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Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention

ABSTRACT: Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention

PROVIDER: PRJNA830473 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18856140.fastq.gz	Fastqsanger.gz
	SRR18856141.fastq.gz	Fastqsanger.gz
	SRR18856142.fastq.gz	Fastqsanger.gz
	SRR18856143.fastq.gz	Fastqsanger.gz
	SRR18856144.fastq.gz	Fastqsanger.gz

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AGO4 slicer activity reveals the passenger strand function of plant 23 nt siRNAs and the post-slicing retention of target RNAs mediating RNA-directed DNA methylation [WGBS]

Project description:In plants, transcriptional silencing by RNA-directed DNA methylation (RdDM) is mediated by ARGONAUTE 4 (AGO4) and 24 nt short-interfering RNAs (siRNAs) that are generated in parallel with 23 nt RNAs of unknown function. We show that 23 nt RNAs serve as the passenger strands of 23/24 nt duplexes loaded into AGO4. The 24 nt siRNAs then guide AGO4 slicing of the passenger strands, generating 11 and 12 nt cleavage products. Unexpectedly, we find that the 12 nt products remain associated with the guide strand-AGO4 complexes. Long noncoding RNAs generated at RdDM loci are similarly sliced and retained by AGO4. These results suggest a model in which RNA POLYMERASE V transcripts at target loci are sliced repeatedly as transcription elongation proceeds, sequentially releasing AGO4-siRNA-scaffold RNA complexes that independently recruit the RdDM machinery. Consistent with this hypothesis, plant lines expressing wild-type versus slicing-defective AGO4 show quantitative variation in cytosine methylation and siRNA levels within RdDM loci.

2023-02-17 | GSE201234 | GEO

AGO4 slicer activity reveals the passenger strand function of plant 23 nt siRNAs and the post-slicing retention of target RNAs mediating RNA-directed DNA methylation [smallRNA-seq]

2023-02-17 | GSE201233 | GEO

Small RNA-seq of ago4 null and slicing-defective mutant in Arabidopsis

Project description:To investigate the effect of AGO4 upon small RNA accumulation in Arabidopsis, we analyzed small RNA accumulation in ago4, ago4/ago6/ago9 triple mutant, stable transgenic lines expressing wild-type AGO4 and slicing-defective AGO4 in ago4 and ago4/ago6/ago9 background.

2017-05-27 | GSE87333 | GEO

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention.

Project description:RNA-directed DNA methylation in plants is guided by 24-nt siRNAs generated in parallel with 23-nt RNAs of unknown function. We show that 23-nt RNAs function as passenger strands during 24-nt siRNA incorporation into AGO4. The 23-nt RNAs are then sliced into 11- and 12-nt fragments, with 12-nt fragments remaining associated with AGO4. Slicing recapitulated with recombinant AGO4 and synthetic RNAs reveals that siRNAs of 21-24 nt, with any 5'-terminal nucleotide, can guide slicing, with sliced RNAs then retained by AGO4. In vivo, RdDM target locus RNAs that copurify with AGO4 also display a sequence signature of slicing. Comparing plants expressing slicing-competent versus slicing-defective AGO4 shows that slicing elevates cytosine methylation levels at virtually all RdDM loci. We propose that siRNA passenger strand elimination and AGO4 tethering to sliced target RNAs are distinct modes by which AGO4 slicing enhances RNA-directed DNA methylation.

| S-EPMC10069450 | biostudies-literature

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

AGO4 slicer activity reveals the passenger strand function of plant 23 nt siRNAs and the post-slicing retention of target RNAs mediating RNA-directed DNA methylation [WGBS]

Project description:AGO4 slicer activity reveals the passenger strand function of plant 23 nt siRNAs and the post-slicing retention of target RNAs mediating RNA-directed DNA methylation [WGBS]

| PRJNA830474 | ENA

AGO4 slicer activity reveals the passenger strand function of plant 23 nt siRNAs and the post-slicing retention of target RNAs mediating RNA-directed DNA methylation [smallRNA-seq]

Project description:AGO4 slicer activity reveals the passenger strand function of plant 23 nt siRNAs and the post-slicing retention of target RNAs mediating RNA-directed DNA methylation [smallRNA-seq]

| PRJNA830475 | ENA

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-02-15 | GSE201235 | GEO

In vivo PIWI slicing in mouse testes deviates from rules established in vitro

Project description:Purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that piRNA target sites with cleavage-site mismatches are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are identified by perfect complementarity base-pairing with the piRNA. Additionally, we find that such pachytene piRNA-guided MIWI/MILI slicing in vivo failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as a pre-piRNA and 17-19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis.

2023-10-23 | GSE219200 | 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), although their function is largely unknown. 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. This indicates co-transcriptional slicing of Pol V transcripts by AGO4-bound siRNAs. Pol V transcript slicing also required the elongation factor SPT5L. These results highlight a novel step in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

2018-01-29 | GSE100010 | GEO

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