Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes

In plants, DNA methylation can be mediated by a class of Argonaute4 (AGO4)-associated heterochromatic siRNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process, as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AGO4 is loaded with hc-siRNAs in the cytoplasm and the formation of mature AGO4/siRNA complexes requires HSP90 and the cleavage activity of AGO4. Intriguingly, siRNA binding facilitates the redistribution of AGO4 into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AGO4/siRNA complexes is a key regulatory point prior to the effector stage of RdDM.     

SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV

DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, domains rearranged methyltransferase 2 (DRM2), is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV). However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of nuclear RNA polymerase D1 (NRPD1), the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA-dependent RNA polymerase 2 (RDR2), CLASSY1 (CLSY1), and RNA-directed DNA methylation 4 (RDM4), suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE homeodomain homolog 1 (SHH1), was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway.     

Cucumber mosaic virus suppressor 2b binds to AGO4-related small RNAs and impairs AGO4 activities

Cucumber mosaic virus suppressor 2b (CMV2b) is a nuclear viral suppressor that interferes with local and systemic silencing and inhibits AGO1 slicer activity. CMV2b-mediated transgene hypomethylation and its localization in Cajal bodies suggests a role of CMV2b in RNA-directed DNA methylation (RdDM). However, its direct involvement in RdDM, or its binding with small RNAs (sRNAs) in vivo is not yet established. Here, we show that CMV2b binds both microRNAs (miRNAs) and small interfering RNAs (siRNAs) in vivo. sRNA sequencing data from the CMV2b immunocomplex revealed its preferential binding with 24-nt repeat-associated siRNAs. We provide evidence that CMV2b also has direct interaction with the AGO4 protein by recognizing its PAZ and PIWI domains. Subsequent analysis of AGO4 functions revealed that CMV2b reduced AGO4 slicer activity and the methylation of several loci, accompanied by the augmented accumulation of 24-nt siRNAs in Arabidopsis inflorescences. Intriguingly, CMV2b also regulated an AGO4-related epiallele independently of its catalytic potential, which further reinforces the repressive effects of CMV2b on AGO4 activity. Collectively, our results demonstrate that CMV2b can counteract AGO4-related functions. We propose that by adopting novel counter-host defense strategies against AGO1 and AGO4 proteins, CMV creates a favorable cellular niche for its proliferation.     

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

Argonaute (AGO) family proteins are conserved key components of small RNA‐induced silencing pathways. In the RNA‐directed DNA methylation (RdDM) pathway in Arabidopsis, AGO6 is generally considered to be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4‐ and AGO6‐dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co‐localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co‐localized. In the nucleoplasm, AGO4 displays a strong co‐localization with Pol II, whereas AGO6 co‐localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V‐dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA‐directed DNA methylation.     

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

Recent studies have identified a conserved WG/GW‐containing motif, known as the Argonaute (AGO) hook, which is involved in the recruitment of AGOs to distinct components of the eukaryotic RNA silencing pathways. By using this motif as a model to detect new components in plant RNA silencing pathways, we identified SPT5‐like, a plant‐specific AGO4‐interacting member of the nuclear SPT5 (Suppressor of Ty insertion 5) RNA polymerase (RNAP) elongation factor family that is characterized by the presence of a carboxy‐terminal extension with more than 40 WG/GW motifs. Knockout SPT5‐like mutants show a decrease in the accumulation of several 24‐nt RNAs and hypomethylation at different loci revealing an implication in RNA‐directed DNA methylation (RdDM). Here, we propose that SPT5‐like emerged in plants as a facultative RNAP elongation factor. Its plant‐specific origin and role in RdDM might reflect functional interactions with plant‐specific RNA Pols required for RdDM.     

Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats

In a number of organisms, transgenes containing transcribed inverted repeats (IRs) that produce hairpin RNA can trigger RNA-mediated silencing, which is associated with 21-24 nucleotide small interfering RNAs (siRNAs). In plants, IR-driven RNA silencing also causes extensive cytosine methylation of homologous DNA in both the transgene "trigger" and any other homologous DNA sequences--"targets". Endogenous genomic sequences, including transposable elements and repeated elements, are also subject to RNA-mediated silencing. The RNA silencing gene ARGONAUTE4 (AGO4) is required for maintenance of DNA methylation at several endogenous loci and for the establishment of methylation at the FWA gene. Here, we show that mutation of AGO4 substantially reduces the maintenance of DNA methylation triggered by IR transgenes, but AGO4 loss-of-function does not block the initiation of DNA methylation by IRs. AGO4 primarily affects non-CG methylation of the target sequences, while the IR trigger sequences lose methylation in all sequence contexts. Finally, we find that AGO4 and the DRM methyltransferase genes are required for maintenance of siRNAs at a subset of endogenous sequences, but AGO4 is not required for the accumulation of IR-induced siRNAs or a number of endogenous siRNAs, suggesting that AGO4 may function downstream of siRNA production.     

RNA介导的植物DNA甲基化研究进展

RNA介导的DNA甲基化作用(RNA-directed DNA Methylation,RdDM)是首次在植物中发现的基因组表观修饰现象,RdDM通过RNA-DNA序列相互作用直接导致DNA甲基化。植物中的RdDM和siRNA介导的mRNA降解现象,都是通过RNA使序列特异性基因发生沉默,它们对于植物的染色体重排、抵御病毒感染、基因表达调控和发育的许多过程起到了非常重要的作用。在植物中有很多的文献报道RdDM现象,但是对于其具体调控机理还不是很清楚。这里对RNA介导的植物DNA甲基化的基本特征进行了简要概述,主要对RdDM机理的研究进展进行了综述,其中包括RdDM过程中的DNA甲基转移酶的种类及其作用机理,DNA甲基化与染色质修饰之间的关系,以及与RdDM相关的重要蛋白质的研究等。在植物中,转录和转录后水平都可能发生RdDM,诱发基因沉默,前者常涉及靶基因启动子的甲基化,后者则牵涉到编码区的甲基化。RdDM的发生依赖于RNAi途径中相似的siRNA和酶,如DCL3、RdR2、SDE4和AGO4。植物中至少含有三类DNA甲基转移酶DRM1/2、MET1和CMT3,其作用部位是与RNA同源的DNA区域中的所有胞嘧啶,而组蛋白H3第九位赖氨酸的甲基化影响着胞嘧啶的甲基化。