Molecular Mechanisms of Gene Silencing by RNAi

Summary

Principal Investigator: Richard W Carthew
Abstract: DESCRIPTION (provided by applicant): A conserved biological response to double-stranded RNA (dsRNA) mediates resistance to parasitic nucleic acids, encoded by transposable elements and viruses. Cleavage of dsRNA into small interfering RNAs (siRNAs) triggers repression of genes related by sequence to the siRNAs. This silencing response is related by mechanism to an endogenous regulatory process that represses the expression of protein-coding genes. The latter process is guided by microRNAs (miRNAs), which are produced from genes within the genome. The long-term goals of our research are to understand the molecular mechanisms of siRNA and miRNA action, and to understand what aspects of cell activities regulate silencing by small RNAs. To this end, we have developed genetic, biochemical and cell biological methods in whole Drosophila and in cells to achieve our goals. In previous work, we conducted screens for Drosophila mutations that perturb siRNA- and miRNA-mediated repression. Genes that were identified from the screens are the basis for research aims in this proposal. Our aims are to: 1) determine how the nuclear Cdk9 kinase recruits a protein complex to co-transcriptionally cleave RNA transcripts into miRNAs, 2) determine how miRNA-protein complexes in the cytoplasm specifically repress the translation of certain mRNAs, 3) learn how these miRNA-protein complexes become associated with trafficking organelles in the cytoplasm, thereby leading to the inactivation of the miRNA-protein complexes, and 4) understand how the Dicer ribonuclease cleaves RNA from infecting viruses to generate antiviral siRNAs. The significance to health is manifold. The ability of small RNAs to regulate virus, transposon, and organismal gene expression suggests that basic understanding will reveal control mechanisms that influence many aspects of biology. It will also have an impact on viral infectious diseases and non-infectious diseases such as cancer. Moreover, small RNAs are used experimentally to probe gene function and are being commercially developed as disease therapeutics, making basic understanding of these molecules an important biomedical research goal.
Funding Period: 2003-07-01 - 2015-07-31
more information: NIH RePORT

Top Publications

  1. pmc RNA interference directs innate immunity against viruses in adult Drosophila
    Xiao Hong Wang
    Graduate Program for Microbiology, University of California, Riverside, CA 92521, USA
    Science 312:452-4. 2006
  2. pmc miR-9a minimizes the phenotypic impact of genomic diversity by buffering a transcription factor
    Justin J Cassidy
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    Cell 155:1556-67. 2013
  3. pmc Functionally diverse microRNA effector complexes are regulated by extracellular signaling
    Pei Hsuan Wu
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    Mol Cell 52:113-23. 2013
  4. pmc Functional specialization of the small interfering RNA pathway in response to virus infection
    Joao Trindade Marques
    Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America
    PLoS Pathog 9:e1003579. 2013
  5. pmc The relationship between long-range chromatin occupancy and polymerization of the Drosophila ETS family transcriptional repressor Yan
    Jemma L Webber
    Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637, USA
    Genetics 193:633-49. 2013
  6. pmc Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors
    Kenji S Nakahara
    Plant Breeding Science, Research Faculty of Agriculture, Hokkaido University, Sapporo, 060 8589, Japan
    Proc Natl Acad Sci U S A 109:10113-8. 2012
  7. pmc Cargo sorting to lysosome-related organelles regulates siRNA-mediated gene silencing
    Dinari A Harris
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    J Cell Biol 194:77-87. 2011
  8. pmc Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila
    Joao Trindade Marques
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, USA
    Nat Struct Mol Biol 17:24-30. 2010
  9. pmc The endo-siRNA pathway is essential for robust development of the Drosophila embryo
    Elena M Lucchetta
    Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, United States of America
    PLoS ONE 4:e7576. 2009
  10. pmc Silencing by small RNAs is linked to endosomal trafficking
    Young Sik Lee
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60201, USA
    Nat Cell Biol 11:1150-6. 2009

Detail Information

Publications19

  1. pmc RNA interference directs innate immunity against viruses in adult Drosophila
    Xiao Hong Wang
    Graduate Program for Microbiology, University of California, Riverside, CA 92521, USA
    Science 312:452-4. 2006
    ..These findings establish a Drosophila model for studying the innate immunity against viruses in animals...
  2. pmc miR-9a minimizes the phenotypic impact of genomic diversity by buffering a transcription factor
    Justin J Cassidy
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    Cell 155:1556-67. 2013
    ..These findings reveal that microRNA-target interactions may be a key mechanism by which the impact of genomic diversity on cell behavior is dampened. ..
  3. pmc Functionally diverse microRNA effector complexes are regulated by extracellular signaling
    Pei Hsuan Wu
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    Mol Cell 52:113-23. 2013
    ..Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities...
  4. pmc Functional specialization of the small interfering RNA pathway in response to virus infection
    Joao Trindade Marques
    Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America
    PLoS Pathog 9:e1003579. 2013
    ..We speculate that this unique framework might be necessary for a prompt and efficient antiviral response. ..
  5. pmc The relationship between long-range chromatin occupancy and polymerization of the Drosophila ETS family transcriptional repressor Yan
    Jemma L Webber
    Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637, USA
    Genetics 193:633-49. 2013
    ....
  6. pmc Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors
    Kenji S Nakahara
    Plant Breeding Science, Research Faculty of Agriculture, Hokkaido University, Sapporo, 060 8589, Japan
    Proc Natl Acad Sci U S A 109:10113-8. 2012
    ..These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco...
  7. pmc Cargo sorting to lysosome-related organelles regulates siRNA-mediated gene silencing
    Dinari A Harris
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    J Cell Biol 194:77-87. 2011
    ..Negative regulation is also not linked to sorting into all LROs but only a specific class of pigment LRO. Thus, regulation of siRNA activity is tied to sorting of specific types of cargo to particular LROs...
  8. pmc Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila
    Joao Trindade Marques
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois, USA
    Nat Struct Mol Biol 17:24-30. 2010
    ..Loqs is primarily required for dsRNA processing, whereas R2D2 is essential for the subsequent loading of siRNAs into effector Ago-RISC complexes...
  9. pmc The endo-siRNA pathway is essential for robust development of the Drosophila embryo
    Elena M Lucchetta
    Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, United States of America
    PLoS ONE 4:e7576. 2009
    ..However, we hypothesized that small RNAs may regulate the embryo's response to temperature, as a ribonucleoprotein complex has been previously shown to mediate mammalian cell response to heat shock...
  10. pmc Silencing by small RNAs is linked to endosomal trafficking
    Young Sik Lee
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60201, USA
    Nat Cell Biol 11:1150-6. 2009
    ..We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm...
  11. pmc Origins and Mechanisms of miRNAs and siRNAs
    Richard W Carthew
    Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208 3500, USA
    Cell 136:642-55. 2009
    ..Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment...
  12. pmc BMP signaling goes posttranscriptional in a microRNA sort of way
    Catherine A Reinke
    Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA
    Dev Cell 15:174-5. 2008
    ..expands our understanding of miRNA biogenesis and maturation, elucidating a mechanism by which extracellular signaling directs cell differentiation via posttranscriptional regulation of miRNA expression...
  13. pmc Cell-type-specific transcription of prospero is controlled by combinatorial signaling in the Drosophila eye
    Takashi Hayashi
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA
    Development 135:2787-96. 2008
    ..The activity level, therefore, is dictated by the proper combination of highly cooperative selector and pre-pattern factors present in the cell...
  14. pmc Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila
    Do Hwan Lim
    Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam Dong, Seongbuk gu, Seoul 136 713, South Korea
    Biochem Biophys Res Commun 371:525-30. 2008
    ..We also found that the carboxyl-terminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure-function relationship of Dicer, which plays a critical role in the siRNA pathway...
  15. pmc Adaptive evolution of newly emerged micro-RNA genes in Drosophila
    Jian Lu
    Department of Ecology and Evolution, University of Chicago, USA
    Mol Biol Evol 25:929-38. 2008
    ..melanogaster is indicative of hitchhiking under positive selection. Thus, a large number of adaptive changes over a long period of time may be essential for the evolution of newly emerged miRNA genes...
  16. pmc Lola regulates cell fate by antagonizing Notch induction in the Drosophila eye
    Limin Zheng
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA
    Mech Dev 125:18-29. 2008
    ..The predicted consequence of this mechanism is to sharpen a cell's responsiveness to Notch signaling by creating a threshold...
  17. pmc Pattern formation in the Drosophila eye
    Richard W Carthew
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, United States
    Curr Opin Genet Dev 17:309-13. 2007
    ..This sorting and culling process results in a sculpted pattern with a precise number and position of cells that is repeated hundreds of times in each compound eye...
  18. ncbi A call to arms: coevolution of animal viruses and host innate immune responses
    Joao T Marques
    Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA
    Trends Genet 23:359-64. 2007
    ..We review how RNAi is used as an antiviral strategy and the mechanisms that viruses have evolved to suppress the RNAi response...
  19. ncbi MicroRNAs and their roles in developmental canalization
    Diana M Posadas
    Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA
    Curr Opin Genet Dev 27:1-6. 2014
    ..Thus, genetic variability is held in check, which is potentially important for both animal evolution and manifestation of disease. ..

Research Grants30

  1. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
    ..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..
  2. Mechanism and functions of small RNA pathways in Neurospora
    Yi Liu; Fiscal Year: 2013
    ..Together, these studies address several fundamental questions in small RNA biogenesis and will expand our current knowledge of sRNA biogenesis pathways and sRNA function. ..
  3. Control of C. elegans Lineage by Heterochronic Genes
    GARY B RUVKUN; Fiscal Year: 2013
    ..The genes identified in this study are likely to be key factors in the function of small RNAs in biology and thus their identification may enable more potent RNAi based drug development. ..
  4. Gene silencing as an antiviral defense in animal cells
    Shou Wei Ding; Fiscal Year: 2013
    ..These studies will likely open up a new area of research on the function of viral piRNAs and establish a mammalian system to investigate the biogenesis and antiviral function of virus-derived small RNAs. ..
  5. Upregulation of ZAP expression as a novel approach for anti-alphavirus therapy
    Margaret R Macdonald; Fiscal Year: 2013
    ..Moreover, the conceptual approach could be applied to treat any infectious disease, as well as any disease or condition that would benefit by up regulation of a specific gene or set of genes. ..
  6. 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. ..
  7. 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. ..
  8. 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. ..
  9. Pacific Southwest RCE for Biodefense &Emerging Infectious Diseases Research
    Alan G Barbour; Fiscal Year: 2013
    ..abstract_text> ..
  10. Rocky Mountain Regional Center of Excellence or Biodefense and Emerging Infectiou
    John T Belisle; Fiscal Year: 2013
    ..abstract_text> ..
  11. Molecular Mechanism of the piRNA Biogenesis
    Alexei A Aravin; Fiscal Year: 2013
    ..Thus, our findings will provide the basis for directing the piRNA pathway in order to make these non-coding RNAs useful tools in epigenetic research and in therapy. ..
  12. UNMC EPPLEY CANCER CENTER SUPPORT GRANT
    Kenneth H Cowan; Fiscal Year: 2013
    ....
  13. Regulating fibrosis and muscle growth in the muscular dystrophies
    Elizabeth M McNally; Fiscal Year: 2013
    ..Core B will provide histopathological assessment of muscular dystrophy after genetic manipulation and treatments, and Core C will perform functional analysis in vivo and provide support to Core B. ..
  14. Electron Microscopy of Biological Macromolecules
    Kenneth H Downing; Fiscal Year: 2013
    ....
  15. Protein homeostasis mechanisms underlying enterovirus replication and evolution
    Raul Andino; Fiscal Year: 2013
    ..Core A: Administrative Core;and Core B: "High-throughput functional genomics and proteomics core. ..