A subgroup of SGS3-like proteins act redundantly in RNA-directed DNA methylation

At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.genomics | mass spectrometry | siRNAs | epigenetics D NA methylation is a stable epigenetic mark that is associated with the repression of genes and transposable elements. In Arabidopsis thaliana, maintenance of DNA methylation at silent loci is carried out by at least three methyltransferases: METH-YLTRANSFERASE 1 (MET1), CHROMOMETHYLTRANS-FERASE 3 (CMT3), and DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). However, DRM2 is solely responsible for establishment of DNA methylation-or de novo methylation-of silent elements (1). DRM2 is guided to chromatin by small interfering RNAs (siRNAs) in a process known as RNAdirected DNA methylation (RdDM) (2). It has been proposed that RdDM also requires intergenic noncoding (IGN) transcripts that are synthesized by RNA Polymerase V (Pol V). These transcripts likely serve as platforms for the recruitment of siRNA-loaded ARGONAUTE 4 (AGO4) to methylated loci (3, 4).Recently, we discovered the requirement of INVOLVED IN DE NOVO 2 (IDN2) for RdDM from a forward genetic screen (5). Alleles of this same gene were later reported from another screen for DNA methylation mutants (6). We previously demonstrated that IDN2 binds to double-stranded RNA through its XS domain, which is also observed in the XS-domain-containing protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) (7). We also found that IDN2 was likely to act in a step downstream of initial siRNA biogenesis. The XS domain is conserved throughout the plant kingdom, and XS-domain-containing proteins are involved in a wide range of processes such as viral defense (8) and stress response (9).To gain a better understanding of the in vivo role of IDN2, we performed affinity purifications from complementing transgenic lines expressing epitope-tagged full-length I...

Section: Discussionsupporting

confidence: 73%

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

Ausín

Greenberg

Simanshu

et al. 2012

“…RNase A treatment abolished the RNA-mediated FDM1-AGO4 interaction (Fig. S3D) (24) but not TGH-DCL1 interaction (Fig. 3 A and B).…”

Section: Tgh Is Required For Accumulation Of Mirnas and Sirnas Inmentioning

confidence: 90%

Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis

Ren

Xie

Dou

et al. 2012

Self Cite

172

203

MicroRNAs (miRNAs) are regulators of gene expression in plants and animals. The biogenesis of miRNAs is precisely controlled to secure normal development of organisms. Here we report that TOUGH (TGH) is a component of the DCL1-HYL1-SERRATE complex that processes primary transcripts of miRNAs [i.e., primary miRNAs (pri-miRNAs)] into miRNAs in Arabidopsis. Lack of TGH impairs multiple DCL activities in vitro and reduces the accumulation of miRNAs and siRNAs in vivo. TGH is an RNA-binding protein, binds pri-miRNAs and precursor miRNAs in vivo, and contributes to pri-miRNA-HYL1 interaction. These results indicate that TGH might regulate abundance of miRNAs through promoting DCL1 cleavage efficiency and/or recruitment of pri-miRNAs.S mall RNAs, including microRNAs (miRNAs) and siRNAs, are sequence-specific regulators of gene expression in plants and animals (1). miRNAs are derived from imperfect stem-loop transcripts, called primary miRNAs (pri-miRNAs), which are predominately produced by DNA-dependent RNA polymerase II, whereas siRNAs are processed from perfect or near-perfect long dsRNAs (2). After generation, miRNA and siRNA are loaded into an RNA-induced silencing complex containing the Argonaute protein to guide posttranscriptional or transcriptional gene silencing (1).In animals, pri-miRNAs are first processed to precursor miRNAs (pre-miRNAs) in the nucleus by the microprocessor containing Drosha and a dsRNA-binding protein DGCR8 (1). The resulting pre-miRNAs are then processed by Dicer in the cytoplasm to produce mature miRNAs (1). It has emerged that the activities of Drosha and Dicer are controlled to regulate miRNA expression in response to developmental and environmental signals (3). In Arabidopsis, DCL1, a dsRNA-binding protein, HYL1, and a zinc finger protein, SERRATE (SE), form a complex to process pri-miRNAs in the nucleus to pre-miRNAs and then to mature miRNAs (4-6). The accumulation of miRNAs in Arabidopsis also requires DDL, which was proposed to stabilize pri-miRNAs and to facilitate their processing (7). Recently, two cap-binding proteins, CBP80/ABH1 and CBP20, were found to be required for pre-mRNA splicing and primiRNA processing (8, 9). Plants also encode several classes of endogenous siRNAs, including the natural antisense transcriptderived siRNA, siRNA derived from repetitive DNA sequences (rasiRNA), and transacting siRNA (ta-siRNA) (10). In Arabidopsis, the generation of these siRNAs from long dsRNAs involves DCL1 homologues DCL2, DCL3, and DCL4, which produce 22-nt, 24-nt, and 21-nt siRNAs, respectively (11-13).In this report, we show that TOUGH (TGH) is an important factor for miRNA and siRNA biogenesis. Loss-of-function TGH in tgh-1 reduces the activity of multiple DCLs in vitro and the accumulation of miRNA and siRNAs in vivo. In the miRNA pathway, TGH associates with the DCL1 complex and binds primiRNAs and pre-miRNAs. TGH is required for the efficient in vivo interaction between pri-miRNA and HYL1. These data suggest that TGH assists DCLs to efficiently process and/or recruit the prec...

“…To test this, we treated the protein extracts with RNase A during the immunoprecipitation. We used this assay previously to show the RNA-dependent factor of DNA methylation 1-AGO4 interaction (31). This treatment did not abolish the AGO1-HESO1 interaction, suggesting that HESO1 may interact with AGO1 in an RNA-independent manner (Fig.…”

Section: Heso1-mediated Uridylation Triggers the Degradation Of The 5′mentioning

confidence: 93%

Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage

Ren

Xie

Zhang

et al. 2014

Self Cite

105

122

In plants, methylation catalyzed by HEN1 (small RNA methyl transferase) prevents microRNAs (miRNAs) from degradation triggered by uridylation. How methylation antagonizes uridylation of miRNAs in vivo is not well understood. In addition, 5′ RNA fragments (5′ fragments) produced by miRNA-mediated RNA cleavage can be uridylated in plants and animals. However, the biological significance of this modification is unknown, and enzymes uridylating 5′ fragments remain to be identified. Here, we report that in Arabidopsis, HEN1 suppressor 1 (HESO1, a miRNA nucleotidyl transferase) uridylates 5′ fragments to trigger their degradation. We also show that Argonaute 1 (AGO1), the effector protein of miRNAs, interacts with HESO1 through its Piwi/Argonaute/Zwille and PIWI domains, which bind the 3′ end of miRNA and cleave the target mRNAs, respectively. Furthermore, HESO1 is able to uridylate AGO1-bound miRNAs in vitro. miRNA uridylation in vivo requires a functional AGO1 in hen1, in which miRNA methylation is impaired, demonstrating that HESO1 can recognize its substrates in the AGO1 complex. On the basis of these results, we propose that methylation is required to protect miRNAs from AGO1-associated HESO1 activity that normally uridylates 5′ fragments. S mall interfering RNAs (siRNAs) and microRNAs (miRNAs), ∼20-25 nucleotides (nt) in size, are important regulators of gene expression. miRNAs and siRNAs are derived from imperfect hairpin transcripts and perfect long double-stranded RNAs, respectively (1, 2). miRNAs and siRNAs are then associated with Argonaute (AGO) proteins to repress gene expression through target cleavage and/or translational inhibition (3). The cleavage of target mRNAs usually occurs at a position opposite the tenth and eleventh nucleotides of miRNAs, resulting in a 5′ RNA fragment (5′ fragment) and a 3′ fragment (4). In Arabidopsis, the major effector protein for miRNA-mediated gene silencing is AGO1, which possesses the endonuclease activity required for target cleavage (5-7). In Drosophila, the exosome removes the 5′ fragments through its 3′-to-5′ exoribonuclease activity (8). How 5′ fragments are degraded in higher plants remains unknown. It has been shown that the 5′ fragments are subject to untemplated uridine addition at their 3′ termini (uridylation) in both animals and plants (9). However, the biological significance of this modification remains unknown because of a lack of knowledge of the enzymes targeting 5′ fragments for uridylation.Uridylation plays important roles in regulating miRNA biogenesis. In animals, TUT4, a terminal uridyl transferase, is recruited by Lin-28 (an RNA binding protein) to the let-7 precursor (prelet-7), resulting in uridylation of prelet-7 (10, 11). This modification impairs the stability of prelet-7, resulting in reduced levels of let-7. In addition, monouridylation has been shown to be required for the processing of some miRNA precursors (12). Deep sequencing analysis reveals that precursor uridylation is a widespread phenomenon occurring in many miRNA families in ani...