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

ABSTRACT: 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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE100010 | GEO | 2018/01/29

REPOSITORIES: GEO

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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: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

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. Examination of siRNA abundance in the trasngenic wild type plant (contains trigger and silencer transgenes) and the ago6-4 mutant.

2011-12-01 | E-GEOD-28537 | biostudies-arrayexpress

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

2011-12-01 | GSE28537 | GEO

A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis

Project description:DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. Twenty-four-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ~20 to 60nt in length, often having same 5' ends but differing in 3' ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on AGO4 and distributive 3'-5' exonucleases. Our findings suggest an alternative route for siRNA biogenesis: precursor transcripts are bound by AGO4 and subsequently subjected to 3'-5' exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.

2015-12-17 | GSE74398 | GEO

RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes

Project description:Transcriptional gene silencing controls transposons and other repetitive elements through RNA-directed DNA methylation (RdDM) and heterochromatin formation. A key component of the Arabidopsis RdDM pathway is ARGONAUTE4 (AGO4), which associates with sizs to mediate DNA methylation. Here, we show that AGO4 preferentially targets transposable elements embedded within promoters of protein-coding genes. This pattern of AGO4 binding cannot be simply explained by the sequences of AGO4-bound siRNAs; instead, AGO4 binding to specific gene promoters is also mediated by long non-coding RNAs (lncRNAs) produced by RNA polymerase V. lncRNA-mediated AGO4 binding to gene promoters directs asymmetric DNA methylation to these genomic regions and is involved in regulating the expression of targeted genes. Finally, AGO4 binding overlaps sites of DNA methylation affected by the biotic stress response. Based on these findings, we propose that the targets of AGO4-directed RdDM are regulatory units responsible for controlling gene expression under specific environmental conditions. ChIP-seq experiments using AGO4 antibody of WT (Col-0) and ago4 or nrpe1 mutant plants of Arabidopsis thaliana

2012-10-17 | E-GEOD-35381 | biostudies-arrayexpress

A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis

2015-12-17 | E-GEOD-74398 | biostudies-arrayexpress

RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes

2012-10-17 | GSE35381 | GEO

Analysis of conjugation-specific 26-32nt siRNAs of Tetrahymena thermophila

Project description:Distinct classes of small RNAs are often selectively sorted to different Argonaute proteins. Various properties of small RNAs, such as length, terminal nucleotide, thermodynamic asymmetry and duplex mismatches, can impact sorting in different RNA silencing pathways in diverse eukaryotes. The developmentally regulated ~26-32 nt siRNAs, which are involved in programmed DNA elimination in Tetrahymena, show a strong bias for uracil at the 5' end. In this study, we analyzed loaded and unloaded populations of ~26-32 nt siRNAs by deep RNA sequencing. We show that the production process is the main determinant of size, whereas the 5' uracil bias is attributed not only to the process of loading siRNAs into the Argonaute protein Twi1p but also significantly to the initial processing of the siRNAs. We also show that both the loaded and the unloaded ~26-32 nt siRNAs have a strong bias for adenine as the 3rd base from the 3' terminus, suggesting that most of these siRNAs are direct Dicer products and little post-processing amplification of this class of siRNAs occurs. Further, we demonstrate that the siRNA-loading process in vivo can be deduced from the fraction of siRNAs with uracil as the first base. These findings provide biochemical bases for the attributes of ~26-32 nt siRNAs, which should help improve our understanding of their production and turnover in vivo.

2012-10-01 | GSE37696 | GEO

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