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A genetics screen uncovers the role of RNA-binding protein RBP45D in flowering time under heat and DCL3-independent RNA-directed DNA methylation (WGBS)

ABSTRACT: RNA-directed DNA methylation (RdDM) is a prominent pathway in plants to defend against invasive nucleic acids. In the canonical RdDM, 24-nt small interfering RNAs (siRNAs) are produced through DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system in which transcriptional gene silencing (TGS) is independent of DLC3. In a forward genetics screen performed with this system, already known components of RdDM and RNA-binding protein RBP45D were identified. RBP45D promotes DNA methylation, and a lack of RBP45D causes late flowering especially under heat, presumably mediated by elevated FLC levels. RBP45D is localized to the nucleus where it is associated with snRNAs and snoRNAs. RBP45D maintains siRNA production originating from the transgene, but does not promote mRNA levels or influence pre-mRNA splicing of known RdDM genes. We hypothesize that RBPD45 facilitates DCL3-independent siRNA production through stabilisation of either the precursor RNA or the – not yet identified – slicer protein.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE178678 | GEO | 2022/05/13

REPOSITORIES: GEO

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A genetics screen uncovers the role of RNA-binding protein RBP45D in flowering time under heat and DCL3-independent RNA-directed DNA methylation

Project description:In the canonical RdDM, small interfering RNAs (siRNAs) are produced through the slicer activity of DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system in which transcriptional gene silencing (TGS) through RdDM is independent of DLC3. To identify players in the DCL3-independent pathway, a forward genetics screen was performed with the prors1 (LUC) system to identify suppressor mutants in which TGS is released. Among already known components of RdDM, we identified RNA-binding protein RBP45D. RBP45D promotes DNA methylation changes, and a lack of RBP45D results in a late flowering phenotype especially under heat, presumably mediated by elevated FLC levels. A lack of RBP45D did not influence the pre-mRNA splicing of known RdDM genes, suggesting that RBP45D involvement in the RdDM might be direct. RBP45D is localized to the nucleus where it is associated with snRNAs and snoRNAs. RBP45D promotes siRNA production originating from the transgene and we hypothesize that this function is direct and not indirect.

2022-05-13 | GSE176315 | GEO

IBM1, a JmjC domain histone demethylase, is involved in the regulation of RNA-directed DNA methylation through epigenetic control of RDR2 and DCL3 expression in Arabidopsis.

Project description:The small RNA mediated DNA methylation (RdDM) is highly elaborated in Arabidopsis as an important epigenetic pathway controls expression of genes and multiple developmental processes. RDR2 and DCL3 are necessary factors to the 24-nt siRNA biogenesis and the RdDM pathway. Here, we find that IBM1 (increase in BONSAI methylation 1), a conserved JmjC family histone demethylase, could directly associated with the chromatin of RDR2 and DCL3. The IBM1 mutation induces hypermethylation on both H3K9 and DNA non-CG sites in these loci, and further represses their expression. The reduction of RDR2 and DCL3 may affect siRNA biogenesis in a locus-specific manner and disrupt the RdDM directed repression. It suggests an indirect way to regulate targets by IBM1 through control of siRNA biogenesis, and demonstrate the relationship between histone methylation and small RNAs.

2012-08-03 | GSE32284 | GEO

IBM1, a JmjC domain histone demethylase, is involved in the regulation of RNA-directed DNA methylation through epigenetic control of RDR2 and DCL3 expression in Arabidopsis.

2012-08-03 | E-GEOD-32284 | biostudies-arrayexpress

RNA-seq analysis of parental (wild type) and dcl3 mutant lines of Chlamydomonas reinhardtii

Project description:DCL3 is the main processor of miRNA precursors in Chlamydomonas reinhardtii. Because of that, and in order identify mRNA targets of miRNA-guided slicer complexes, we used RNAseq of the transcriptome in dcl3 mutant and parental lines.

2016-08-28 | E-MTAB-3852 | biostudies-arrayexpress

flagellin experiment in dcl2-dcl3-dcl4 mutant background-Gene profiling upon biotic stress treatment.

Project description:rs06-06_mirna - flagellin experiment in dcl2-dcl3-dcl4 mutant background - Time course : What are the genes (incuding microRNA precursors) that are differentially regulated upon flg22 treatment? miRNA : What are the genes (including miRNA precursors) that are differentially regulated in a set of microRNA mutants? siRNA : What are the genes (including miRNA precursors) that are differentially regulated in a set of siRNA mutants? - What are the genes (including primary miRNA transcripts) that are differentially regulated upon flg22 treatment? 14-day old seedlings (Col-0 and dcl2-dcl3-dcl4) treated with flg22 (flagellin- derived peptide). Keywords: treated vs untreated comparison

2008-06-02 | GSE10354 | GEO

Generation and characterization of a tomato DCL3 silencing mutant

Project description:DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide siRNA and long miRNA biogenesis and mutants have been characterized from Arabidopsis and rice. Here, a tomato DCL3 mutant was generated through the use of trans-activated artificial microRNA and characterized. Global tomato DCL3 (SlDCL3) silencing was induced by crossing the generated responder line (OP:amiRDCL3) with constitutive driver line. Constitutive trans-activation knocked down SlDCL3 levels by ~77%, resulting in dramatically decreased 24-nucleotide small RNA levels, but a significant increase in 21- and 22- nucleotide small RNAs, which was correlated with specific upregulation of SlDCL4 and SlDCL2b. Moreover, in the majority of small RNA-generating loci an almost complete overlap between the control and 35S>>amiRSlDCL3 siRNA signatures, was observed, strongly suggesting that the reduction in 24-nucleotide siRNAs was compensated by increased biogenesis of 21-22 nt siRNAs from the same genomic sequences. Collectively, these results suggest the requirement of SlDCL3 for the biogenesis of 23-24 nt sRNAs and its substitution by SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt small RNAs. In addition, differential expression between control and SlDCL3-silenced small RNAs indicated a significant reduction in the abundance of 24-nt putative long miRNAs, which was validated by northern blots, implicating SlDCL3 in their biogenesis.

2014-03-31 | GSE52265 | GEO

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.

2014-08-05 | GSE47852 | GEO

flagellin experiment in dcl2-dcl3-dcl4 mutant background-Gene profiling upon biotic stress treatment.

2008-06-01 | E-GEOD-10354 | biostudies-arrayexpress

High-throughput sequencing of Pol IV and RDR2 transcripts, and their DCL3-diced products, generated from an M13mp18 (+) strand DNA template

Project description:In plants, the biogenesis of 24 nt and 23 nt small interfering RNAs (siRNAs) requires NUCLEAR RNA POLYMERASE IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). We show that single-stranded M13 bacteriophage DNA can be used as a template for siRNA synthesis in vitro. Deep sequencing of RNAs produced from the in vitro reactions of Pol IV, RDR2 and DCL3 shows that Pol IV transcribes the DNA into first-strand RNAs which RDR2 then uses as templates to synthesize complementary second strands. These siRNA precursor transcripts made by Pol IV and RDR2 are mostly 30-50 nt. An untemplated 3' terminal nucleotide is a characteristic of RDR2 transcripts. DCL3 dicing of double-stranded precursor RNAs synthesized by Pol IV and RDR2 generates siRNAs that are mostly 24 nt, with a smaller population of 23 nt also produced.

2019-07-01 | GSE126086 | GEO

Generation and characterization of a tomato DCL3 silencing mutant

2014-03-31 | E-GEOD-52265 | biostudies-arrayexpress

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