YEAST RIBOSOME BIOGENESIS

Carnegie-Mellon University, Pittsburgh PA

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Abstract

We want to understand the regulation of ribosomal protein gene expression and the roles of nonribosomal-nucleolar proteins in ribosome assembly in yeast. We use the ribosomal protein gene RPS14B as a model system to study feedback regulation of ribosomal protein gene expression in eukaryotes. rpS14 represses expression of RPS14B by binding to a stem-loop structure in the 5' splice-site of RPS14B pre- mRNA. Repression occurs either by inhibition of splicing and subsequent degradation of the pre-mRNA, or by turnover of the pre-mRNA before it can be spliced, rpS14 also binds to 185 rRNA. Expression of RPS14B also requires sequences in the intron that can basepair with each other, a potential splicing enhancer. Our working model is that expression of RPS14B is coupled with ribosome assembly by competition between rpS14 binding to its pre-mRNA and to rRNA in assembling ribosomes. Binding of rpS14 may stabilize the stem-loop that represses splicing and thus prevent formation of the mutually exclusive splicing enhancer structure. We will test predictions of this model by asking (l) Is splicing blocked or turnover promoted by the 5' stem-loop in the RPS14B intron? (2) Does RPS14B indeed contain a splicing enhancer? (3) Do the repressor and enhancer exist in alternative conformers within the RPS14B intron? (4) Does binding of rpS14 to the intron mediate a conformational switch between the repressor and the enhancer? (5) How does rpS14 bind to and discriminate between RPS14B pre-mRNA and 18S rRNA? What features in the protein and in the RNAs are important for binding? We want to define the steps in pre-rRNA processing and ribosome assembly specifically leading to production of 60S ribosomal subunits. Although many proteins are necessary to produce 60S subunits, their precise functions and the order of these functions are unknown. We are focusing on the DEAD-box protein Drs1p and the RRM-containing protein Nop4p, both nucleolar proteins necessary for synthesis of 60S subunits. Recently we identified two nucleolar proteins that interact with Drs1p - the nuclease Mtr3p and a potential scaffolding protein Nop7p. We will use genetic and biochemical approaches to (1) characterize interactions between Drs1p and Mtr3p or Nop7p, (2) search for proteins that interact with Nop4p and Nop7p and for nucleolar complexes containing them, (3) collaborate with the groups of Patrick Linder and John Aris to systematically catalogue interactions among proteins necessary for 60S subunit biogenesis, and (4) search for RNA ligands of Drs1p and Nop4p.