Review
The RNAs of RNA-directed DNA methylation

https://doi.org/10.1016/j.bbagrm.2016.08.004Get rights and content

Highlights

  • RNA-directed DNA methylation in plants involves siRNAs and long noncoding RNAs.

  • Double-stranded RNA precursors of 24 nt siRNAs have recently been identified.

  • Properties of long noncoding RNAs made by RNA Polymerase V remain enigmatic.

  • Pol V transcripts have 5′ triphosphate groups and lack poly-A tails.

  • Facts, missing information and controversies are reviewed.

Abstract

RNA-directed chromatin modification that includes cytosine methylation silences transposable elements in both plants and mammals, contributing to genome defense and stability. In Arabidopsis thaliana, most RNA-directed DNA methylation (RdDM) is guided by small RNAs derived from double-stranded precursors synthesized at cytosine-methylated loci by nuclear multisubunit RNA Polymerase IV (Pol IV), in close partnership with the RNA-dependent RNA polymerase, RDR2. These small RNAs help keep transposons transcriptionally inactive. However, if transposons escape silencing, and are transcribed by multisubunit RNA polymerase II (Pol II), their mRNAs can be recognized and degraded, generating small RNAs that can also guide initial DNA methylation, thereby enabling subsequent Pol IV-RDR2 recruitment. In both pathways, the small RNAs find their target sites by interacting with longer noncoding RNAs synthesized by multisubunit RNA Polymerase V (Pol V). Despite a decade of progress, numerous questions remain concerning the initiation, synthesis, processing, size and features of the RNAs that drive RdDM. Here, we review recent insights, questions and controversies concerning RNAs produced by Pols IV and V, and their functions in RdDM. We also provide new data concerning Pol V transcript 5′ and 3′ ends. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Section snippets

Overview of RNA-directed DNA methylation (RdDM) and multisubunit RNA Polymerases IV and V

RNA-directed chromatin modification is used throughout eukaryotes as a means to prevent the transcription and movement of transposable elements, thereby protecting the genome from mutation and instability. In eukaryotes that do not methylate their DNA, such as fission yeast, fruit flies or nematodes, small noncoding RNAs (RNAs that do not encode proteins) can guide histone modifications that help establish chromatin states refractive to transcription by RNA polymerases I, II or III. These same

Pol IV transcript function: precursors for siRNAs

The requirement of Pol IV for the accumulation of 24 nt siRNAs in vivo was one of the first phenotypes identified in the initial characterization of this enzyme [35], [73]. However, the Pol IV-dependent precursors that give rise to siRNAs have only been described recently, based on their accumulation when DICER-LIKE 3 (DCL3), the endonuclease primarily responsible for 24 nt siRNA processing, is mutated [8], [60], [98], [99], [101]. Mutations in DCL2 and DCL4 further increase the abundance of

How do Pols IV and V know where to transcribe?

Given the importance of Pols IV and V in RNA-directed DNA methylation and gene silencing, a critical question is: how do these polymerases target specific loci? DNA sequences associated with Pol IV and Pol V have been identified genome-wide by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) [56], [92], [109]. No consensus sequences that are highly correlated with Pol IV or Pol V-associated regions have been identified. This suggests that Pol IV and Pol V recruitment may not

P4R2 and Pol V transcript characteristics

Because Pols IV and V are evolutionarily derivatives of Pol II, might their transcripts undergo processing like Pol II derived mRNAs? Major processing steps for Pol II transcripts include the addition of a 7-methylguanosine cap on the 5′ end, a 3′ poly-A tail, and splicing of intronic sequences. All of these modifications are mediated by protein-protein interactions between Pol II and processing enzymes. Of particular importance is the C-terminal domain (CTD) of the Pol II largest subunit,

Outstanding questions for Pol IV and Pol V dependent RNAs

As noted above, Pol IV and RDR2 are both capable of transcription independently of one another in vitro, yet both are equally required for the accumulation of P4R2 RNAs in vivo. Thus, a major question remaining for P4R2 RNAs is the extent to which Pol IV and RDR2 each contribute to the population of precursor RNAs present in vivo. The presence of a 5′ monophosphate on P4R2 RNAs strongly suggests there are additional steps in P4R2 RNA processing that are yet to be discovered. Indeed, a recent

RNA extraction and RT-PCR

Total RNA was extracted from 2–2.5 week old above-ground tissue of Col-0 WT or nrpe111 [77] using Trizol and RT-PCR was conducted as described in [93]. Primers for IGN22 (RT: 5′ CGGGTCCTTGGACTCCTGAT 3′; PCR: 5′ TCGTGACCGGAATAATTAAATGG 3′), IGN23 (RT: 5′ GCCATTAGTTTTAGATGGACTGCAA 3′; PCR: 5′ GGGCGAACCTGGAGAAAGTT 3′), IGN25 (RT: 5′ CTTCTTATCGTGTTACATTGAGAACTCTTTCC 3′; PCR: 5′ ATTCGTGTGGGCTTGGCCTCTT 3′), and IGN26 (RT: 5′ CGTGACATTAGAAGCTCTACGAGAA 3′; PCR: 5′ TTCCTGGCCGTTGATTGGT 3′) are from [83].

Acknowledgements

Research in the Pikaard lab is supported by National Institutes of Health grant GM077590 (to C.S.P.) and support to C.S.P. as an Investigator of the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation. JMW received support from the NIH departmental training Grant, T32GM007757, and the National Institute of General Medical Sciences of the NIH under Award Number F31GM116346. The content of this work is solely the responsibility of the authors and does not necessarily

References (111)

  • K. Kollen et al.

    The zinc-finger protein SPT4 interacts with SPT5L/KTF1 and modulates transcriptional silencing in Arabidopsis

    FEBS Lett.

    (2015)
  • J.A. Law et al.

    A protein complex required for polymerase V transcripts and RNA- directed DNA methylation in Arabidopsis

    Curr. Biol.

    (2010)
  • J. Li et al.

    Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis

    Curr. Biol.

    (2005)
  • Z.J. Lorkovic et al.

    Involvement of a GHKL ATPase in RNA-directed DNA methylation in Arabidopsis thaliana

    Curr. Biol.

    (2012)
  • F.W. Martinez-Rucobo et al.

    Molecular basis of transcription-coupled pre-mRNA capping

    Mol. Cell

    (2015)
  • P. Mourrain et al.

    Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance

    Cell

    (2000)
  • C. Notredame et al.

    T-coffee: a novel method for fast and accurate multiple sequence alignment

    J. Mol. Biol.

    (2000)
  • Y. Onodera et al.

    Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation

    Cell

    (2005)
  • E. Pearson et al.

    The evolutionarily conserved Pol II flap loop contributes to proper transcription termination on short yeast genes

    Cell Rep.

    (2014)
  • O. Pontes et al.

    The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center

    Cell

    (2006)
  • D. Pontier et al.

    NERD, a plant-specific GW protein, defines an additional RNAi-dependent chromatin-based pathway in Arabidopsis

    Mol. Cell

    (2012)
  • T.S. Ream et al.

    Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II

    Mol. Cell

    (2009)
  • M.H. Suh et al.

    A dual interface determines the recognition of RNA polymerase II by RNA capping enzyme

    J. Biol. Chem.

    (2010)
  • W. Wei et al.

    A role for small RNAs in DNA double-strand break repair

    Cell

    (2012)
  • A.T. Wierzbicki et al.

    Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes

    Cell

    (2008)
  • R. Ye et al.

    A dicer-independent route for biogenesis of siRNAs that direct DNA methylation in Arabidopsis

    Mol. Cell

    (2016)
  • A. Zemach et al.

    The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin

    Cell

    (2013)
  • W. Aufsatz et al.

    HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA

    EMBO J.

    (2002)
  • I. Ausin et al.

    INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

    Proc. Natl. Acad. Sci. U. S. A.

    (2012)
  • I. Ausin et al.

    IDN1 and IDN2 are required for de novo DNA methylation in Arabidopsis thaliana

    Nat. Struct. Mol. Biol.

    (2009)
  • B. Batts-Young et al.

    Triphosphate residues at the 5′ ends of rRNA precursor and 5S RNA from Dictyostelium discoideum

    Proc. Natl. Acad. Sci. U. S. A.

    (1978)
  • D.L. Bentley

    Coupling mRNA processing with transcription in time and space

    Nat. Rev. Genet.

    (2014)
  • N. Bies-Etheve et al.

    RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family

    EMBO Rep.

    (2009)
  • T. Blevins et al.

    Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

    Elife

    (2015)
  • G. Bohmdorfer et al.

    RNA-directed DNA methylation requires stepwise binding of silencing factors to long non-coding RNA

    Plant J.

    (2014)
  • X. Cao et al.

    Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes

    Proc. Natl. Acad. Sci. U. S. A.

    (2002)
  • C. Chandrasekhara et al.

    Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis

    Genes Dev.

    (2016)
  • P. Cramer et al.

    Structure of eukaryotic RNA polymerases

    Annu. Rev. Biophys.

    (2008)
  • K.M. Creasey et al.

    miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis

    Nature

    (2014)
  • A. Deleris et al.

    Involvement of a Jumonji-C domain-containing histone demethylase in DRM2-mediated maintenance of DNA methylation

    EMBO Rep.

    (2010)
  • C.G. Duan et al.

    Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation

    EMBO J.

    (2015)
  • K.W. Earley et al.

    Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation

    Genes Dev.

    (2010)
  • M.L. Ebbs et al.

    H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases

    Mol. Cell. Biol.

    (2005)
  • M.L. Ebbs et al.

    Locus-specific control of DNA methylation by the Arabidopsis SUVH5 histone methyltransferase

    Plant Cell

    (2006)
  • M. El-Shami et al.

    Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components

    Genes Dev.

    (2007)
  • E. Elvira-Matelot et al.

    Arabidopsis RNASE THREE LIKE2 modulates the expression of protein-coding genes via 24-nucleotide small interfering RNA-directed DNA methylation

    Plant Cell

    (2016)
  • C. Eun et al.

    AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana

    PLoS One

    (2011)
  • E.J. Finnegan et al.

    Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana

    Nucleic Acids Res.

    (1993)
  • Z. Gao et al.

    An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation

    Nature

    (2010)
  • J.R. Haag et al.

    Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing

    Nat. Rev. Mol. Cell Biol.

    (2011)
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