Arabidopsis thaliana
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ABSTRACT:
PROVIDER: PRJNA261075 | ENA |
REPOSITORIES: ENA
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Project description:Detection of Pol IV-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis
| PRJNA245920 | ENA
Project description:24 nucleotide siRNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes DNA methylation at transposable elements to ensure genome stability. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is thought to transcribe RdDM loci to produce primary transcripts that serve as precursors to siRNAs. Yet, no such transcripts have ever been reported. Here, through RNA sequencing and double-stranded RNA sequencing in genotypes that compromise the dicing of siRNA precursors, we were able to identify Pol IV-dependent transcripts from tens of thousands of loci. We show that Pol IV-dependent transcripts correspond to both DNA strands, while the Pol II-dependent transcripts produced upon de-repression of the loci are derived from primarily one strand. We show that Pol IV-dependent transcripts have a 5’ monophosphate, lack a polyA tail at the 3’ end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Moreover, RDR2 is shown to play similar roles with Pol IV in both the abundance of siRNA precursors and siRNAs as well as the CHH DNA methylation. The decreased CHH methylation at dcl234 can inhibit the transcription of Pol IV at DRM2-target sites. Finally, the regulations of siRNA biogenesis were explored.
2014-11-11 | GSE57215 | GEO
Project description:Canonically, Arabidopsis RNA Polymerase IV (Pol IV) is known to produce heterochromatic small RNAs (typically 23-24 nucleotide in length) that maintain silencing of transposable elements (TEs). In contrast, when TEs are transcriptionally activated and produce Pol II transcripts, these transcripts are degraded into 21-22 nt small RNAs (sRNAs) known to participate in post transcriptional gene silencing. Recently, it was reported that Pol IV can also produce 21-22 nt sRNAs in pollen. We investigated the mechanism of Pol IV dependent 21-22 nt sRNAs and show that this mechanism is not specific to pollen. Additionally, we show that these 21-22 nt sRNAs can participate in RNA-directed DNA methylation, are incorporated into ARGONAUTE 1 (AGO1) and may guide AGO1 to cleave genic mRNAs to regulate their expression.
2020-02-24 | GSE133618 | GEO
Project description:In Arabidopsis thaliana, DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA (sRNA) transcripts. These transcripts are processed by DICER-LIKE3 into 24-nt small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nt epigenetically activated siRNAs (easiRNAs), which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernable pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV in the Brassicaceae species Capsella rubella, caused post-meiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were 2 depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nt and 24-nt siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella rubella compared to Arabidopsis thaliana, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.
2020-01-23 | GSE136847 | GEO
Project description:24 nucleotide siRNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes DNA methylation at transposable elements to ensure genome stability. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is thought to transcribe RdDM loci to produce primary transcripts that serve as precursors to siRNAs. Yet, no such transcripts have ever been reported. Here, through RNA sequencing and double-stranded RNA sequencing in genotypes that compromise the dicing of siRNA precursors, we were able to identify Pol IV-dependent transcripts from tens of thousands of loci. We show that Pol IV-dependent transcripts correspond to both DNA strands, while the Pol II-dependent transcripts produced upon de-repression of the loci are derived from primarily one strand. We show that Pol IV-dependent transcripts have a 5â monophosphate, lack a polyA tail at the 3â end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Moreover, RDR2 is shown to play similar roles with Pol IV in both the abundance of siRNA precursors and siRNAs as well as the CHH DNA methylation. The decreased CHH methylation at dcl234 can inhibit the transcription of Pol IV at DRM2-target sites. Finally, the regulations of siRNA biogenesis were explored. To detect siRNA precursors transcribed by RNA polymerase IV, the genome wide profiling of RNA were carried out at dcl234 and dcl234 nrpd1. Different types of RNA (including Total RNA, polyA+ RNA, polyA- RNA, double stranded RNA) libraries were built to detect different transcripts. RDR2 is a RNA-dependent RNA polymerase in Pol IV complex, so the RNA-seq libraries with the mutation of RDR2 were also built. In addition, smRNA libraries with mutations blocking siRNA biogenesis were also built
2014-11-11 | E-GEOD-57215 | biostudies-arrayexpress
Project description:Epigenetic and biochemical determinants of Pol IV-dependent genome surveillance in plants
| PRJNA510791 | ENA
Project description:RNA polymerases IV and V (Pol IV and Pol V) are plant-specific polymerases required for the generation of noncoding RNAs in RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Their subunit compositions largely resemble that of Pol II. However, the mechanism and accessory factors involved in their assembly remain largely unknown. In this study, we performed a forward genetic screen and identified novel mutant alleles of MINIYO (IYO), QUATRE-QUART 2 (QQT2) and NUCLEAR RNA POLYMERASE B/D/E 11 (NRPB/D/E11) that are defective in RdDM. We found that Pol IV-dependent small interfering RNAs (siRNAs) and Pol V-dependent transcripts were greatly reduced in the mutants. NRPE1, the largest subunit of Pol V, dissociated from other Pol V subunits in the iyo and qqt2 mutants, suggesting the involvement of IYO and QQT2 in Pol V assembly. Furthermore, we found that IYO and QQT2 were mutually dependent for their binding to the NRPE3 subassembly to facilitate the assembly of Pol V holoenzyme. Our findings reveal IYO and QQT2 as cofactors for Pol V assembly and provide mechanistic insights into how RNA polymerases are assembled in Arabidopsis.
2018-04-09 | GSE103326 | GEO
Project description:Epigenetic memory mediated by HDA6, Pol IV and Pol V in Arabidopsis.
| PRJNA210297 | ENA
Project description:Assessment of the Pol IV largest subunit, NRPD1, DeCL domain deletion construct to rescue Pol IV-dependent siRNAs in the nrpd1-3 mutant (sRNA).
| PRJNA378529 | ENA
Project description:In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SM-NM-^T35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SM-NM-^T35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SM-NM-^T35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter. Examination of whole-genome DNA methylation status in transgenic T+S Arabidopsis plant
2013-05-30 | E-GEOD-47453 | biostudies-arrayexpress