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AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana

ABSTRACT: RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of AROGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing (TGS) of homologous promoter sequences. AGO proteins act in silencing effector complexes by anchoring the 3â and 5â ends of the guide siRNAs at their N-terminal PAZ domain and MID domain, respectively. In addition, many AGO proteins cleave complementary target RNAs through an endonuclease (âslicerâ) activity in their C-terminal PIWI domain. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and TGS in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points and the preferential association of Pol V with AGO6. Examination of siRNA abundance in the trasngenic wild type plant (contains trigger and silencer transgenes) and the ago6-4 mutant.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Blake Meyers

PROVIDER: E-GEOD-28537 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana

Project description:RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of AROGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing (TGS) of homologous promoter sequences. AGO proteins act in silencing effector complexes by anchoring the 3â and 5â ends of the guide siRNAs at their N-terminal PAZ domain and MID domain, respectively. In addition, many AGO proteins cleave complementary target RNAs through an endonuclease (âslicerâ) activity in their C-terminal PIWI domain. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and TGS in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points and the preferential association of Pol V with AGO6.

2011-12-01 | GSE28537 | GEO

Redundant AGO-hook platforms and AGO4-DNA interaction in polV-directed RdDM

Project description:Small RNA-dependent pairing of AGO/PIWI-containing effector complexes to chromatin-bound nascent transcripts has been proposed as a universal mechanism for guiding RNA-mediated TGS in eukaryotes. Likewise, Pol V-dependent transcripts have been implicated in the targeting of AGO4 to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we show that the AGO hook platforms of PolV and SPT5L, another component of the transcriptional complex, are functionally redundant yet essential for RdDM at a genome-wide level. Synthesis of Pol V transcripts is uncoupled from AGO4 recruitment in AGO hook-minus plants, challenging the prevailing RNA-based mechanism of AGO4 targeting to chromatin in RdDM. Transcription is essential to lock the PolV transcription complex into a stable and productive DNA-bound state potentiating interactions of AGO4 with DNA. Consistent with this idea, laser UV-assisted crosslinking shows specific AGO4-DNA interaction at RdDM loci, suggesting a revised model for Pol V-mediated DNA methylation in plants, which explains the exquisite specificity of methylation.

2015-06-01 | GSE67216 | GEO

Deep sequencing of small RNAs in the trasngenic wild type plant and the IWR1-type transcription factor mutant, dms4

Project description:Full title: Deep sequencing of small RNAs in the trasngenic wild type plant (contains trigger and silencer transgenes) and the IWR1-type transcription factor mutant, dms4 RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase II (PolII)-related RNA polymerases named PolIV and PolV. A genetic screen designed to reveal factors important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype.DMS4 is similar to yeast IWR1, a conserved putative transcription factor that interacts with PolII subunits. In the transgenic system studied, mutations in DMS4 directly or indirectly affect PolIV-dependent secondary siRNAs, PolV-mediated RdDM, PolV-dependent synthesis of intergenic noncoding RNA, and expression of many PolII-driven genes. These data suggest that DMS4 may be a regulatory factor for multiple RNA polymerases, thus explaining its diverse roles in the plant. Examination of siRNA abundance in the trasngenic wild type plant (contains trigger and silencer transgenes) and the dms4 mutant.

2010-03-31 | E-GEOD-18302 | biostudies-arrayexpress

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

Project description:In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs). We also examined small RNA abundance at individual CRP genes in the wild type plant, nrpd1, and rdr2 mutant plants.

2012-11-22 | E-GEOD-34207 | biostudies-arrayexpress

Structural Variant of a Tandem Repeat-containing Silencer Locus Reveals Distinct Pol II and Pol IV-dependent Pathways of siRNA Biogenesis

Project description:We used a two-component transgene system to study the RNA-dependent DNA methylation (RdDM) and transcriptional gene silencing (TGS) in Arabidopsis. By profiling the small RNA population in mutants defected in RdDM or RNA polymerase II-transcribed trigger for generating silencing siRNA, we investigated how repetitive loci such as tandem repeats were regulated transcriptionally through the action of RNA polymerase IV. Genome-wide small RNA profiling was done by Illumina TruSeq sample preparation followed by high-throughput sequencing with the Illumina HiSeq 2000 platform. The six samples represent mutants and their corresponding control lines.

2014-08-05 | E-GEOD-47852 | biostudies-arrayexpress

Characterization of small RNA-generating regions targeted by RNA polymerase V in Arabidopsis

Project description:In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (Pol) Pol IV and Pol V. Pol IV plays a major role in siRNA biogenesis, while Pol V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions which are dependent on both Pol IV and Pol V have been identified previously, the genomic loci targeted Pol V for siRNA accumulation and silencing have not been described extensively. To characterize the Pol V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and Pol V mutant plants. Furthermore, we characterized the siRNA-generating regions which were dependent on RdDM effectors and examined their dependency on Pol V. Small RNA libraries were generated and deeply sequenced from mutant alleles dms4-1, drd1-1, dms3-1, and rdm1-4, along with their control library (“Wt(T+S)”) which has been described previously (Kanno et al. 2004 Current Biology). More than 2,000 small RNA-generating loci were identified which were greatly suppressed in Pol V mutants. The Pol V-dependent, heterochromatic siRNA-generating regions were characterized in the Arabidopsis genome by deep sequencing the small RNA populations of wild-type and Pol V mutant plants. Deep SBS sequencing was used for small RNA profiling of immature inflorescence tissues from RNA polymerase V and RdDM mutants.

2012-06-11 | E-GEOD-36424 | biostudies-arrayexpress

3'RACE analyses of Pol V transcript 3' end lengths in mutants of the exonuclease RRP6L1

Project description:RNA Polymerase V transcription recruits siRNA-Argonaute protein complexes to chromatin, thereby specifying sites of RNA-directed DNA methylation (RdDM) and transcriptional gene silencing in plants. The Pol V largest subunit, NRPE1, has an extensive carboxyl-terminal domain (CTD) that is dispensable for catalytic activity in vitro, yet essential in vivo. A CTD subdomain, DeCL, named for its similarity to a chloroplast protein, DEFECTIVE CHLOROPLASTS AND LEAVES, is required for Pol V function at virtually all loci, similar to mutants defective for Pol V recruitment. Deletions removing three other CTD subdomains affect overlapping subsets of loci, similar to mutants lacking proteins that bind Pol V or its transcripts. A yeast two-hybrid screen for CTD-interactors identified the 3-prime -> 5-prime exoribonuclease, RRP6L1 as an interactor with the DeCL subdomain and the adjacent QS subdomain, named for its numerous glutamine-serine (QS) repeats. These RRP6L1-binding subdomains immediately follow the Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3-prime ends of Pol V transcripts sliced by ARGONAUTE 4 (AGO4), suggesting a model whereby the adjacent CTD subdomains enable the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

2017-02-25 | GSE93354 | GEO

Functional analysis of the Arabidopsis thaliana Pol V largest subunit, NRPE1, carboxy-terminal domain by whole genome bisulfite sequencing

2017-02-25 | GSE93358 | GEO

sRNA expression in the Pol V largest subunit mutant, nrpe1-11

2017-02-25 | GSE93355 | GEO

RNA-directed DNA methylation involves co-transcriptional small RNA-guided slicing of Pol V transcripts in Arabidopsis.

Project description:Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide (nt) small RNAs (siRNAs) that bind ARGONAUTE4 (AGO4) and target genomic regions for silencing. It also requires non-coding RNAs transcribed by RNA POLYMERASE V (Pol V), that likely serve as scaffolds for binding of AGO4/siRNA complexes. Here we utilized a modified global nuclear run-on (GRO) protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5’ adenine found in many AGO4-bound 24-nt siRNAs and was eliminated in an siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expressing wild-type AGO4 in ago4/6/9 was able to restore slicing of Pol V transcripts but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required the little understood elongation factor SPT5L. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

2018-01-29 | GSE108078 | GEO

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