Ribosome Export in Eukaryotes

Brown University, Providence RI

Investigators

Abstract

The ribosome is a cellular organelle that is required for protein synthesis and is essential for life in all kingdoms. It is composed of structural RNAs (ribosomal RNAs - rRNAs) complexed with a set of ribosomal proteins. Because of the integral role of the ribosome in basic cellular mechanisms, it is important to understand how this crucial molecular machine functions. Recent work has identified conserved nucleotide elements (CNEs) within rRNAs that are perfectly conserved in all eukaryotic rRNAs but not in those from bacteria. CNEs are likely to carry out important eukaryotic-specific functions for the ribosome, such as nuclear export. Preliminary data support the idea that CNE-1 contains nuclear export information, which may be mediated by the proteins that are bound there. This project has two specific aims. The first aim focuses on characterization of CNEs in 28S rRNA and their role in mediating ribosome export. Nuclear export will be assayed for yeast ribosomes that are mutated in CNE-1; these mutant ribosomes will contain an MS2 coat protein (cp) binding site that will bind a green fluorescent protein (GFP)-cp fusion protein to facilitate visualization of the export process. The eight CNEs in large subunit (LSU) rRNA will be screened for nuclear export information, using GFP for live cell imaging in budding yeast. The data will indicate which CNEs in addition to CNE-1 contain sufficient information for nuclear export. Leptomycin B (LMB) inhibition in yeast will test Crm1/Xpo1 as the transport receptor for nuclear export of the CNE chimera. The second aim of the project focuses on CNE binding proteins, which will be identified by three complementary approaches. One involves streptavidin-bead pull-down of biotinylated CNE after incubation with yeast extracts, with subsequent identification of the bound proteins by gel electrophoresis and mass spectrometry. In a second approach, a yeast three-hybrid screen will identify genes which encode proteins that interact with CNE-1. As a third approach, a high copy suppressor screen of rDNA with mutated CNE-1 will be used to identify plasmids with genes that are high copy suppressors. In any remaining time of the three year project period, experiments will be initiated to explore the function of proteins that bind to CNE-1. The intellectual significance of this project is the elucidation of a nuclear export function for a eukaryotic-specific conserved nucleotide element. The project also has broader significance for science and society. It will advance discovery and understanding while promoting teaching, training and learning, by involvement of undergraduates in the research. It will broaden participation by underrepresented groups, as some of the undergraduates working on this project will be women and others will be minority students from Brown or from minority-serving institutions that participate in the Leadership Alliance which is housed at Brown. The data from these experiments will be published in widely read journals so that it is readily accessible to the scientific community.