RNA Dynamics of Transient Macromolecular Complexes in Cancer Cells

Summary

Principal Investigator: Jack D Keene
Abstract: DESCRIPTION (provided by applicant): In this proposal, we seek to understand RNA dynamics in transient cellular complexes and to use that information to identify candidate drugs that may modulate the pathways to malignancy. While protein components of RNP granules have been studied in mammalian cells, quantitative RNA population dynamics have not been analyzed for lack of suitable technologies. Our laboratory focuses on multi-targeting of populations of mRNAs regulated by RNA-binding proteins (RBPs) and microRNAs. We have demonstrated that sequence specific RBPs coordinately regulate groups of functionally related mRNAs and these RNPs are remodeled during activation of cells with small molecule drugs. For example, the mRNAs associated with several RBPs have been shown to change coordinately following treatment of embryonic carcinoma cells with retinoic acid or leukemia-derived immune T cells with phorbol esters plus mitogens. We hypothesize that dynamic changes in RNA populations within these transient RNP complexes coordinate functionally-related subsets of mRNAs that encode proteins whose synchronized expression is required for oncogenic transformation. Here, we will use a probabilistic approach that quantifies dynamic RNA changes en masse to analyze mRNAs associated with RBPs during progression to malignancy. We will examine the transition from a precancerous state to a cancerous state in primary epithelial cells using methods pioneered in the laboratory of Robert Weinberg with RAS, telomerase and four other transforming proteins quantify dynamic changes in RNAs associated with HuR (early response gene mRNAs), TIAR (stress granule RNAs) and AGO2/RISC (processing "P" body RNAs). HuR shift to the cytoplasm alters its mRNA targeting and has been claimed as a prognostic factor in hereditary breast cancer. We will quantify the levels of mRNAs and microRNAs in these complexes and determine how they change in response to progressive development of a transformed phenotype, as well as after inducing oxidative stress and hypoxia. The RNP-Immunoprecipitation microarray (RIP-Chip) procedure will be used to identify and quantify mRNAs associated with specific RBPs;an ultraviolet light crosslinking procedure with high specificity and efficiency termed PAR-CLIP recently developed in the laboratory of our collaborator, Thomas Tuschl, will be used to identify the precise binding sites of these RBPs and microRNAs. Dynamic changes in sites of microRNA binding to mRNAs will be globally integrated with RBP binding sites. We will construct a quantitative dynamic model of these events and use these data to query the drug-genome Connectivity Map for drugs that affect these processes as demonstrated in our recent publications. These compounds will be used to further investigate the underlying biology of carcinogenesis in this system. Microscopic visualization at each stage of progression will be used to confirm the RNA/RBP localization and phenotypic effects of drug treatments. It is our long-term plan to use this quantitative probabilistic approach of RNA targeting to investigate these and other transient macromolecular RNP complexes involved in posttranscriptional gene expression using animal models of cancer.
Funding Period: 2011-09-01 - 2016-07-31
more information: NIH RePORT

Top Publications

  1. pmc Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation
    Kyle D Mansfield
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27701, USA
    Nucleic Acids Res 40:2734-46. 2012
  2. pmc Mechanisms coordinating ELAV/Hu mRNA regulons
    Laura E Simone
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, United States
    Curr Opin Genet Dev 23:35-43. 2013
  3. ncbi Post-transcriptional RNA regulons affecting cell cycle and proliferation
    Jeff G Blackinton
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Box 3020, Durham, NC 27710, USA
    Semin Cell Dev Biol 34:44-54. 2014

Detail Information

Publications4

  1. pmc Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation
    Kyle D Mansfield
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27701, USA
    Nucleic Acids Res 40:2734-46. 2012
    ....
  2. pmc Mechanisms coordinating ELAV/Hu mRNA regulons
    Laura E Simone
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, United States
    Curr Opin Genet Dev 23:35-43. 2013
    ....
  3. ncbi Post-transcriptional RNA regulons affecting cell cycle and proliferation
    Jeff G Blackinton
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Box 3020, Durham, NC 27710, USA
    Semin Cell Dev Biol 34:44-54. 2014
    ....

Research Grants30

  1. Translational Control by Osmotically Active Solutes
    Maria Hatzoglou; Fiscal Year: 2013
    ..These studies will increase our understanding of stress-induced human diseases and generate biological markers that can be used for drug development. ..
  2. Superfund Metal Mixtures, Biomarkers and Neurodevelopment
    David C Bellinger; Fiscal Year: 2013
    ..Aim 4- To promote rapid dissemination of significant research findings;and Aim 5- Compliance- To ensure compliance with NIH requirements for data and resource-sharing and the human and animal institutional review board requirements ..
  3. Semi-volatile PCBs: Sources, Exposures, Toxicities
    Larry W Robertson; Fiscal Year: 2013
    ..These data and dietary studies in the last Aim will provide a scientific basis for risk assessment and advice for stakeholders with the ultimate goal to protect highly-exposed individuals and populations. ..
  4. Role of 11q23 Chromosome Abnormalities in the Causation of Acute Leukemia
    Carlo M Croce; Fiscal Year: 2013
    ..abstract_text> ..
  5. Interrogating Epigenetic Changes in Cancer Genomes
    Tim H M Huang; Fiscal Year: 2013
    ..abstract_text> ..
  6. UNMC EPPLEY CANCER CENTER SUPPORT GRANT
    Kenneth H Cowan; Fiscal Year: 2013
    ....
  7. Model-based predictions of responses RTK Pathway therapies
    Joe W Gray; Fiscal Year: 2013
    ..abstract_text> ..