNAs) in eukaryotic rRNA processing. Most of the proposed experiments focus on U3 snoRNA, which is the most abundant of the snoRNAs and is essential for cleavage at several sites in prerRNA. Our recent dissection of U3 has identified functionally important areas in the molecule. The studies described here will explore interactions of U3 with pre-rRNA. The Xenopus oocyte will be used as an "in vivo test tube" to deduce rRNA processing mechanisms. Yeast will be used for a genetic screen. (1) Analysis of point mutations in the 5' region of U3 snoRNA will be completed; pseudouridine (w) formation at nt 8 and 12 in Xenopvs U3 snoRNA will be confirmed for endogenous U3 and analyzed for injected U3 mutants. (2) Our recent results suggest a set of dynamic base-pair interactions between U3 snoRNA and prerRNA. These will be assessed by compensatory changes in yeast and in Xenopus. (3) Yeast suppressor screens will be carried out to identify intermolecular contacts of U3 Box A' with RNA and protein. (4) Site-directed mutagenesis of Xenopus pre-rRNA will reveal the sequences and structures required for cleavage at sites AO, I and 2. (5) snoRNAs may modulate the conformation of rRNA during its biogenesis. Chemical modification or pre-rRNAs after depletion of U3 snoRNA or after depletion and subsequent injection of mutant U3 snoRNAs will be used to analyze if U3 snoRNA influences the conformation of pre-RNA. (6) Antisense oligonucleotide depletion in Xenopus oocytes will demonstrate if Ui 3 and 7-2IMRP snoRNAs are required for cleavage at sites 2 and 3, respectively. The proposed studies should elucidate the importance of dynamic RNA-RNA interactions during ribosome biogenesis, a fundamental process essential for cell growth and viability.