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Small RNA-Mediated Epigenetic Modifications in Plants Available online at www.sciencedirect.com Small RNA-mediated epigenetic modifications in plants Stacey A Simon and Blake C Meyers Epigenetic modifications in plants can be directed and of which are involved in post-transcriptional silencing. mediated by small RNAs (sRNAs). This regulation is composed Small interfering RNAs (siRNAs) typically 24 nt in of a highly interactive network of sRNA-directed DNA length are involved in heterochromatin formation and methylation, histone, and chromatin modifications, all of which transcriptional gene silencing by guiding sequence- control transcription. Identification and functional specific DNA and histone methylation through a path- characterization of components of the siRNA-directed DNA way termed RNA-directed DNA methylation (RdDM) methylation pathway have provided insights into epigenetic [1,10,11,12,13]. Targeted RdDM begins with siRNAs pathways that form heterochromatin and into chromatin-based produced by the RNAi pathway. At different steps, this pathways for gene silencing, paramutation, genetic imprinting, pathway utilizes both of the RNA polymerases Pol IV and and epigenetic reprogramming. Next-generation sequencing Pol V. RNA polymerase IV acts upstream of Pol V, technologies have facilitated new discoveries and have helped functioning in a complex with CLASSY1 (CLSY), a create a basic blueprint of the plant epigenome. As the multiple SNF2-like chromatin remodeling factor [14]and layers of epigenetic regulation in plants are dissected, a more RDR2, which copies single-stranded RNA (ssRNA) into comprehensive understanding of the biological importance of double-stranded RNA (dsRNA). The dsRNA molecules epigenetic marks and states has been developed. are cleaved by DCL3 [4,15]into24ntheterochromatic siRNAs that are recruited by an effector complex con- Address taining either AGO4 or AGO6 to help guide chromatin Department of Plant and Soil Sciences & Delaware Biotechnology modifications to homologous DNA sequences [6–10,16]. Institute, University of Delaware, Newark, DE 19711, United States Pol V acts downstream in a complex termed DDR [17] Corresponding author: Meyers, Blake C ([email protected]) composed of DEFECTIVE IN RNA DIRECTED DNA METHYLATION1 (DRD1), another SNF2-like chromatin remodeling factor [18], DEFECTIVE IN Current Opinion in Plant Biology 2011, 14:148–155 MERISTEM SILENCING 3 (DMS3), a structural- This review comes from a themed issue on maintenance-of-chromosomes hinge domain-containing Genome studies and molecular genetics protein [19] and RNA-DIRECTED DNA METHYL- Edited by Jeffrey L. Bennetzen and Jian–Kang Zhu ATION1(RDM1),anovelprotein[17]. Pol V with the DDR complex functions to amplify and reinforce siRNA Available online 13th December 2010 production and to mediate de novo methylation at the 1369-5266/$ – see front matter target sites of siRNAs [1,13]. Pol V, with the above- # 2010 Elsevier Ltd. All rights reserved. mentioned accessory factors, is believed to transcribe genomic sequences that have been targeted to interact DOI 10.1016/j.pbi.2010.11.007 with siRNAs [1]. The AGO4-bound siRNA complex can either interact with a nascent Pol V-derived RNA or the target DNA to facilitate recruitment of effectors of de Introduction novo DNA methylation and histone modifying com- Small RNAs (sRNAs) are now known to be a core com- plexes to the target loci [1,20–22,23]. ponent of a signaling network that mediates epigenetic modifications in plants. Epigenetic regulation can be The Pol IV-mediated production of siRNAs described mediated through a dynamic interplay between sRNAs, above reflects primary RdDM (18 RdDM), and the siR- DNA methylation, and histone modifications, which NAs produced by Pol IV form the most abundant class of together modulate transcriptional silencing of DNA. sRNAs (Figure 1). The siRNAs produced at this stage can Regulatory sRNAs are short (approximately 20–24 nt in be amplified by a turnover mechanism in which Pol IV length), noncoding RNAs produced through the RNA transcribes the methylated DNA template, thereby pro- interference (RNAi) pathway that involves the plant- ducing an aberrant or perhaps atypically processed RNA specific DNA-dependent RNA polymerases Pol IV and that can be copied by RDR2 leading to the production of Pol V [1,2], the RNA-dependent RNA polymerase RDR2 additional 18 siRNAs that can trigger methylation at the [3,4], the double-stranded RNA endonuclease DICER- target region (Figure 2)[11,24,25]. Another important LIKE3 (DCL3) [4,5], and at least two Argonautes, AGO4 aspect of RdDM utilizes 28 siRNAs to trigger the spread- and AGO6 [6–9]. ing of methylation into areas adjacent and beyond the 18 siRNA-targeted sites [19,23]. It is possible that some of sRNAs 21 nt in length are typically microRNAs (miR- the Pol IV 18 sRNAs may act in trans at distal, related NAs) or trans-acting small interfering (ta-siRNAs), both sites, to direct 28 RdDM in a Pol V-dependent manner. Current Opinion in Plant Biology 2011, 14:148–155 www.sciencedirect.com Small RNAs, RdDM, and the epigenome of plants Simon and Meyers 149 Figure 1 Transposons, repeats, etc. Pol IV + CLSY1 ss RNA Pol IV transcript RDR2 dsRNA DCL3 24 nt siRNA AGO4 AGO4-bound AGO4 siRNA complex + Pol V + DDR Complex Target Locus Nascent RNA Nascent RNA AGO4 Pol V Pol V PolV or AGO4 M M M M M M Recruitment of complexes for de novo DNA methylation (DRM2) and histone modifications Current Opinion in Plant Biology Heterochromatin formation via de novo DNA methylation and the recruitment of histone modifying enzymes. Data suggest that RNA polymerase IV transcribes transposons and other genomic regions, recruits RDR2 to make a double-stranded RNA that is cleaved by DCL3 into 24 nt sRNAs. These are loaded into an AGO4 complex, and this complex is then either recruited to function with the Pol V-DDR complex, or the AGO4 complex associates with Pol V-derived nascent transcripts. The activity of these proteins recruits de novo DNA methyltransferases including DRM2, as well as other chromatin remodeling enzymes. For 28 RdDM, Pol IV is believed to transcribe a meth- region of primary RdDM. Notably, the establishment and ylated target template and the downstream sequence. maintenance of 18 RdDM is independent of 28 RdDM The result is an aberrant RNA that gets copied and [23]. cleaved by RDR2 and DCL3, respectively, to produce 28 siRNAs that induce methylation downstream of the The progress made in identifying the machinery associ- target site [23](Figure 2). In primary RdDM, the ated with siRNA biogenesis and siRNA-directed DNA synthesis and amplification of 18 siRNAs target and methylation in plants has also revealed a fairly complex reinforce methylation at the original siRNA generating repertoire of RNA-mediated epigenetic regulatory mech- locus. Whereas, in 28 RdDM, 28 siRNAs are produced to anisms. The contribution of sRNAs is discussed here, facilitate the spreading of methylation adjacent to the with an emphasis on the epigenetic aspects of sRNAs in www.sciencedirect.com Current Opinion in Plant Biology 2011, 14:148–155 150 Genome studies and molecular genetics Figure 2 Heterochromatic Region from Figure 1 Aberrant RNA Pol IV Pol IV M M M M M M Reinforce 1º RdDM RDR2 Aberrant RNA 1º siRNAs 2º siRNAs RDR2 + DCL3 24 nt siRNA 24 nt siRNA Methylated Template 2º RdDM M M M M M M MMM M M M M M M M Current Opinion in Plant Biology RNA polymerase IV-dependent production of small RNAs (18 siRNAs) reinforces existing heterochromatic regions by primary RNA-directed DNA methylation (18 RdDM). Primary RdDM can lead to the production of 28 siRNAs which trigger the spreading of methylation into adjacent regions, resulting in 28 RdDM. Secondary RdDM results from a Pol IV-derived aberrant RNA transcribed from methylated target templates. Some of these sRNAs may act in trans to direct 28 RdDM in a Pol V-dependent manner. the context of RdDM, heterochromatin formation and important since sRNAs are impacted upon loss of meth- chromatin-based gene silencing, RNA-mediated chroma- ylation [32,33,34]. DNA demethylation in plants is tin silencing in paramutation, development, genetic known to result from the activity of the DNA glycosy- imprinting, and heritable epigenetic changes by way of lase/lyase proteins REPRESSOR OF SILENCING 1 and mobile siRNAs. 3 (ROS1 and ROS3), DEMETER (DME) and DME-like (DML) [33–35]. Small RNA-directed DNA methylation DNA methylation is one of the most well-studied epige- A recently identified regulator of RdDM, RNA- netic modifications. In plants, methylation can occur at DIRECTED DNA METHYLATION 1 (RDM1) was any cytosine and in three different sequence contexts. shown to be associated with the accumulation of 24 nt ‘Symmetric methylation’ corresponds to CG and CHG siRNAs, DNA methylation, and silencing at target loci sites, while ‘asymmetric’ methylation corresponds to [12]. RDM1 was found to encode a protein that can bind CHH sites [26]; in each case, the H represents A, C, or single-stranded, methylated DNA. In addition, RDM1 was T. As described above, the recruitment of the siRNA- shown to associate with RNA polymerase II, AGO4 and producing machinery is the first step in the de novo DNA DRM2, which makes it a strong candidate for being a part methylation of cytosines, and the second step is the of the AGO4-effector complex of RdDM [12]. RDM1 targeted siRNA-directed DNA methylation at the hom- was found to copurify with DRD1 and DMS3, forming the ologous DNA region. There are multiple DNA methyl- DDR complex (DRD1–DMS3–RDM1) [17]. Gao et al. transferases involved in the establishment and [12] proposed that the single-stranded, methyl-DNA- maintenance of RdDM, including DOMAINS binding activity of RDM1 could facilitate AGO4 targeting. REARRANGED METHYLTRANSFERASE 1 and 2 Additionally, Gao et al. [12] showed that RDM1 and Pol V (DRM1 and DRM2), which establish CHH methylation, are colocalized in the perinucleolar processing center and CHROMOMETHYLASE 3 (CMT3), which establishes that RDM1 is required for Pol V transcripts.
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