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DNA Methylation Mediated by a MicroRNA Pathway

ABSTRACT: In plants, the known microRNAs (miRNAs) are produced as ~21 nucleotide (nt) duplexes from their precursors by Dicer like 1 (DCL1). They are incorporated into Argonaute 1 (AGO1) protein to regulate target gene expression primarily through mRNA cleavage. We report here the discovery of a new class of miRNAs in the model monocot rice (Oryza sativa). These are 24 nt in length and require another member of the Dicer family, DCL3, for their biogenesis. The 24 nt long miRNAs (lmiRNAs) are loaded into AGO4 clade proteins according to hierarchical rules, depending on the upstream biogenesis machinery and the 5’ terminal nucleotide. We demonstrated that lmiRNAs direct DNA methylation at loci from which they are produced as well as in trans at their target genes and play roles in gene regulation. Considered together, our findings define a novel miRNA pathway that mediates DNA methylation.

ORGANISM(S): Oryza sativa

PROVIDER: GSE20748 | GEO | 2010/03/12

SECONDARY ACCESSION(S): PRJNA124993

REPOSITORIES: GEO

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DNA Methylation Mediated by a MicroRNA Pathway

Project description:In plants, the known microRNAs (miRNAs) are produced as ~21 nucleotide (nt) duplexes from their precursors by Dicer like 1 (DCL1). They are incorporated into Argonaute 1 (AGO1) protein to regulate target gene expression primarily through mRNA cleavage. We report here the discovery of a new class of miRNAs in the model monocot rice (Oryza sativa). These are 24 nt in length and require another member of the Dicer family, DCL3, for their biogenesis. The 24 nt long miRNAs (lmiRNAs) are loaded into AGO4 clade proteins according to hierarchical rules, depending on the upstream biogenesis machinery and the 5’ terminal nucleotide. We demonstrated that lmiRNAs direct DNA methylation at loci from which they are produced as well as in trans at their target genes and play roles in gene regulation. Considered together, our findings define a novel miRNA pathway that mediates DNA methylation. Small RNAs were prepared from Rice total extract in wide type, dcl1, dcl3, rdr2 dbsRNA mutant and AGO4a, AGO4b, and AGO16 complexes, ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of solexa cloning primers and then provided for sequencing. For technical details, see Wu, L., Zhang, Q., Zhou, H., Ni, F., Wu, X., and Qi, Y. (2009). Rice microRNA effector complexes and targets. The Plant Cell, 21: 3421-3435.

2010-03-12 | E-GEOD-20748 | biostudies-arrayexpress

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

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

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

Generation and characterization of a tomato DCL3 silencing mutant

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

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

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

Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation

Project description:In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are central to sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nt small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation (RdDM) pathway. Here, we determined structure of a DCL3-pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated-A1 of the guide strand and the 1-nt 3′-overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated-end of the guide RNA and dual cleavage sites formed by the paired RNaseIII domains. These structural studies, complemented by functional data, reveal insights into the dicing principle for Dicers in general.

2021-10-14 | GSE179616 | GEO

Small RNAs in monocots

Project description:In monocots other than the cereals maize and rice, the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs (sRNAs) from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, siRNAs and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of tRNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th and 3’ end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nt reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE 5 (DCL5), important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs is the largest collection to date, and should facilitate continued exploration of small RNA diversification in flowering plants.

2020-04-17 | GSE148762 | GEO

Arabidopsis AGO3 predominantly recruits 24-nucleotide small RNAs to regulate epigenetic silencing [miRNA-seq]

Project description:AGO3 predominantly bound 24-nt sRNAs with 5’ terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4 and over 60% of AGO3-associated 24-nt sRNA enriched loci were identical to those of AGO4. In addition, expression of AGO3 driven by AGO4 native promoter partially complemented AGO4 function and rescued DNA methylation defect in ago4-1 background.

2016-05-31 | GSE78088 | GEO

Arabidopsis AGO3 predominantly recruits 24-nucleotide small RNAs to regulate epigenetic silencing [Bisulfite-seq]

2016-05-31 | GSE78089 | GEO

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