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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077590-07
Application #
8204558
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2006-05-01
Project End
2014-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
7
Fiscal Year
2012
Total Cost
$300,988
Indirect Cost
$105,541
Name
Indiana University Bloomington
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
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
Pikaard, Craig S; Mittelsten Scheid, Ortrun (2014) Epigenetic regulation in plants. Cold Spring Harb Perspect Biol 6:a019315
Haag, Jeremy R; Brower-Toland, Brent; Krieger, Elysia K et al. (2014) Functional diversification of maize RNA polymerase IV and V subtypes via alternative catalytic subunits. Cell Rep 9:378-90
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 (2013) Methylating the DNA of the most repressed: special access required. Mol Cell 49:1021-2
Dekker, Job; Wysocka, Joanna; Mattaj, Iain et al. (2013) Nuclear biology: what's been most surprising? Cell 152:1207-8
Pontvianne, Frederic; Blevins, Todd; Chandrasekhara, Chinmayi et al. (2013) Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes Dev 27:1545-50
Pontes, Olga; Vitins, Alexa; Ream, Thomas S et al. (2013) Intersection of small RNA pathways in Arabidopsis thaliana sub-nuclear domains. PLoS One 8:e65652
Pontvianne, Frédéric; Blevins, Todd; Chandrasekhara, Chinmayi et al. (2012) Histone methyltransferases regulating rRNA gene dose and dosage control in Arabidopsis. Genes Dev 26:945-57

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