FUNCTIONS OF SMALL NUCLEOLAR RNAS

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Ribosome biogenesis is a fundamental process in cell biology but little is known about its mechanism. E1, E2 and E3 small nucleolar RNAs (snoRNAs) do not belong to the best known class of snoRNAs since they lack the sequences that are present in those snoRNAs. These three snoRNAs interact directly with unique segments of ribosomal RNA precursor (pre-rRNA) in vivo. Degradation of each of these three snoRNAs produces a unique frog oocyte pre-rRNA processing phenotype that is reversed by the corresponding snoRNA. El and E2 RNAs are essential for 18S rRNA formation. Each of these snoRNAs has unique conserved sequences. Based on the available results, we propose that these snoRNAs: a) are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites, E1 at the 5' end and E2 at the 3' end of the 18S rRNA sequence, and E3 at the 5' end of the 5.8S rRNA sequence; and b) have novel, and probably unique, cis-acting domains for rRNA processing that may interact with novel proteins for their functions. Long-term goal: To determine the molecular mechanisms of the functions of the E1, E2, and E3 small nucleolar ribonucleoprotein particles in ribosome formation. Our Specific Aims are 1) to identify the snoRNA cis-acting elements needed for the functions of these snoRNAs in pre-rRNA processing, 2) to identify the interactions of these snoRNA domains with proteins, pre- rRNA and other snoRNAs, that are required for function, 3) to test the models of these functions focused on these snoRNA elements and these interactions, and 4) to develop cell-free pre-rRNA processing systems that are dependent on exogenous El or E2 RNA. This will be done by snoRNA mutation analysis, mobility shift assay, RNA-protein photocrosslinking , antisense 2'-0-methyloligoribonucleotide competition, and snoRNA and pre-rRNA compensatory mutation analysis, in injected frog oocytes and transfected mammalian cells. The E2 RNA gene lies in an intron of the gene for a lamininbinding protein that is important in tumor invasion and metastasis.