Genetic Analysis of U14 snoRNP Core Protein Function

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9727945 MAXWELL Eukaryotic ribosome biogenesis occurs in the nucleolus where rRNA is synthesized as a large precursor transcript that undergoes processing and subsequent assembly into new ribosomal subunits. The eukaryotic nucleolus contains a large population of small nucleolar RNAs (snoRNAs) which are critical for ribosome biogenesis. Investigations have demonstrated that the snoRNAs are important for pre-rRNA folding, rRNA processing, and rRNA base modification. U14 is one such eukaryotic snoRNA species, essential for pre-rRNA processing and 18S rRNA accumulation in both vertebrates and yeast. Previous investigations have defined the RNA sequences and structures within the U14 snoRNA:pre-mRNA precursor required for U14 excision from its pre-mRNA intron. These essential processing elements include nucleotide boxes C and D, consensus sequences important not only for processing but also for snoRNA transport to the nucleolus and pre-rRNA methylation. A "box C/D core motif" has been defined within this U14 snoRNA element and shown to serve as a binding site for nucleolar proteins that are required for snoRNA synthesis. The widespread occurrence of the box C/D core motif indicates that this snoRNA element as well as its associated nucleolar proteins are critical for the biogenesis and function of the box C/D snoRNA family. While the essential snoRNA elements are well defined, the nucleolar proteins that bind this core motif remain uncharacterized. Therefore, this project will isolate and define these critical nucleolar proteins, characterize their genes, and then genetically examine in yeast the role individual proteins play in snoRNA processing, nucleolar transport, and rRNA methylation. Ribosomes are essential components of all living cells, necessary for the synthesis of proteins crucial for cell structure, growth, and function. Eukaryotic ribosome biogenesis occurs in the nucleolus where numerous RNAs and proteins participate in a coordinated series of events necessary for ribos ome assembly. U14 snoRNA is an essential snoRNA species required for pre-rRNA processing and the accumulation of 18S rRNA. Strikingly, U14 was the first of what is now a rapidly-growing class of snoRNA species that are encoded within interruptions in the protein coding regions of genes (introns) and synthesized via an intron-processing pathway. Analysis of mouse U14 snoRNA biosynthesis has demonstrated that an RNA structural motif is essential for processing. Additional experiments have shown that nuclear proteins bind this core motif and are also crucial for snoRNA synthesis and function. These experiments will define these important but as yet uncharacterized proteins. These results will provide new and fundamental insight into the role these proteins play in snoRNA biosynthesis as well as snoRNP function during ribosome biogenesis.