Ultrastructural Analysis of RNA Synthesis and Processing

University Of Virginia Main Campus, Charlottesville VA

Investigators

Abstract

0094561 Ann Beyer The long term goal of the project is to contribute to an understanding of the complex process of ribosome biogenesis. Ribosomes are of obvious interest and importance as the protein synthesis machinery of the cell. In an actively growing yeast cell (Saccharomyces cerevisiae), ribosomes are made at a rate of ~2000 per minute in a process that has turned out to be surprisingly complex. Indeed, more than 60 trans-acting components are known to function in yeast ribosome biogenesis, and it has not proven possible to assemble ribosomes from purified components. This research will provide a new perspective on early events in ribosome synthesis, which are important to the successful assembly of the mature ribosome. The project combines two powerful approaches that have not previously been exploited simultaneously. Specifically, these are yeast genetics and the Miller chromatin spreading technique for electron microscope (EM) visualization of active genes. The basic idea is to visualize ribosomal RNA genes in yeast cells that have mutations in the trans-acting factors that function in early steps in ribosomal RNA processing. The three largest rRNAs are synthesized by RNA polymerase I as a single large 35S precursor RNA and are extensively modified (mainly by pseudouridylation and ribose methylation) and separated by nucleolytic cleavages. During the 5-6 minutes required to synthesize this rRNA, the nascent transcripts are accessible for EM visualization in the characteristic tandemly repeated 'Christmas-tree' gene configuration. These rRNA transcripts are packaged into large ribonucleoprotein complexes that occur at nonrandom positions. By asking if and how this RNP structure changes as various components are deleted or mutated, combined with knowledge as to effects of the mutation on rRNA processing, information will be obtained regarding the structural framework of processing, molecular interactions in processing, and the timing and order of these events. In addition, three rRNA processing events can be visualized co-transcriptionally, at low frequency but reproducibly. The three events visualized are thought to represent removal of the extreme 5' end of the transcript (the 5' ETS), separation of the bulk of the transcript from the extreme 3' end (the 3' ETS), and cleavage about midway through the transcript, which presumably corresponds to cleavage in ITS1 separating the 18S rRNA precursor from the rest of the precursor. These predictions will be tested using mutations in known required components, and if correct, effects on these specific processing steps will be tested in a structural setting. Components to be tested include both RNA and protein components of the two major classes of snoRNPs involved in rRNA modification (Box H/ACA and Box C+D), with an emphasis on the snoRNPs required for the early RNA cleavage steps, as well as non-snoRNP proteins involved in these steps. Completion of these experiments will provide important new information on early steps in ribosome biogenesis.