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As sessile organisms, plants need to counteract different biotic and abiotic stresses to survive. RNA interference provides natural immunity against various plant pathogens, especially against viral infections via inhibition of viral genome replication or translation. In plants, DRB3, a multi-domain protein containing two N-terminal dsRNA binding domains (dsRBD), plays a vital role in RNA-directed DNA methylation of the geminiviral genome. Additionally, DRB3 arrests the replication of the viral genome in the viral replication complex of RNA viruses through a mechanism that has yet to be fully deciphered. Therefore, as a first step towards exploring the structural details of DRB3, we present a nearly complete backbone and side chain assignment of the two N-terminal dsRBD domains. [ABSTRACT FROM AUTHOR]

In plants, the RNA-directed DNA methylation (RdDM) pathway plays a major role in establishing DNA methylation. At least some components of the RdDM machinery, including the central component AGO4, are known to concentrate in a subnuclear compartment called the Cajal body in the model plant Arabidopsis thaliana. The molecular underpinnings of Cajal body localization, however, have remained elusive so far. Here, we found that Arabidopsis AGO4 (AtAGO4) fused to GFP does not present its typical Cajal body localization, when transiently expressed in Nicotiana benthamiana. Nevertheless, the endogenous AGO4 protein from N. benthamiana shows a clear accumulation in the Cajal body. Thus, our results suggest that the Cajal body localization of AtAGO4 requires specific molecular machinery that cannot be replaced by orthologues in N. benthamiana. This study presents an experimental system that could lead to mechanistic insights into the targeting of proteins to and localization in the Cajal body in plants.

Abstract Background Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). Methods RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3’UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. Results Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. Conclusion Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively. Graphical abstract

Summary: Methylation at the MdMYB1 promoter in apple sports has been reported as a regulator of the anthocyanin pathway, but little is known about how the locus is recognized by the methylation machinery to regulate anthocyanin accumulation. In this study, we analysed three differently coloured 'Fuji' apples and found that differences in the transcript levels of MdMYB1, which encodes a key regulator of anthocyanin biosynthesis, control the anthocyanin content (and therefore colour) in fruit skin. The CHH methylation levels in the MR3 region (−1246 to −780) of the MdMYB1 promoter were found to be negatively correlated with MdMYB1 expression. Thus, they were ideal materials to study DNA methylation in apple sports. The protein of RNA‐directed DNA methylation (RdDM) pathway responsible for CHH methylation, MdAGO4, was found to interact with the MdMYB1 promoter. MdAGO4s can interact with MdRDM1 and MdDRM2s to form an effector complex, fulfilling CHH methylation. When MdAGO4s and MdDRM2s were overexpressed in apple calli and Arabidopsis mutants, those proteins increase the CHH methylation of AGO4‐binding sites. In electrophoretic mobility shift assays, MdAGO4s were found to specifically bind to sequence containing ATATCAGA. Knockdown of MdNRPE1 did not affect the binding of MdAGO4s to the c3 region of the MdMYB1 promoter in 35S::AGO4 calli. Taken together, our data show that the MdMYB1 locus is methylated through binding of MdAGO4s to the MdMYB1 promoter to regulate anthocyanin biosynthesis by the RdDM pathway. [ABSTRACT FROM AUTHOR]

The transcriptional and posttranscriptional AGO-mediated control of gene expression may play important roles during male monocot gametophyte development. In this report, we demonstrated dynamic changes in the spatiotemporal distribution of AGO1 and AGO4, which are key proteins of the RNA-induced silencing complex (RISC) in Hyacinthus orientalis male gametophyte development. During maturation of the bicellular pollen grains and in vitro pollen tube growth, the pattern of AGO1 localization was correlated with previously observed transcriptional activity of the cells. During the period of high transcriptional activity, AGO1 is associated with chromatin while the clustered distribution of AGO1 in the interchromatin areas is accompanied by condensation of chromatin and the gradual transcriptional silencing of both cells in mature, dehydrated pollen. During pollen tube growth and the restarting of RNA synthesis in the vegetative nucleus, AGO1 is dispersed in the chromatin. Additionally, the gradual increase in the cytoplasmic pool of AGO1 in the elongating pollen tube indicates the activation of the posttranscriptional gene silencing (PTGS) pathway. During pollen tube growth in the generative cell and in the sperm cells, AGO1 is present mainly in the areas between highly condensed chromatin clusters. Changes in the distribution of AGO4 that indicated the possibility of spatiotemporal organization in the RNA-directed DNA methylation (RdDM) process (cytoplasmic and nuclear steps) were also observed during hyacinth male gametophyte development. Based on our findings, we propose that in the germinating pollen tube, the cytoplasmic assembly of AGO4/siRNA takes place and that the mature complexes could be transported to the nucleus to carry out their function during the next steps of pollen tube growth. [ABSTRACT FROM AUTHOR]

Background: Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). Methods: RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. Results: Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. Conclusion: Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively. [ABSTRACT FROM AUTHOR]

The Cucumber mosaic virus (CMV) suppressor 2b co-localizes with AGO4 in cytoplasmic and nuclear fractions of Arabidopsis thaliana. Biochemical fractionation of A. thaliana cellular extracts revealed that 2b and AGO4 coexist in multiple size exclusions. 2b transgenic A. thaliana exhibited an enhanced accumulation of 24nt siRNAs from flowering wageningen (FWA) and other heterochromatic loci. These plants also exhibited hypo-methylation of an endogenous- as well as transgene-FWA promoter at non-CG sites. In corroboration, both transgenic 2b and CMV infection affected the regulation of transposons which mimics the ago4 phenotype. In conclusion, 2b perturbs plant defense by interfering with AGO4-regulated transcriptional gene silencing.

By controlling gene expression, DNA methylation contributes to key regulatory processes during plant development. Genomic methylation patterns are dynamic and must be properly maintained and/or re-established upon DNA replication and active removal, and therefore require sophisticated control mechanisms. Here we identify direct interplay between the DNA repair factor DNA damage-binding protein 2 ( DDB2) and the ROS1-mediated active DNA demethylation pathway in Arabidopsis thaliana. We show that DDB2 forms a complex with ROS1 and AGO4 and that they act at the ROS1 locus to modulate levels of DNA methylation and therefore ROS1 expression. We found that DDB2 represses enzymatic activity of ROS1. DNA demethylation intermediates generated by ROS1 are processed by the DNA 3′-phosphatase ZDP and the apurinic/apyrimidinic endonuclease APE1L, and we also show that DDB2 interacts with both enzymes and stimulates their activities. Taken together, our results indicate that DDB2 acts as a critical regulator of ROS1-mediated active DNA demethylation. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA)-mediated gene silencing is a key mechanism regulating numerous biological processes such as development of organs and tumorigenesis. The expression of miRNA machinery genes linked to miRNA biogenesis and processing is finely regulated. Despite accumulating evidence for chicken miRNA in the female reproduction system, precise regulatory mechanisms are largely unknown. Therefore, the objective of this study was to determine changes in expression levels of miRNA machinery genes in developmental stages of the oviduct and ovarian carcinogenesis of laying hens. In the present study, differential expression of miRNA machinery genes during ovarian carcinogenesis was determined using cancerous and normal ovaries collected from normal laying hens and hens with cancer. Our results showed that 3 miRNA machinery genes (Dicer1, AGO3, and AGO4) were differentially expressed as laying hens' reproductive organs developed. These genes were simultaneously upregulated in cancerous ovaries compared with those in normal ovaries. Their transcripts were abundantly localized in glandular epithelial cells of cancerous ovaries. Our results indicate that Dicer1, AGO3, and AGO4 play critical roles in the development of reproductive organs and ovarian carcinogenesis in laying hens, suggesting that simultaneous overexpression of these genes might serve as a prognostic factor for ovarian cancer. [ABSTRACT FROM AUTHOR]

RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation mechanism that requires long noncoding RNA (lncRNA) as scaffold to define target genomic loci. While the role of RdDM in maintaining genome stability is well established, how it regulates protein-coding genes remains poorly understood and few RdDM target genes have been identified. In this study, we obtained sequences of RdDM-associated lncRNAs using nuclear RNA immunoprecipitation against ARGONAUTE 4 (AGO4), a key component of RdDM that binds specifically with the lncRNA. Comparison of these lncRNAs with gene expression data of RdDM mutants identified novel RdDM target genes. Surprisingly, a large proportion of these target genes were repressed in RdDM mutants suggesting that they are normally activated by RdDM. These RdDM-activated genes are more enriched for gene body lncRNA than the RdDM-repressed genes. Histone modification and RNA analyses of several RdDM-activated stress response genes detected increased levels of active histone mark and short RNA transcript in the lncRNA-overlapping gene body regions in the ago4 mutant despite the repressed expression of these genes. These results suggest that RdDM, or AGO4, may play a role in maintaining or activating stress response gene expression by directing gene body chromatin modification preventing cryptic transcription. [ABSTRACT FROM AUTHOR]

The RNA-directed DNA methylation (RdDM) pathway is important for the transcriptional repression of transposable elements and for heterochromatin formation. Small RNAs are key players in this process by regulating both DNA and histone methylation. Taking into account that methylation underlies gene silencing and that there are genes with meiosis-specific expression profiles, we have wondered whether genes involved in RdDM could play a role during this specialized cell division. To address this issue, we have characterized meiosis progression in pollen mother cells from Arabidopsis thaliana mutant plants defective for several proteins related to RdDM. The most relevant results were obtained for ago4-1. In this mutant, meiocytes display a slight reduction in chiasma frequency, alterations in chromatin conformation around centromeric regions, lagging chromosomes at anaphase I, and defects in spindle organization. These abnormalities lead to the formation of polyads instead of tetrads at the end of meiosis, and might be responsible for the fertility defects observed in this mutant. Findings reported here highlight an involvement of AGO4 during meiosis by ensuring accurate chromosome segregation at anaphase I. [ABSTRACT FROM AUTHOR]

Background: DNA methylation ensures genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs through a process known as RNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. Once established, DNA methylation at each of the three sequence contexts is maintained through different mechanisms. Although some players involved in RdDM and maintenance methylation have been identified, the underlying molecular mechanisms are not fully understood. To aid the comprehensive identification of players in DNA methylation, we generated a transgenic reporter system that permits genetic and chemical genetic screens in Arabidopsis. Results: A dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal level of luciferase activity, was obtained. LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context. Methylation was present throughout the promoter and LUC coding region. Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Using LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor. Conclusion: We developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis. The low basal level of LUCL expression provides an easy readout in genetic and chemical genetic screens that will dissect the mechanisms of RdDM and methylation maintenance. [ABSTRACT FROM AUTHOR]

Background: DNA sequences around HMGB1-produced DNA gaps are hypermethylates. DNA methylation of interspersed repetitive sequences (IRS) such as Alu elements can be established through AGO4-mediating, RNA-directed DNA methylation (RdDM). HMGB1 depletion, DNA gap reduction and global hypomethylation promote genomic instability. Methods: HMGB1, SIRT1, AGO4 and DNA gap colocalizations were evaluated. Then, Alu methylation was analyzed in HMGB1-deficient or HMGB1-overexpressing cells and Alu siRNA-transfected HMGB1-deficient cells. Results: HMGB1, SIRT1, AGO4 and DNA gap are colocalized in the nucleus. Moreover, HMGB1 or Alu siRNA increased Alu methylation, whereas Alu siRNA could not methylate HMGB1-deficient cells. Conclusion: AGO4 play a role in methylating DNA sequence around HMGB1-produced DNA gaps and localize DNA gap in IRS, and loss of intranuclear HMGB1 causes global hypomethylation.

Objectives: It is a common practice in Arabidopsis to transfer a mutation generated in one genetic background to other genetic background via crossing. However, the drawback of this methodology is unavoidable presence of genomic fragments from the donor parent being often replacing desirable genomic fragments of the recurrent parent. Here, we highlighted problem of Arabidopsis mutants being recombinant introgression lines that can lead to unreliable and misinterpreted results. Results: We studied the regulation of low copy number transposable elements Tag1 and Evelknievel (EK), located at the end of the bottom arm of chromosome 1 and both are present in the Arabidopsis Landsberg erecta (Ler) but not in Columbia (Col) ecotype. Using various epigenetic mutants (cmt3, ddm1, kyp2, ago4, rdr2 hen1 etc.), we found that certain mutants in the Ler background are deficient of Tag1 or EK or both and represent recombinant introgression lines whereby chromosomal regions from Col have been recombined into the Ler genome. Our data support a recent proposal calling for formulating standards for authentication of plant lines that are used in plant research. Most important is to verify that a given trait or genomic locus under study is correctly identified, particularly when using mutants generated by crossing. [ABSTRACT FROM AUTHOR]

Argonaute proteins bind small RNAs and mediate cleavage of complementary target RNAs. The human Argonaute protein Ago4 is catalytically inactive, although it is highly similar to catalytic Ago2. Here, we have generated Ago2-Ago4 chimeras and analyzed their cleavage activity in vitro. We identify several specific features that inactivate Ago4: the catalytic center, short sequence elements in the N-terminal domain, and an Ago4-specific insertion in the catalytic domain. In addition, we show that Ago2-mediated cleavage of the noncanonical miR-451 precursor can be carried out by any catalytic human Ago protein. Finally, phylogenetic analyses establish evolutionary distances between the Ago proteins. Interestingly, these distances do not fully correlate with the structural changes inactivating them, suggesting functional adaptations of individual human Ago proteins., (© 2014 Hauptmann et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)