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. 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. Assembly must be efficient to conserve cellular resources and rapidly respond to cells' needs, and accurate to avoid making error-prone ribosomes. The many steps of subunit assembly are made more efficient and more accurate by the activities of more than 200 assembly factors that are present in nascent eukaryotic ribosomes, required for their assembly, and conserved across eukaryotes. To enable an in-depth study of the mechanisms driving ribosome assembly in vivo, we are focusing on one particular stage of assembly of the yeast large ribosomal subunit: just prior to, during, and immediately after the exit of large ribosomal subunit precursors from the nucleolus into the nucleoplasm. We want to understand the multiple remodeling steps enabling assembly of functional centers of the large subunit during these transitions. These functional centers are the peptidyltransferase center (PTC), where peptide bonds are formed, the GTPase activating center (GAC), where translation factor GTPases bind to ribosomes and enable protein synthesis, and the polypeptide exit tunnel (PET), through which all nascent polypeptides travel to emerge from ribosomes. In particular, we want to understand the roles of several assembly factor enzymes in these steps, namely the RNA helicases Drs1 and Has1 and the GTPase Nog1. Our working hypothesis is that Drs1 and Has1 use ATP binding and hydrolysis to trigger remodeling events required for construction of functional centers, removal of the ITS2 spacer RNA, and exit of pre-ribosomes from the nucleolus. We think that Nog1 uses GTP binding and hydrolysis to enable assembly of the GAC and PTC, and inserts its C- terminal tail into the PET to enable or inspect assembly of this tunnel. Experiments are described to test these hypotheses. Our experimental approaches to address these questions include using structural biology (cryo- EM), molecular and classical genetics, biochemistry, proteomics, and genomics.

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Reddy, Michael K
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Carnegie-Mellon University
Schools of Arts and Sciences
United States
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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
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
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
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

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