Monitoring mechanisms in mammalian ribosome biogenesis

Summary

Principal Investigator: Dimitri G Pestov
Abstract: DESCRIPTION (provided by applicant): Ribosomes are biological nanomachines that carry out synthesis of the entire cellular proteome. Cells require a large number of ribosomes to make proteins, especially during periods of active growth and proliferation. Each ribosome in eukaryotes is manufactured through an elaborate assembly pathway that requires more than 200 accessory protein factors. Like any other complex assembly process, biosynthesis of ribosomes generates a certain fraction of defective products and kinetically trapped intermediates. How do cells distinguish between ribosomes that are built correctly and those that are not? The main objective of the proposed research is to answer this question by elucidating the mechanisms underlying quality control of ribosome synthesis in mammalian cells. We use mouse cells in our studies because surveillance mechanisms in mammals differ in many aspects from those in other model organisms such as yeast. One of such differences is that defects in ribosome formation in mammals induce a p53-mediated nucleolar stress response, which is mechanistically not completely understood. Because the framework of preribosomes, like the ribosome itself, is made of RNA, ribonucleases play a key role in dismantling defective ribosome precursors. Here, we wish to establish the pathway through which exoribonucleases start the process of elimination of the defective preribosomes. Our project has three specific aims. 1. Determine the role of the mammalian exosome in the degradation of misassembled pre-60S subunits. We will determine whether the exosome functions in primary surveillance of misassembled pre-60S subunits or acts as a scavenger and how these activities may be regulated through candidate adaptors. 2. Identify structural features of the pre-60S subunit that control whether it will be processed or degraded. Our model is that certain components of preribosomes act as gatekeepers that control nuclease access to pre-rRNA. This will be tested by dissecting the interactions between the exonuclease Xrn2 and the 5.8S RNA-ribosomal protein complex in pre-60S subunits. 3. Determine if pre-rRNA decay products play a role in the nucleolar stress response induced by mutations in ribosome assembly factors and by anticancer drugs that block ribosome maturation, both of which significantly increase pre-rRNA breakdown by nucleases. Together, these studies will test the hypothesis that pre-rRNA surveillance by mammalian exoribonucleases serves the dual function of enabling accurate synthesis of ribosomes under normal circumstances, and initiating stress signaling when the system becomes overloaded.
Funding Period: 2005-02-01 - 2015-06-30
more information: NIH RePORT

Top Publications

  1. pmc Restricting conformational flexibility of the switch II region creates a dominant-inhibitory phenotype in Obg GTPase Nog1
    Yevgeniya R Lapik
    Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60607, USA
    Mol Cell Biol 27:7735-44. 2007
  2. pmc Rapid cytoplasmic turnover of yeast ribosomes in response to rapamycin inhibition of TOR
    Dimitri G Pestov
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey, USA
    Mol Cell Biol 32:2135-44. 2012
  3. pmc The ubiquitin ligase Rsp5 is required for ribosome stability in Saccharomyces cerevisiae
    Natalia Shcherbik
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA
    RNA 17:1422-8. 2011
  4. pmc 5'-end surveillance by Xrn2 acts as a shared mechanism for mammalian pre-rRNA maturation and decay
    Minshi Wang
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, NJ 08084, USA
    Nucleic Acids Res 39:1811-22. 2011
  5. pmc Mammalian DEAD box protein Ddx51 acts in 3' end maturation of 28S rRNA by promoting the release of U8 snoRNA
    Leena Srivastava
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, 2 Medical Center Drive, Stratford, NJ 08084, USA
    Mol Cell Biol 30:2947-56. 2010
  6. pmc Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells
    Natalia Shcherbik
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, 2 Medical Center Drive, Stratford, New Jersey 08084, USA
    EMBO Rep 11:106-11. 2010
  7. pmc The 5' external transcribed spacer in mouse ribosomal RNA contains two cleavage sites
    Tatyana Kent
    Department of Cell Biology, School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA
    RNA 15:14-20. 2009
  8. ncbi Assays for ribosomal RNA processing and ribosome assembly
    Dimitri G Pestov
    University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Department of Cell Biology, Stratford, New Jersey, USA
    Curr Protoc Cell Biol . 2008
  9. pmc Separation of long RNA by agarose-formaldehyde gel electrophoresis
    Farrah H Mansour
    Department of Cell Biology, Rowan University, School of Osteopathic Medicine, Stratford, NJ 08084, USA
    Anal Biochem 441:18-20. 2013

Detail Information

Publications10

  1. pmc Restricting conformational flexibility of the switch II region creates a dominant-inhibitory phenotype in Obg GTPase Nog1
    Yevgeniya R Lapik
    Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60607, USA
    Mol Cell Biol 27:7735-44. 2007
    ....
  2. pmc Rapid cytoplasmic turnover of yeast ribosomes in response to rapamycin inhibition of TOR
    Dimitri G Pestov
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey, USA
    Mol Cell Biol 32:2135-44. 2012
    ..We propose that ribosome content is regulated dynamically in eukaryotes by TOR through both ribosome synthesis and the cytoplasmic turnover of mature ribosomes...
  3. pmc The ubiquitin ligase Rsp5 is required for ribosome stability in Saccharomyces cerevisiae
    Natalia Shcherbik
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA
    RNA 17:1422-8. 2011
    ..Our results suggest that functional Rsp5p is required to maintain the integrity of cytoplasmic ribosomes under rich nutrient conditions...
  4. pmc 5'-end surveillance by Xrn2 acts as a shared mechanism for mammalian pre-rRNA maturation and decay
    Minshi Wang
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, NJ 08084, USA
    Nucleic Acids Res 39:1811-22. 2011
    ..We propose that probing of pre-rRNA maturation intermediates by exonucleases serves the dual function of generating mature rRNAs and suppressing suboptimal processing paths during ribosome assembly...
  5. pmc Mammalian DEAD box protein Ddx51 acts in 3' end maturation of 28S rRNA by promoting the release of U8 snoRNA
    Leena Srivastava
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, 2 Medical Center Drive, Stratford, NJ 08084, USA
    Mol Cell Biol 30:2947-56. 2010
    ..These data demonstrate the emergence of a novel factor that facilitates a pre-rRNA processing event specific for higher eukaryotes...
  6. pmc Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells
    Natalia Shcherbik
    Department of Cell Biology, University of Medicine and Dentistry of New Jersey, 2 Medical Center Drive, Stratford, New Jersey 08084, USA
    EMBO Rep 11:106-11. 2010
    ..The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD...
  7. pmc The 5' external transcribed spacer in mouse ribosomal RNA contains two cleavage sites
    Tatyana Kent
    Department of Cell Biology, School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA
    RNA 15:14-20. 2009
    ..We discuss the finding of a second processing site in mammalian 5'ETS in relation to the involvement of the U3 snoRNA in pre-rRNA processing and present a revised map of the mouse 18S rRNA processing pathway...
  8. ncbi Assays for ribosomal RNA processing and ribosome assembly
    Dimitri G Pestov
    University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Department of Cell Biology, Stratford, New Jersey, USA
    Curr Protoc Cell Biol . 2008
    ..Separation of preribosomal particles by sucrose gradient centrifugation is suitable for the analysis of proteins associated with preribosomes during their assembly and maturation in the cell nucleus...
  9. pmc Separation of long RNA by agarose-formaldehyde gel electrophoresis
    Farrah H Mansour
    Department of Cell Biology, Rowan University, School of Osteopathic Medicine, Stratford, NJ 08084, USA
    Anal Biochem 441:18-20. 2013
    ..The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting...

Research Grants31

  1. RNA quality control and environmental stress
    Sandra L Wolin; Fiscal Year: 2013
    ....
  2. The Architecture and Function of RNPs Required for Ribosome Biogenesis
    Susan J Baserga; Fiscal Year: 2013
    ..A more thorough understanding of these processes will help us design better therapies for malignancies. ..
  3. Visualizing Genetic Activity in Normal &Mutant Yeast
    Ann L Beyer; Fiscal Year: 2013
    ....
  4. The Pathogenesis of Dyskeratosis Congenita
    Philip J Mason; Fiscal Year: 2013
    ....
  5. A New E3 Ligase Implicated in Protein Quality Control and Neurodegeneration
    Claudio A P Joazeiro; Fiscal Year: 2013
    ..abstract_text> ..
  6. TOR, Translation and Aging
    Brian K Kennedy; Fiscal Year: 2013
    ..Through achieving a better understand of the modulatory role played by regulated protein translation in aging, we will be able to pinpoint key targets for pharmacological interventions. ..
  7. A S. cerevisiae high-coverage high-quality protein-protein binary interactome map
    Marc Vidal; Fiscal Year: 2013
    ....
  8. Expanding Excellence in Developmental Biology in Oklahoma
    Linda F Thompson; Fiscal Year: 2013
    ..abstract_text> ..
  9. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  10. New England Regional Center of Excellence in Biodefense and Emerging Infectious D
    Dennis L Kasper; Fiscal Year: 2013
    ..NERCE will also continue its Developmental Projects program and Career Development in Biodefense program in an effort to initiate new research efforts and to attract new investigators to this field. ..
  11. Northeast Biodefense Center
    W Ian Lipkin; Fiscal Year: 2013
    ..As a Center based in a School of Public Health and a State Department of Health, the NBC has a firm commitment to and practical understanding of Emergency Preparedness. ..
  12. Time-resolved hydroxyl radical footprinting of RNA
    Sarah A Woodson; Fiscal Year: 2013
    ....
  13. Assembling an understanding of Ribosome Biogenesis
    Gloria M Culver; Fiscal Year: 2013
    ..coli may result in the identification of novel targets for antibiotic action and may aid in alleviating the problem of bacterial drug resistance as a health concern. ..
  14. Age-Induced Impairment of Nutrient Signaling Results in Bone Loss
    CARLOS MIGUEL ISALES; Fiscal Year: 2013
    ..Ultimately, we expect the Program Project to identify new therapeutic strategies and develop specific countermeasures for age-associated declines in musculoskeletal function. ..
  15. Protein Dynamics in Enzymatic Catalysis
    Robert Callender; Fiscal Year: 2013
    ..The Equipment Core (Core A) supports the specialized comprehensive suite of instrumentation for the Program. The Administrative Core (Core B) administers the Program Project. ..
  16. RNA decay and processing activities of the RNA exosome
    Ambro van Hoof; Fiscal Year: 2013
    ..Our proposed experiments seek to clarify how these helicases assist the exosome in its many function. ..
  17. Structure and function of 5'to 3'exoribonucleases
    Liang Tong; Fiscal Year: 2013
    ..PUBLIC HEALTH RELEVANCE: The proteins being studied in this proposal have not been linked to human diseases based on current information. ..