An Effector of RNA-Directed DNA Methylation in Arabidopsis Is an ARGONAUTE 4- and RNA-Binding Protein

He, Xin; Hsu, Yi‐Feng; Zhu, Shihua; Wierzbicki, Andrzej; Pontes, Olga; Pikaard, Craig S.; Liu, Hailiang; Wang, Co Shine; Jin, Hailing; Zhu, Jian Kang

doi:10.1016/j.cell.2009.04.028

Cited by 241 publications

(252 citation statements)

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“…The AGO4/siRNA complexes are recruited to target loci via base-pairing with scaffold transcripts generated by RNA polymerase Pol V (Pontier et al, 2005;Mosher et al, 2008;Wierzbicki et al, 2008Wierzbicki et al, , 2009). The recruitment of AGO4 to chromatin may also be facilitated by its interaction with the GW/WGrich extensions of NRPE1 (the largest subunit of Pol V) Pontes et al, 2006;El-Shami et al, 2007;Li et al, 2008) and KTF1/SPT5L (a transcription elongation factor) (Bies-Etheve et al, 2009;He et al, 2009). The conserved GW/WG motif, defining an "Ago hook," is widely present in factors implicated in AGO actions (Azevedo et al, 2011;Garcia et al, 2012;Pontier et al, 2012).…”

Section: Dna Methylationmentioning

confidence: 99%

“…In plants, DNA methylation can be mediated by AGO4-bound hc-siRNAs through a specialized RNAi pathway termed RNA-directed DNA methylation (RdDM). RdDM entails the generation of double-stranded RNAs (dsRNAs) by coordinated activities of RNA Polymerase IV (Pol IV) and RNAdependent RNA polymerase 2 (RDR2) (Xie et al, 2004;Herr et al, 2005;Kanno et al, 2005;Onodera et al, 2005;Li et al, 2008;He et al, 2009;Haag et al, 2012). dsRNAs are then processed by DCL3 to produce 24-nucleotide hc-siRNAs (Xie et al, 2004).…”

Section: Dna Methylationmentioning

confidence: 99%

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RNAi in Plants: An Argonaute-Centered View

2016

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Argonaute (AGO) family proteins are effectors of RNAi in eukaryotes. AGOs bind small RNAs and use them as guides to silence target genes or transposable elements at the transcriptional or posttranscriptional level. Eukaryotic AGO proteins share common structural and biochemical properties and function through conserved core mechanisms in RNAi pathways, yet plant AGOs have evolved specialized and diversified functions. This Review covers the general features of AGO proteins and highlights recent progress toward our understanding of the mechanisms and functions of plant AGOs.

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Section: Dna Methylationmentioning

confidence: 99%

Section: Dna Methylationmentioning

confidence: 99%

RNAi in Plants: An Argonaute-Centered View

2016

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“…ROS1 is a key DNA demethylation component in vegetative tissues (9), and its transcript level is regulated by MET1 and several RdDM components (10). Screening for second-site suppressors of ros1 mutants by using silenced RD29A-LUC has enabled researchers to identify the main components of the RdDM pathway and has further indicated that RdDM and ROS1 antagonistically regulate DNA methylation (11)(12)(13)(14).…”

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confidence: 99%

Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

Qian

Zhao

et al. 2012

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

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REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.transcriptional gene silencing | epigenetic | gene silencing | histone modification D NA cytosine methylation is an important epigenetic modification in plants and animals. The molecular mechanism for RNA-directed DNA methylation (RdDM) has been well-established by both genetic and biochemical studies in Arabidopsis (1-4). Small RNAs (∼24 bp) and long noncoding RNAs produced by a complex transcriptional machinery composed of polymerase (Pol) IV and Pol V, together with other proteins, target specific sequences for de novo DNA methylation (1-4). RdDM mainly occurs in transposons, retrotransposons, rDNA arrays, and centromeric repeat regions, and accounts for about 30% of the DNA methylation in the genome of Arabidopsis (5, 6).DNA methylation can be removed passively during DNA replication or actively by the DNA glycosylase REPRESSOR OF SILENCING 1 (ROS1) and its homologs DEMETER (DME), DEMETER-LIKE 2 (DML2), and DML3 (1, 2, 7, 8). ROS1 was identified in a genetic screen in which the originally active RD29A-LUC was silenced in ros1 mutants (9). ROS1 is a key DNA demethylation component in vegetative tissues (9), and its transcript level is regulated by MET1 and several RdDM components (10). Screening for second-site suppressors of ros1 mutants by using silenced RD29A-LUC has enabled researchers to identify the main components of the RdDM pathway and has further indicated that RdDM and ROS1 antagonistically regulate DNA methylation (11)(12)(13)(14).ros1 mutations also cause the silencing of another transgene, 35S-NPTII, which is in the same T-DNA construct as RD29A-LUC (9). RD29A-LUC has been used to identify ROS1 and ROS3 genes (9, 15). By screening for second-site suppressors of ros1 using silenced 35S-NPTII, we previously identified genes that are involved in DNA replication and maint...

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“…The 24-nt siRNAs are loaded onto ARGONAUTE proteins AGO4 and/or AGO6 (Zheng et al 2007;Matzke et al 2009) and target genomic regions, possibly by base-pairing with nascent Pol V transcripts from the target loci. The Pol V-dependent RNA transcripts are tethered to AGO4 by the action of KTF1, forming an effector complex containing AGO4, KTF1, Pol V-dependent transcripts, and siRNAs (He et al 2009a). The effector complex directs DRM2 for de novo methylation of target genomic sequences.…”

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confidence: 99%

A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development

He¹,

Hsu²,

Zhu³

et al. 2009

Genes Dev.

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RNA-directed DNA methylation (RdDM) is a conserved mechanism for epigenetic silencing of transposons and other repetitive elements. We report that the rdm4 (RNA-directed DNA Methylation4) mutation not only impairs RdDM, but also causes pleiotropic developmental defects in Arabidopsis. Both RNA polymerase II (Pol II)-and Pol V-dependent transcripts are affected in the rdm4 mutant. RDM4 encodes a novel protein that is conserved from yeast to humans and interacts with Pol II and Pol V in plants. Our results suggest that RDM4 functions in epigenetic regulation and plant development by serving as a transcriptional regulator for RNA Pol V and Pol II, respectively. DNA methylation and histone modifications are important epigenetic silencing mechanisms in eukaryotic cells (Chan et al. 2005;Matzke and Birchler 2005). In plants, DNA methylation is catalyzed by DNA methytransferase MET1, CMT3, and DRM2 (Chan et al. 2005). MET1 and CMT3 mainly function in maintaining DNA hypermethylation at CG and CHG sites (H is A, T, or C) during DNA duplication, while the function of DRM2 is involved in de novo DNA methylation at CHH sites directed by RNA (Chan et al. 2005). RNA-directed DNA methylation (RdDM) was first discovered in plants (Wassenegger et al. 1994), and plays important roles in transgene silencing, genome integrity, and transposon stability (Matzke et al. 2009).In the RdDM pathway, both 24-nucleotide (nt) siRNAs and long noncoding RNA transcripts are essential for de novo DNA methylation (Wierzbicki et al. 2008). The 24-nt siRNAs are generated in a pathway involving the putative DNA-directed RNA polymerase IV (Pol IV), RDR2 (RNA-dependent RNA polymerase 2), and DCL3 (Dicer-like RNA Pol IV and Pol V have distinct largest subunits-NRPD1 and NRPE1, respectively-but share some common subunits such as NRPD/E2 and NRPD/E4 (He et al. 2009b). Some subunits of Pol IV and Pol V are also shared with Pol II (Huang et al. 2009;Ream et al. 2009). It appears that the plant-specific RNA Pol IV and Pol V evolved from an ancestral RNA polymerase to function specifically in RdDM. The transcription activity of Pol II is tightly regulated with the help of a series of general transcription factors (Kornberg 2007). Although many general transcription factors of Pol II have been studied extensively, nothing is known about the regulation of transcription by Pol IV and Pol V.In the present study, we carried out a forward genetic screen for second site suppressors of the DNA demethylase mutant ros1, and identified a transcription factor, RDM4 (RNA-directed DNA Methylation4), that is required for RdDM in Arabidopsis. Interestingly, unlike other RdDM mutants, the rdm4 mutant plants display pleiotropic developmental phenotypes. RDM4 encodes a novel protein that is conserved in eukaryotic organisms. We found that RDM4 interacts with Pol II and Pol V in plants. Our results suggest that RDM4 is a regulator of Pol II and Pol V transcription, and thereby contributes to both development and RdDM. Results and DiscussionThe rdm4 mutation suppresses transcr...

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An Effector of RNA-Directed DNA Methylation in Arabidopsis Is an ARGONAUTE 4- and RNA-Binding Protein

Cited by 241 publications

References 44 publications

RNAi in Plants: An Argonaute-Centered View

RNAi in Plants: An Argonaute-Centered View

Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development

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