Review Article | Published:

RNA-directed DNA methylation: an epigenetic pathway of increasing complexity

Nature Reviews Genetics volume 15, pages 394408 (2014) | Download Citation

  • A Corrigendum to this article was published on 18 July 2014

This article has been updated


RNA-directed DNA methylation (RdDM) is the major small RNA-mediated epigenetic pathway in plants. RdDM requires a specialized transcriptional machinery that comprises two plant-specific RNA polymerases — Pol IV and Pol V — and a growing number of accessory proteins, the functions of which in the RdDM mechanism are only partially understood. Recent work has revealed variations in the canonical RdDM pathway and identified factors that recruit Pol IV and Pol V to specific target sequences. RdDM, which transcriptionally represses a subset of transposons and genes, is implicated in pathogen defence, stress responses and reproduction, as well as in interallelic and intercellular communication.

Key points

  • RNA-directed DNA methylation (RdDM) is prevalent in flowering plants and induces transcriptional silencing at repetitive DNA, including all types of transposons.

  • During RdDM, RNA polymerase IV (Pol IV) initiates production of 24-nucleotide small interfering RNAs (siRNAs) that direct transcriptionally repressive DNA methylation to homologous Pol V-transcribed loci. Pol IV and Pol V are recruited to genomic regions that contain transcriptionally repressive epigenetic marks, thereby reinforcing and maintaining the silent state.

  • Recent research has uncovered variations on the canonical RdDM pathway, including the involvement of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and NEEDED FOR RDR2-INDEPENDENT DNA METHYLATION (NERD), which might allow 'young' (that is, recently acquired) transposons to come under the control of RdDM.

  • In additional to transposon control, RdDM might help hosts to respond to biotic or abiotic challenges, or to faithfully transmit DNA methylation patterns to their offspring. RdDM might also affect germ cell specification and parent-specific gene expression.

  • There is increasing evidence that siRNAs are used to communicate epigenetic states between homologous sequences within a nucleus or indeed between nuclei.

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Change history

  • 18 July 2014

    In this article, on page 394 and in the footnote of Table 1 (page 397) the sequence contexts of DNA methylation were incorrectly defined. For CHG and CHH sequences, H represents A, C or T (not A, T or G as originally written). The article has been corrected online. The authors and editors apologize for these errors.


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M.A.M. thanks Academia Sinica for financial support and C. Ying for editorial assistance. R.A.M. is supported by the US National Science Foundation under grant MCB-1243608. The authors apologize to colleagues whose publications are not cited owing to space limitations.

Author information


  1. Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan.

    • Marjori A. Matzke
  2. School of Plant Sciences, University of Arizona, Tucson, Arizona 85721–0036, USA.

    • Rebecca A. Mosher


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Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Marjori A. Matzke or Rebecca A. Mosher.



(DCR). A ribonuclease III enzyme that cleaves double-stranded RNA precursors into small RNAs of 20–30 nucleotides. In plants, homologues of Dicer are referred to as DICER-LIKE (DCL). Of the four DCL enzymes in Arabidopsis thaliana, DCL3 produces 24-nucleotide small interfering RNAs (siRNAs) that act in the canonical RNA-directed DNA methylation pathway.


(AGO). A family of effector proteins of RNA interference that bind to small interfering RNAs (siRNAs) through their PAZ (PIWI–AGO–ZWILLE) and MID (middle) domains and, in some cases, slice RNA through their PIWI domain. Of the ten AGOs in Arabidopsis thaliana, AGO4, AGO6 and AGO9 act in canonical RNA-directed DNA methylation and/or transcriptional gene silencing.


Invasive genetic elements that move within a genome and that are sometimes associated with replicative movement which produces many copies. Transposons include retrotransposons, DNA transposons and helitrons.

de novo methylation

Methylation of a previously unmodified DNA sequence. Small interfering RNAs (siRNAs) in the RNA-directed DNA methylation pathway are well-known triggers of sequence-specific de novo methylation of cytosines in all sequence contexts.

Silencing effector complex

A multiprotein complex that elicits RNA interference and related small RNA-mediated gene silencing pathways. It is composed of an Argonaute protein (which binds to the small RNA guide) and, in the case of RNA-directed DNA methylation, cofactors that aid in directing DNA methylation to the small RNA-targeted region of the genome.


Pertaining to the region surrounding the centromere, which is the chromosomal region where two sister chromatids are joined.

RNA-dependent RNA polymerase

(RDR). A cellular enzyme that copies single-stranded RNAs to produce double-stranded RNA precursors, which are processed by Dicer-like proteins to generate small interfering RNAs (siRNAs). Of the six RDRs in Arabidopsis thaliana, RDR2 is associated with the canonical RdDM pathway.

Structural maintenance of chromosomes

(SMC). A large family of ATPases that can manipulate chromosome-sized molecules and that contribute to higher-order chromatin structure and dynamics.

Symmetrical methylation

Cytosine methylation at CG:GC and CHG:GHC nucleotide groups in both DNA strands. As a result of complementary base pairing, CG and CHG are base-paired to GC and GHC, respectively, on the opposite DNA strand and hence considered symmetrical.

Maintenance methylation

The preservation of pre-existing methylation at symmetrical CG and CHG sites after DNA replication by the DNA methyltransferases MET1 and CMT3, which recognize hemimethylated substrates (that is, those methylated on one strand but not the other).


(miRNAs). Small non-coding RNAs (~21–23 nucleotides) that silence gene expression by mRNA degradation or translational repression through complementarity with the target transcripts.

Trans-acting siRNAs

(tasiRNAs). A class of small interfering RNAs (siRNAs) that silences gene expression in land plants by targeting complementary mRNAs for cleavage. Their biogenesis depends on microRNA (miRNA)-mediated cleavage of longer TAS RNA precursors that are further acted on by RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DICER-LIKE 4 (DCL4). The miRNA-triggered initiation followed by DCL4 cleavage results in a phased pattern of accumulation, in which small RNAs are in an exact head-to-tail arrangement. tasiRNAs are one category of 'phased' siRNA (phasiRNA).


The process of mobilizing a retrotransposon. It involves transcription, processing of the RNA, translation, reverse transcription of the transposon RNA and integration of the reverse-transcribed DNA into a new genomic location.


Alleles that differ in transcriptional level from other genetically identical alleles, frequently owing to DNA methylation. Some epialleles are faithfully transmitted to the progeny.

Lamarckian inheritance

The hypothesis that an organism can pass on traits acquired during its lifetime to its progeny.


The multicellular structure formed through mitosis from a single haploid spore. Male and female gametophytes contain sperm and egg cells, respectively.

Diplosporous apomixis

A process of reproduction whereby failure of meiosis produces an unreduced female gametophyte. An embryo then develops from the diploid egg cell and forms a clone of the maternal plant.


Pertaining to an evolutionary change in the timing of a developmental process so that a character or process occurs earlier or later in ontogeny, or grows at a different rate.

Genomic imprinting

A phenomenon whereby differential epigenetic marks on maternally and paternally derived alleles result in uniparental gene expression.


A process whereby a transcriptionally silent allele confers meiotically heritable silencing on an active sister allele.


Crossing of two different plant varieties to combine valuable traits from each variety.

Vegetative cell

A haploid cell in the male gametophyte (that is, the pollen grain) that assists fertilization but that does not directly contribute to the zygote.


A tissue in the seed that supports the growth of the embryo. Endosperm is produced after fertilization of the diploid (2N) central cell by a haploid (1N) sperm cell, which creates a maternal:paternal genome ratio of 2:1.


Regions of undifferentiated cells at the shoot or root apex that is responsible for cell division and organogenesis. All aerial tissues, including the germ line, arise from the shoot meristem, and all root tissues arise from the root meristem.

Additive gene expression

Gene expression in a hybrid that is the average of the expression levels in the two parental lines.

Interspecific hybrids

Crosses between two closely related but distinct species.

Hybrid vigour

Increase in fitness associated with crosses between distinct inbred strains.

Introgression lines

Lines into which defined DNA segments have been introduced from a different line through backcrossing.

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