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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

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

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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.

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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.

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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.

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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

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Generation and characterization of a tomato DCL3 silencing mutant

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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.

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

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

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2019-07-01 | GSE126086 | GEO

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