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. 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. ncbi 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
  3. ncbi 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
  4. ncbi 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
  5. ncbi 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
  6. ncbi 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
  7. ncbi 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
  8. ncbi 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
  9. ncbi 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
  10. ncbi 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

Scientific Experts

  • Craig S Pikaard
  • Olga Pontes
  • Andrzej T Wierzbicki
  • Jeremy R Haag
  • Thomas S Ream
  • Jian Kang Zhu
  • Xin Jian He
  • Xianwu Zheng
  • Yasuyuki Onodera
  • Lisa M Smith
  • Hye Ryun Woo
  • Carey Fei Li
  • Thomas J Guilfoyle
  • Angela D Norbeck
  • Ljiljana Pasa-Tolic
  • Gretchen Hagen
  • Shihua Zhu
  • Co Shine Wang
  • Yi Feng Hsu
  • Hailing Jin
  • Hai liang Liu
  • Carrie D Nicora
  • Tetsuo Mikami
  • Jianhua Zhu
  • Wen Xue Li
  • Diane Pikaard
  • Fei Zhang
  • Daisuke Miki
  • Thomas Ream
  • Kei Iida
  • Kosuke Nakagawa
  • Avnish Kapoor
  • Yusuke Ito
  • Nataliya Yelina
  • Alan J Herr
  • Eric J Richards
  • Iain Searle
  • David C Baulcombe
  • Faridoon K Yousafzai
  • Mahmoud El-Shami
  • Thierry Lagrange
  • Simon W-L Chan
  • Ian R Henderson
  • Simon W L Chan
  • Yana V Bernatavichute
  • Steven E Jacobsen

Detail Information

Publications16

  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. ncbi 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
    ....
  3. ncbi 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...
  4. ncbi 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...
  5. ncbi 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...
  6. ncbi 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...
  7. ncbi 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...
  8. ncbi 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...
  9. ncbi 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...
  10. ncbi 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...
  11. ncbi 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...
  12. ncbi 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...
  13. 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...
  14. ncbi 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...
  15. 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...