Roles of RNA Polymerases IV and V in siRNA-mediated gene silencing

Summary

Principal Investigator: CRAIG STUART PIKAARD
Abstract: DESCRIPTION (provided by applicant): All eukaryotes use three essential DNA-dependent RNA polymerases to decode the genetic information in chromosomal DNA, namely RNA Polymerases I, II and III. Remarkably, plants have evolved two additional RNA polymerases, abbreviated as Pol IV and Pol V. These novel RNA polymerases play non- redundant roles in RNA-directed DNA methylation, silencing of transposable elements, large-scale heterochromatin organization, long-distance spreading of silencing signals, and proper temporal and morphological development. Affinity purification and mass spec analyses revealed that Pol IV and Pol V are specialized forms of RNA Polymerase II, with half of their twelve subunits encoded by the same genes. Three subunits differ between Pol IV and Pol V and presumably account for their unique functions. Pol IV and Pol V are best understood with respect to their roles in the Arabidopsis siRNA-directed DNA methylation pathway. Pol IV acts early in the pathway, generating transcripts that are required for the biogenesis of 24 nt short interfering RNAs (siRNAs) that are loaded into ARGONAUTE 4 (AGO4). Independent of siRNA biogenesis, Pol V generates noncoding transcripts at target loci. siRNA-AGO4 complexes bind to these Pol V transcripts, facilitating recruitment of AGO4 to the adjacent chromatin. In subsequent steps that are not understood, the de novo DNA methyltransferase, DRM2 and histone modifying activities are recruited to target loci, generating heterochromatin that is refractive to transcription by conventional polymerases such as Pol II and Pol III. Major questions in need of answers include: what are the templates used by Pol IV and Pol V?;how are Pol IV and Pol V recruited to these templates?;are Pol IV and Pol V transcription units specified by conventional promoters or by chromatin structures?;how are Pol IV and Pol V coordinated with other proteins of the gene silencing machinery;and how do the unique subunits of Pol II, Pol IV and Pol V confer the unique functions of these novel polymerases? Using genetics and genomics as well as cell biological and biochemical approaches, our specific aims are designed to find answers to these questions. In diverse eukaryotes, including humans, flies, worms and fission yeast, siRNAs and noncoding RNAs essential processes through chromatin modifications. Examples include transposon silencing, centromere maintenance, X-chromosome inactivation and imprinting of maternal or paternal alleles. DNA methylation and chromatin modifications are also implicated in Rett, ICF, Prader-Willi, Beckwith-Wiedemann and Fragile X syndromes, as well as numerous forms of cancer. By understanding how noncoding RNAs and siRNAs specify sites of DNA methylation and gene silencing, our study will contribute to the long-term goal of understanding these processes with respect to human disease. PUBLIC HEALTH RELEVANCE: Large non-coding RNAs and small RNAs are critical for X-chromosome inactivation and dosage control, imprinting of genes expressed from only one parent, defense against retrotransposons and viruses, heterochromatin formation at centromeres and genome stability. Alterations in DNA methylation and heterochromatin are involved in multiple human diseases and genetic disorders, including cancer, and targeted gene silencing using small RNA interference (RNAi) technologies are being used in medical and agricultural biotechnology applications. Our studies of RNA polymerases IV and V are significant with respect to all of these areas, especially the roles of intergenic noncoding RNAs and siRNAs in chromatin-mediated gene silencing.
Funding Period: 2006-05-01 - 2014-11-30
more information: NIH RePORT

Top Publications

  1. ncbi The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center
    Olga Pontes
    Biology Department, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA
    Cell 126:79-92. 2006
  2. pmc An effector of RNA-directed DNA methylation in arabidopsis is an ARGONAUTE 4- and RNA-binding protein
    Xin Jian He
    Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, 92521, USA
    Cell 137:498-508. 2009
  3. pmc RNA polymerase V functions in Arabidopsis interphase heterochromatin organization independently of the 24-nt siRNA-directed DNA methylation pathway
    Olga Pontes
    Biology Department, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA
    Mol Plant 2:700-10. 2009
  4. pmc Posttranscriptional gene silencing in nuclei
    Paul Hoffer
    Monsanto, Calgene Campus, Davis, CA 95616, USA
    Proc Natl Acad Sci U S A 108:409-14. 2011
  5. ncbi Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing
    Jeremy R Haag
    Department of Biology, Indiana University, Bloomington, Indiana 47405, USA
    Nat Rev Mol Cell Biol 12:483-92. 2011
  6. pmc Functional consequences of subunit diversity in RNA polymerases II and V
    Ek Han Tan
    Department of Biology, Indiana University, Bloomington, IN 47405, USA
    Cell Rep 1:208-14. 2012
  7. pmc In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing
    Jeremy R Haag
    Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
    Mol Cell 48:811-8. 2012
  8. ncbi A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation
    C S Pikaard
    Department of Biology, Indiana University, Bloomington, Indiana 47405, USA
    Cold Spring Harb Symp Quant Biol 77:205-12. 2012
  9. pmc Intersection of small RNA pathways in Arabidopsis thaliana sub-nuclear domains
    Olga Pontes
    Department of Biology, University of New Mexico, Albuquerque, New Mexico, United States of America
    PLoS ONE 8:e65652. 2013
  10. pmc RNA polymerase V transcription guides ARGONAUTE4 to chromatin
    Andrzej T Wierzbicki
    Biology Department, Washington University, St Louis, Missouri, USA
    Nat Genet 41:630-4. 2009

Research Grants

  1. Coupling of Transcription with Pre-mRNA Metabolism
    DAVID LEONARD BENTLEY; Fiscal Year: 2013

Detail Information

Publications24

  1. ncbi The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center
    Olga Pontes
    Biology Department, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA
    Cell 126:79-92. 2006
    ..We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers...
  2. pmc An effector of RNA-directed DNA methylation in arabidopsis is an ARGONAUTE 4- and RNA-binding protein
    Xin Jian He
    Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, 92521, USA
    Cell 137:498-508. 2009
    ..The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA-silencing effector complexes...
  3. pmc RNA polymerase V functions in Arabidopsis interphase heterochromatin organization independently of the 24-nt siRNA-directed DNA methylation pathway
    Olga Pontes
    Biology Department, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA
    Mol Plant 2:700-10. 2009
    ....
  4. pmc Posttranscriptional gene silencing in nuclei
    Paul Hoffer
    Monsanto, Calgene Campus, Davis, CA 95616, USA
    Proc Natl Acad Sci U S A 108:409-14. 2011
    ..Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants...
  5. ncbi Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing
    Jeremy R Haag
    Department of Biology, Indiana University, Bloomington, Indiana 47405, USA
    Nat Rev Mol Cell Biol 12:483-92. 2011
    ..However, their subunit compositions reveal that they evolved as specialized forms of RNA polymerase II, which provides the unique opportunity to study the functional diversification of a eukaryotic RNA polymerase family...
  6. pmc Functional consequences of subunit diversity in RNA polymerases II and V
    Ek Han Tan
    Department of Biology, Indiana University, Bloomington, IN 47405, USA
    Cell Rep 1:208-14. 2012
    ..Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V...
  7. pmc In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing
    Jeremy R Haag
    Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
    Mol Cell 48:811-8. 2012
    ..These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis...
  8. ncbi A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation
    C S Pikaard
    Department of Biology, Indiana University, Bloomington, Indiana 47405, USA
    Cold Spring Harb Symp Quant Biol 77:205-12. 2012
    ..These considerations are discussed and incorporated into a "transcription fork" model for Pol IV and Pol V-dependent RNA-directed DNA methylation. ..
  9. pmc Intersection of small RNA pathways in Arabidopsis thaliana sub-nuclear domains
    Olga Pontes
    Department of Biology, University of New Mexico, Albuquerque, New Mexico, United States of America
    PLoS ONE 8:e65652. 2013
    ..The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place...
  10. pmc RNA polymerase V transcription guides ARGONAUTE4 to chromatin
    Andrzej T Wierzbicki
    Biology Department, Washington University, St Louis, Missouri, USA
    Nat Genet 41:630-4. 2009
    ..Collectively, our data suggest that AGO4 is guided to target loci through base-pairing of associated siRNAs with nascent Pol V transcripts...
  11. pmc Metal A and metal B sites of nuclear RNA polymerases Pol IV and Pol V are required for siRNA-dependent DNA methylation and gene silencing
    Jeremy R Haag
    Department of Biology, Washington University, St Louis, MO, USA
    PLoS ONE 4:e4110. 2009
    ..Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active...
  12. ncbi An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana
    Carey Fei Li
    Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA
    Cell 126:93-106. 2006
    ..Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci...
  13. pmc VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization
    Hye Ryun Woo
    Department of Biology, Washington University, St Louis, Missouri 63130, USA
    Genes Dev 21:267-77. 2007
    ..VIM1 associates with methylated genomic loci in vivo and is enriched in chromocenters. Our findings suggest that VIM1 acts at the DNA methylation-histone interface to maintain centromeric heterochromatin...
  14. ncbi Cell biology of the Arabidopsis nuclear siRNA pathway for RNA-directed chromatin modification
    C S Pikaard
    Biology Department, Washington University, St Louis, Missouri 63130, USA
    Cold Spring Harb Symp Quant Biol 71:473-80. 2006
    ..Therefore, assembly and trafficking of chromatin-modifying RISCs may share similarities with other nuclear RNPs...
  15. pmc An SNF2 protein associated with nuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis
    Lisa M Smith
    Sainsbury Laboratory, John Ines Centre, Norwich NR4 7UH, United Kingdom
    Plant Cell 19:1507-21. 2007
    ..We interpret the effect of polymerase IVa and trans-acting siRNA pathway mutations in terms of a modular property of RNA silencing pathways...
  16. pmc siRNA and miRNA processing: new functions for Cajal bodies
    Olga Pontes
    Biology Department, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA
    Curr Opin Genet Dev 18:197-203. 2008
    ..Collectively, these observations suggest interesting variations in the way siRNAs and miRNAs can accomplish their similar functions in plants and animals...
  17. pmc Roles of RNA polymerase IV in gene silencing
    Craig S Pikaard
    Department of Biology, Washington University, 1 Brookings Drive, St Louis, MO 63130, USA
    Trends Plant Sci 13:390-7. 2008
    ..Although biochemical details concerning Pol IV enzymatic activities are lacking, genetic evidence suggests several alternative models for how Pol IV might function...
  18. pmc Sex-biased lethality or transmission of defective transcription machinery in Arabidopsis
    Yasuyuki Onodera
    Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan
    Genetics 180:207-18. 2008
    ..As a result, the expected stringent selection against nonfunctional essential genes in the haploid state occurs in the female lineage but is relaxed in the male lineage...
  19. pmc ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis
    Xianwu Zheng
    Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA
    Nature 455:1259-62. 2008
    ..These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3...
  20. pmc Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes
    Andrzej T Wierzbicki
    Biology Department, Washington University, St Louis, MO 63130, USA
    Cell 135:635-48. 2008
    ..Pol IVb/Pol V function provides a solution to a paradox of epigenetic control: the need for transcription in order to transcriptionally silence the same region...
  21. pmc Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II
    Thomas S Ream
    Biology Department, Washington University, St Louis, MO 63130, USA
    Mol Cell 33:192-203. 2009
    ..Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense...
  22. pmc A two-step process for epigenetic inheritance in Arabidopsis
    Todd Blevins
    Howard Hughes Medical Institute, Indiana University, Bloomington, IN 47405, USA Department of Biology and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
    Mol Cell 54:30-42. 2014
    ..Collectively, these observations indicate that silent locus specification and silencing are separable steps that together account for epigenetic inheritance of the silenced state. ..

Research Grants30

  1. Coupling of Transcription with Pre-mRNA Metabolism
    DAVID LEONARD BENTLEY; Fiscal Year: 2013
    ..This study will help identify defects in the function of the 'mRNA factory" that may play a major role in corrupting splicing and cleavage/polyadenylation in cancer cells. ..