Role of RNA polymerase IV in the heterochromatic siRNA pathway

Summary

Principal Investigator: CRAIG STUART PIKAARD
Affiliation: Indiana University
Country: USA
Abstract: In organisms as diverse as humans, flies, nematodes, fission yeast and plants, small interfering RNAs (siRNAs) regulate essential processes that include viral defense, silencing of retrotransposons, inactivation of specific mRNAs and centromere maintenance. The mechanisms by which siRNAs target homologous RNAs for degradation in Dicer and RISC-mediated RNA interference (RNAi) pathways are fairly well understood. By contrast, the mechanisms by which siRNAs direct histone modifications and/or cytosine methylation events that silence and maintain heterochromatic loci are poorly understood. A newly discovered activity required in the heterochromatic siRNA pathway in Arabidopsis thaliana is Nuclear RNA polymerase IV (pol IV). The two largest subunits of pol IV are similar to the catalytic subunits of E. coli DNA-dependent RNA polymerase and to the corresponding subunits of eukaryotic nuclear RNA polymerases I, II, and III. Disruption of pol IV subunits causes heterochromatin to disperse coincident with losses in cytosine methylation and siRNA production at repetitive genes and retrotransposons. The simplest hypothesis is that pol IV generates precursor transcripts for Dicer-generated siRNAs that direct heterochromatin modifications to homologous sequences. Interestingly, pol IV appears to lack conventional RNA polymerase activity using double-stranded DNA as the template. Pol IV largest subunits also have non- canonical amino acids at the presumptive active site, suggesting the hypothesis that pol IV has a novel polymerase activity. The finding that pol IV-dependent siRNA production also requires RNA-dependent RNA polymerase 2 (RDR2) and cytosine methyltransferase activity (DRM or MET1) suggests that double- stranded RNA or methylated DNA could be specialized templates transcribed by pol IV. The aims of the proposed research are: 1) To determine the templates, products and subunits of pol IV; 2) To deduce the step at which pol IV acts in the heterochromatic siRNA pathway and 3) To test the hypothesis that two distinct forms of pol IV act non-redundantly in contributing to global heterochromatin organization. Chromatin modifications are increasingly linked to siRNA involvement and are implicated in numerous cancers as well as Rett, ICF, Prader-Willi, Beckwith-Wiedemann and Fragile X syndromes. Chromosome instability, also linked to siRNA involvement at centromeres, can lead to trisomies or embryo lethality. Moreover RNA interference shows promise as a gene-therapy tool with which to combat disease. Our studies, which make use of Arabidopsis unique genetic resources, may therefore shed light on mechanisms of siRNA production and chromatin modification that have relevance to human health.
Funding Period: 2006-05-01 - 2010-04-30
more information: NIH RePORT

Top Publications

  1. 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
  2. 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
  3. doi 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
  4. 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
  5. 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
  6. doi 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
  7. 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
  8. 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
  9. 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
  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

Scientific Experts

  • C S Pikaard
  • Olga Pontes
  • Jeremy R Haag
  • Andrzej T Wierzbicki
  • Thomas S Ream
  • Todd Blevins
  • Jian Kang Zhu
  • Ek Han Tan
  • Paul Hoffer
  • Carrie D Nicora
  • Angela D Norbeck
  • Ljiljana Pasa-Tolic
  • Xin Jian He
  • Xianwu Zheng
  • Yasuyuki Onodera
  • Hye Ryun Woo
  • Lisa M Smith
  • Carey Fei Li
  • Brandon Lee
  • Doug Rusch
  • Chinmayi Chandrasekhara
  • Haixu Tang
  • Chris Braun
  • Frederic Pontvianne
  • Keithanne Mockaitis
  • Ram Podicheti
  • Satwica Yerneni
  • Ross Cocklin
  • Michelle Marasco
  • Andrew Mroczka
  • Nicholas Wagner
  • Sergey Ivashuta
  • Alexa Vitins
  • Toni Voelker
  • Co Shine Wang
  • Hailing Jin
  • Hai liang Liu
  • Gretchen Hagen
  • Yi Feng Hsu
  • Thomas J Guilfoyle
  • Shihua Zhu
  • Diane Pikaard
  • Fei Zhang
  • Tetsuo Mikami
  • Yusuke Ito
  • Kei Iida
  • Jianhua Zhu
  • Wen Xue Li
  • Kosuke Nakagawa
  • Avnish Kapoor
  • Thomas Ream
  • Daisuke Miki
  • David C Baulcombe
  • Eric J Richards
  • Iain Searle
  • Faridoon K Yousafzai
  • Nataliya Yelina
  • Alan J Herr
  • Yana V Bernatavichute
  • Steven E Jacobsen
  • Ian R Henderson
  • Mahmoud El-Shami
  • Simon W L Chan
  • Thierry Lagrange

Detail Information

Publications24

  1. 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. ..
  2. 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...
  3. doi 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. ..
  4. 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...
  5. 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...
  6. doi 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...
  7. 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...
  8. 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
    ....
  9. 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...
  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. 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...