Targeted suppression of siRNA biogenesis in Arabidopsis pollen [small RNA-seq]
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE231668 | GEO | 2023/05/07
REPOSITORIES: GEO
Similar Datasets
Project description:We used the RNaseIII-like protein RTL1 to suppress siRNA biogenesis specifically in Arabidopsis pollen, and found distinct siRNA subsets produced in the sperm and vegetative cell lineages via the RNA polymerase IV (Pol IV).
2023-05-07 | GSE231669 | GEO
Project description:We used the RNaseIII-like protein RTL1 to suppress siRNA biogenesis specifically in Arabidopsis pollen, and found distinct siRNA subsets produced in the sperm and vegetative cell lineages via the RNA polymerase IV (Pol IV).
2023-05-07 | GSE231667 | GEO
Project description:Small RNAs play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode DICER-LIKE (DCL) RNaseIII enzymes, including DCL1, which produces miRNAs, and DCL2, DCL3 and DCL4, which produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Small RNA sequencing in plants over-expressing RTL1 or RTL2 or lacking RTL2 revealed that RTL1 over-expression inhibits the accumulation of all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. By contrast, RTL2 plays minor, if any, role in the small RNA repertoire. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by degrading dsRNA before they are processed by DCL2, DCL3 and DCL4. The substrate of RTL1 cleavage is likely long perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from short imperfect dsRNA. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. These results reveal another level of complexity in the evolutionary battle between viruses and plant defenses. Flower sRNA profiles in diverse conditions involving RTL1 and RTL2
2013-09-10 | E-GEOD-49866 | biostudies-arrayexpress
Project description:Small RNAs play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode DICER-LIKE (DCL) RNaseIII enzymes, including DCL1, which produces miRNAs, and DCL2, DCL3 and DCL4, which produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Small RNA sequencing in plants over-expressing RTL1 or RTL2 or lacking RTL2 revealed that RTL1 over-expression inhibits the accumulation of all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. By contrast, RTL2 plays minor, if any, role in the small RNA repertoire. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by degrading dsRNA before they are processed by DCL2, DCL3 and DCL4. The substrate of RTL1 cleavage is likely long perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from short imperfect dsRNA. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. These results reveal another level of complexity in the evolutionary battle between viruses and plant defenses.
2013-09-10 | GSE49866 | GEO
Project description:The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that would transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with down-regulation of the heterochromatin remodeler DECREASE IN DNA METHYLATION 1 and of most TE siRNAs. However, 21 nucleotide siRNA from Athila retrotransposons is generated in pollen and accumulates in sperm, indicating that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes.
2014-09-04 | GSE61028 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2023-05-07 | GSE231670 | GEO
Project description:The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that would transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with down-regulation of the heterochromatin remodeler DECREASE IN DNA METHYLATION 1 and of most TE siRNAs. However, 21 nucleotide siRNA from Athila retrotransposons is generated in pollen and accumulates in sperm, indicating that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes. Mature pollen was collected from Columbia reference strain plants by vacuum filtration (Johnson-Brousseau and McCormick, 2004). DNA and RNA were isolated from a ddm1-2 plant in the Columbia reference background. Small RNAs of 19–28 nt were size selected by denaturing 15% PAGE, and cloned as in Brennecke et al. (2007). Additional details regarding the cloning of small RNAs are found in the Supplemental Data. The small RNA libraries were sequenced on Illumina 1G sequencer. The total number of sequences perfectly matching the Arabidopsis genome were as follows: WT inflorescence, 4,158,848 (2,286,133 unique); WT pollen, 1,034,665 (437,984 unique); ddm1 inflorescence, 4,098,772 (1,637,771 unique); and WT sperm, 760,651 (429,972 unique).
2014-09-04 | E-GEOD-61028 | biostudies-arrayexpress
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:To assay siRNA movement in the pollen grain, we took advantage of the known behavior of 22nt siRNAs to target transcripts for cleavage and initiate secondary siRNA biogenesis. Previous research has demonstrated that when targeted by a 22nt microRNA, a transcript containing a truncated non-fluorescent ~400 bp version of GFP (trGFP) will produce GFP secondary siRNAs that can target a second copy of a functional full-length GFP mRNA in trans. Therefore, we created a ~400 bp non-fluorescent version of trGFP with a 5’ 22nt microRNA target site driven by the vegetative cell-specific KRP6 promoter and transformed this construct into a line homozygous for the sperm-specific functional full-length GFP driven by the MGH3 promoter. If cleaved by a 22nt microRNA, the vegetative cell-driven trGFP will be cleaved into secondary siRNAs, and if the RNA components of this system are mobile and transferred into the sperm cells, the sperm-specific fluorescence of GFP will be reduced. We used two versions of the microRNA target site, which included a mock target site that is not targeted by small RNAs, the target site of microRNA173 (which is a 22nt known to induce the production of secondary siRNAs). We demonstrate that when the 22nt microRNA173 target site is used in the vegetative cell transcript, a corresponding decrease in sperm cell fluorescence is observed. In the mock small RNA target site this reduction in fluorescence was not observed. To ensure the transitive silencing sperm cell transcripts is occurring via secondary small RNAs, we transferred this trGFP experiment into a dcl2/dcl4 mutant background that is defective in TE 21-22nt siRNA silencing. In dcl2/dcl4 double mutants with the trGFP system, we find that the GFP fluorescence of the mock target site transgene does not change, while the repression found in wild-type pollen with the 22nt microRNA173 target site is alleviated. The data for microRNA173 is particularly important, as DCL2 and DCL4 are not necessary for microRNA173 production, and therefore this demonstrates that specifically secondary siRNAs acting downstream of microRNA action are required to silence the sperm cell GFP. We further analyzed the production of sRNAs derived from GFP in pollen grains of transgenic lines expressing the 22nt miR173 or 22nt mock target sites inserted on the 5’ region of the trGFP transgene in both wt and dcl2/dcl4 backgrounds.
2016-03-24 | GSE68154 | 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 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