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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077590-02
Application #
7220652
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2006-05-01
Project End
2010-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
2
Fiscal Year
2007
Total Cost
$255,843
Indirect Cost
Name
Washington University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Wendte, Jered M; Pikaard, Craig S (2017) The RNAs of RNA-directed DNA methylation. Biochim Biophys Acta Gene Regul Mech 1860:140-148
Blevins, Todd; Wang, Jing; Pflieger, David et al. (2017) Hybrid incompatibility caused by an epiallele. Proc Natl Acad Sci U S A 114:3702-3707
Wang, Jing; Blevins, Todd; Podicheti, Ram et al. (2017) Mutation of Arabidopsis SMC4 identifies condensin as a corepressor of pericentromeric transposons and conditionally expressed genes. Genes Dev 31:1601-1614
Marasco, Michelle; Li, Weiyi; Lynch, Michael et al. (2017) Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination. Nucleic Acids Res 45:11315-11326
Wendte, Jered M; Haag, Jeremy R; Singh, Jasleen et al. (2017) Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation. Cell Rep 19:2796-2808
Wendte, Jered M; Pikaard, Craig S (2016) Targeting Argonaute to chromatin. Genes Dev 30:2649-2650
Blevins, Todd; Podicheti, Ram; Mishra, Vibhor et al. (2015) Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. Elife 4:e09591
Ream, Thomas S; Haag, Jeremy R; Pontvianne, Frederic et al. (2015) Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit. Nucleic Acids Res 43:4163-78
Blevins, Todd; Pontvianne, Frédéric; Cocklin, Ross et al. (2014) A two-step process for epigenetic inheritance in Arabidopsis. Mol Cell 54:30-42
Pikaard, Craig S; Mittelsten Scheid, Ortrun (2014) Epigenetic regulation in plants. Cold Spring Harb Perspect Biol 6:a019315

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