Roles of Ribosomal Proteins in Eukaryotic Ribosome Assembly In Vivo

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

The long-term goal is to discover principles of dynamic interactions among proteins and RNAs leading to construction of an essential cellular nanomachine, the ribosome, whose function is intimately connected with regulation of cell growth and proliferation. Assembly of the two subunits of the ribosome, containing 79 different ribosomal proteins and four rRNAs, is an essential, evolutionarily conserved process. The experimental system is the yeast Saccharomyces cerevisiae, in which eukaryotic ribosome biogenesis has been most thoroughly characterized, using genetic, molecular biological, and proteomic tools. Biogenesis begins in the nucleolus, where rRNA is transcribed and folds into specific conformations that enable its site-specific modification, nucleolytic processing, and binding to ribosomal proteins to form productive assembly intermediates. Maturation continues as preribosomal particles transit from the nucleolus through the nucleoplasm to the cytoplasm, to form mature, functional subunits. These processes require more than 180 trans-acting assembly factors. When and how these assembly factors function are beginning to be understood, yet the roles of ribosomal proteins in ribosome biogenesis in vivo remain largely unexplored. This project involves systematic investigation of the function in subunit assembly of each of the 46 ribosomal proteins found in the large (60S) subunit of yeast ribosomes. Strains conditional for synthesis of each of these ribosomal proteins in 60S ribosomal subunits are being constructed to investigate their roles in processing of pre-rRNA, nucleocytoplasmic transit of preribosomes, and particle maturation. The order of assembly of ribosomal proteins into 60S ribosomes, and the ribosomal proteins or assembly factors with which they associate during assembly will be determined. Broader impact of the proposed research will arise from mentoring of six to eight undergraduate and two to three graduate students. Ribosome biogenesis offers outstanding opportunities to learn how to approach systems biology at a molecular level, by integrating training in molecular genetics and proteomics. In addition, the proposed research should provide mutant yeast strains and an extensive dataset to enable the larger community to develop a complete catalogue of roles of ribosomal proteins in ribosome assembly and ribosome function.