Ribosomes are ribonucleoprotein particles that contain 50-80 different proteins and 3-4 RNAs. These complex machines catalyze protein synthesis in almost all cells in nature. The long-term goal of this project is to understand how eukaryotic ribosomes are assembled in vivo. We use the yeast Saccharomyces cerevisae, to facilitate molecular genetic, biochemical, and proteomic approaches. Production of ribosomes is tightly linked to cell growth and proliferation. Consequently dysregulation of ribosome biogenesis is linked to many diseases such as cancer or developmental disorders. Because pathways of ribosome biogenesis are very conserved, our studies in yeast will help understand mechanisms of regulation and dysregulation of ribosome production in humans. In addition to ribosomal proteins and rRNAs, more than 200 assembly factors are present in nascent eukaryotic ribosomes and are required for their assembly. Production of these complex ribonucleoprotein machines requires a dynamic series of remodeling steps in which protein-protein, protein-RNA, and RNA-RNA interactions are established and reconfigured to produce functional ribosomes. Because most assembly factors are predicted to play a structural rather than enzymatic role, we need to understand in more detail how the networks of interactions among them and with r proteins and pre-rRNAs are established and reorganized to drive assembly. Potential conformational switches of RNA-RNA interactions that are likely to play an important role to regulate the timing and fidelity of assembly also need to be explored in more detail. We focus on twelve assembly factors and nearby ribosomal proteins that exhibit physical and functional interactions with each other and with pre-rRNA in precursors to 60S ribosomal subunits. We want to understand how this interaction network enables formation of stable precursor particles able to undergo processing of 27S precursors to 5.8S and 25S ribosomal RNAs. We will use genetic selections and site-directed mutagenesis to generate mutations in these proteins or pre-rRNAs, designed to disrupt interactions, including potential conformational switches in pre-rRNAs. We will then employ a battery of assays, and adapt new ones, to investigate effects of these mutations on pre-rRNA folding and processing, and assembly of ribosomal proteins into preribosomes.

Public Health Relevance

This proposal describes research to study how ribosomal RNA, ribosomal proteins, and ribosome assembly factors interact with each other to drive assembly of ribosomes in yeast. As the ribonucleoprotein machines that catalyze protein synthesis in all cells, ribosomes are essential for and closely linked to the growth, proliferation, and adaptation of cells. Consequently dysregulation of ribosome synthesis results in cancers, anemias, mental retardation, and a variety of developmental disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM028301-32A1
Application #
8696227
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1980-08-01
Project End
2018-02-28
Budget Start
2014-05-01
Budget End
2015-02-28
Support Year
32
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Carnegie-Mellon University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Biedka, Stephanie; Micic, Jelena; Wilson, Daniel et al. (2018) Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes. J Cell Biol 217:2503-2518
Wu, Shan; Tan, Dan; Woolford Jr, John L et al. (2017) Atomic modeling of the ITS2 ribosome assembly subcomplex from cryo-EM together with mass spectrometry-identified protein-protein crosslinks. Protein Sci 26:103-112
Konikkat, Salini; Woolford Jr, John L (2017) Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast. Biochem J 474:195-214
Biedka, Stephanie; Wu, Shan; LaPeruta, Amber J et al. (2017) Insights into remodeling events during eukaryotic large ribosomal subunit assembly provided by high resolution cryo-EM structures. RNA Biol 14:1306-1313
Ma, Chengying; Wu, Shan; Li, Ningning et al. (2017) Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1. Nat Struct Mol Biol 24:214-220
Liu, Zheng; Gutierrez-Vargas, Cristina; Wei, Jia et al. (2016) Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit. Proc Natl Acad Sci U S A 113:12174-12179
Ramesh, Madhumitha; Woolford Jr, John L (2016) Eukaryote-specific rRNA expansion segments function in ribosome biogenesis. RNA 22:1153-62
Tutuncuoglu, Beril; Jakovljevic, Jelena; Wu, Shan et al. (2016) The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly. RNA 22:1386-99
Talkish, Jason; Biedka, Stephanie; Jakovljevic, Jelena et al. (2016) Disruption of ribosome assembly in yeast blocks cotranscriptional pre-rRNA processing and affects the global hierarchy of ribosome biogenesis. RNA 22:852-66
Kormuth, Karen A; Woolford Jr, John L; Armitage, Bruce A (2016) Homologous PNA Hybridization to Noncanonical DNA G-Quadruplexes. Biochemistry 55:1749-57

Showing the most recent 10 out of 63 publications