Functional Analysis of Scp160p: A Polyribosome-Associated Multiple KH-domain Protein in Yeast

Emory University, Atlanta GA

Investigators

Abstract

One of the fundamental questions in molecular biology concerns the role of RNA-binding proteins in regulating the processing, transport, stability, subcellular localization, and translation of specific messages in eukaryotic cells. The long-term goal of this project is to elucidate the role of Scp160p, a 160kDa RNA-binding protein in Saccharomyces cerevisiae, as a factor contributing to the post-transcriptional regulation of gene expression in yeast. The specific objectives of this three-year project are: (1) to identify and characterize yeast proteins that interact, physically and/or functionally, with Scp160p, (2) to characterize the mRNA components of Scp160p-containing complexes, and to explore the biological consequence of Scp160p interaction with these messages, and (3) to initiate structure/function studies of Scp160p by using deletion mutagenesis to define regions of the protein that are essential for function. All three objectives will be pursued using a combination of genetic and biochemical approaches well-suited to the yeast model system. The results of these studies will be significant in at least five ways. First, in the course of defining yeast proteins that associate with Scp160p, novel factors not previously recognized to play a role in gene expression may be identified. Second, the specific mRNA components of Scp160p complexes to be identified in Aim 2 will represent not only targets of Scp160p function, but also may provide insight into the variety of cellular processes whose disruption leads to the scp160-null phenotype. Third, the structure/function studies described in Aim 3 will address the long-standing question of whether individual KH domains in a multiple KH domain protein function independently or dependently of one another. Fourth, a number of important reagents will be developed in the course of this work, including a panel of truncated alleles of Scp160p, and both wild-type and mutant clones of genes with which Scp160p interacts. Finally, by elucidating the function and interactions of Scp160p in yeast, this project will provide a foundation to enable studies of other RNA-binding proteins in more complex systems, including humans. The long-term goal of this project is to elucidate the role of Scp160p as a factor contributing to the post-transcriptional regulation of gene expression in yeast. Studies will include both genetic and biochemical approaches to (1) identify and characterize yeast proteins that interact, physically and/or functionally, with Scp160p, (2) characterize the mRNA components of Scp160p-containing complexes, and explore the biological consequence of Scp160p interaction with these messages, and (3) initiate structure/ function studies of Scp160p by using deletion mutagenesis to define regions of the protein that are essential for function. Because Scp160p is a member of a large family of RNA-binding proteins found in both eukaryotes and prokaryotes, the results of these studies are not only relevant to Scp160p in yeast; they may also provide a foundation for future studies of other RNA-binding proteins in more complex systems, including humans.