scholarly journals Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block

2020 ◽  
Author(s):  
Zhenxing Wang ◽  
Nicolas Butel ◽  
Juan Santos-González ◽  
Lauriane Simon ◽  
Cecilia Wärdig ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Early Stage ◽  
Suppressive Effect ◽  
Transgenerational Effect ◽  
Pol Iv ◽  
Short Rnas ◽  
Methylation Pathway ◽  
Rna Polymerase Iv ◽  
Triploid Block

AbstractHybridization of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. Mutation in NRPD1, encoding the largest subunit of the plant-specific RNA Polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates short RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. In this study, we found that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While nrpd1 is able to suppress in the first homozygous generation, mutants in RDR2, NRPE1, and DRM2 require at least one additional round of inbreeding to exert a suppressive effect. Inbreeding of nrpd1 was connected with a transgenerational loss of non-CG DNA methylation on sites jointly regulated by CHROMOMETHYLASES 2 and 3. Our data thus reveal that loss of RdDM function differs in its effect in early and late generations and that Pol IV acts at an early stage of triploid block establishment.One-sentence summaryInbreeding of mutants impaired in RdDM components transgenerationally enhanced their ability to suppress the triploid block.

scholarly journals Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

2019 ◽  
Author(s):  
Jasleen Singh ◽  
Vibhor Mishra ◽  
Feng Wang ◽  
Hsiao-Yun Huang ◽  
Craig S. Pikaard
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Dna Transcription ◽  
Pol Iv ◽  
Methylation Pathway ◽  
Terminal Nucleotide ◽  
Passenger Strand ◽  
Rna Polymerase Iv ◽  
Rna Pathways ◽  
Template Dna

SummaryIn eukaryotes with multiple small RNA pathways the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the siRNA biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV) whose atypical termination mechanism, induced by nontemplate DNA basepairing, channels transcripts to the associated RNA-dependent RNA polymerase, RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs then typically adds an extra untemplated 3’ terminal nucleotide to the second strands. The dicer endonuclease, DCL3 cuts resulting duplexes to generate 24 and 23nt siRNAs. The 23nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling and RNA channeling from template DNA transcription to siRNA guide strand/passenger strand discrimination.

scholarly journals Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Todd Blevins ◽  
Ram Podicheti ◽  
Vibhor Mishra ◽  
Michelle Marasco ◽  
Jing Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Transferase Activity ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

scholarly journals Reaction Mechanisms of Pol IV, RDR2, and DCL3 Drive RNA Channeling in the siRNA-Directed DNA Methylation Pathway

2019 ◽  
Vol 75 (3) ◽  
pp. 576-589.e5 ◽  
Author(s):  
Jasleen Singh ◽  
Vibhor Mishra ◽  
Feng Wang ◽  
Hsiao-Yun Huang ◽  
Craig S. Pikaard
Keyword(s):  
Dna Methylation ◽  
Reaction Mechanisms ◽  
Pol Iv ◽  
Methylation Pathway

scholarly journals A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation

2021 ◽  
Author(s):  
Andrew Loffer ◽  
Jasleen Singh ◽  
Akihito Fukudome ◽  
Vibhor Mishra ◽  
Feng Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Transferase Activity ◽  
Rna Dependent Rna Polymerase ◽  
Dna Viruses ◽  
Selfish Genetic Elements ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In plants, selfish genetic elements including retrotransposons and DNA viruses are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of ~30 bp synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV initiating nucleotide choice, RDR2 initiation 1-2 nt internal to Pol IV transcript ends and RDR2 terminal transferase activity collectively yield a code that influences which end of the precursor is diced and whether 24 or 23 nt siRNAs are generated from the Pol IV or RDR2-transcribed strands. By diversifying the size, sequence, and strand polarity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow maximal siRNA coverage at methylated target loci.

scholarly journals Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin

2016 ◽  
Vol 90 (16) ◽  
pp. 7529-7540 ◽  
Author(s):  
Jamie N. Jackel ◽  
Jessica M. Storer ◽  
Tami Coursey ◽  
David M. Bisaro
Keyword(s):  
Dna Methylation ◽  
Small Rnas ◽  
Cytosine Methylation ◽  
De Novo ◽  
Rna Polymerases ◽  
Epigenetic Marks ◽  
Content Type ◽  
Pol Iv ◽  
Pol V ◽  
Virus Genomes

ABSTRACTIn plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirusBeet curly top virus(BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formedde novoin infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to triggerde novodeposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin.IMPORTANCEIn plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible.

scholarly journals RNA Pol IV has antagonistic parent-of-origin effects on Arabidopsis endosperm

2021 ◽  
Author(s):  
P.R. V. Satyaki ◽  
Mary Gehring
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Regulatory System ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Gene Regulatory System ◽  
Rna Polymerase Iv ◽  
Parent Of Origin ◽  
Origin Effects ◽  
Maternal Resources

Gene expression in endosperm, a seed tissue that mediates transfer of maternal resources to offspring, is under complex epigenetic control. We show here that plant-specific RNA Polymerase IV mediates parental control of endosperm gene expression. Pol IV is required for the production of small interfering RNAs that typically direct DNA methylation. We compared small RNAs, DNA methylation, and mRNAs in A. thaliana endosperm from reciprocal heterozygotes produced by crossing wildtype plants to Pol IV mutants. We find that maternally and paternally acting Pol IV have divergent effects on endosperm with loss of maternal and paternal Pol IV impacting sRNAs and DNA methylation at different genomic sites. Strikingly, maternally and paternally-acting Pol IV have antagonistic impacts on gene expression at some loci, divergently promoting or repressing endosperm gene expression. Antagonistic parent-of13 origin effects have only rarely been described and are consistent with a gene regulatory system evolving under parental conflict.

scholarly journals Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

2015 ◽  
Vol 112 (3) ◽  
pp. 917-922 ◽  
Author(s):  
Donna M. Bond ◽  
David C. Baulcombe
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Gene Silencing ◽  
De Novo ◽  
Feedback Mechanism ◽  
Epigenetic Silencing ◽  
Virus Induced Gene Silencing ◽  
Epigenetic Modifiers ◽  
Rna Polymerase Iv ◽  
Genetic Mutants

In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active FLOWERING WAGENINGEN epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM.

scholarly journals A Null Allele of the Pol IV Second Subunit is Viable in Oryza sativa

2021 ◽  
Author(s):  
Tania Chakraborty ◽  
Joshua T Trujillo ◽  
Timmy Kendall ◽  
Rebecca A Mosher
Keyword(s):  
Dna Methylation ◽  
Small Rna ◽  
Null Allele ◽  
De Novo ◽  
Land Plants ◽  
Pol Ii ◽  
Pol Iv ◽  
Pol V ◽  
Grass Family ◽  
Adult Plants

All eukaryotes possess three DNA-dependent RNA polymerases, Pols I-III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24-nt siRNAs that interact with Pol V transcripts to target de novo DNA methylation and silence transcription of transposons. Members of the grass family encode additional duplicated subunits of Pol IV and V, raising questions regarding the function of each paralog. In this study, we identify a null allele of the putative Pol IV second subunit, NRPD2, and demonstrate that NRPD2 is the sole subunit functioning with NRPD1 in small RNA production and CHH methylation in leaves. Homozygous nrpd2 mutants have neither gametophytic defects, nor embryo lethality, although adult plants are dwarf and sterile.

scholarly journals Arabidopsis RNA Polymerase IV generates 21-22 nucleotide small RNAs that can participate in RNA-directed DNA methylation and may regulate genes

2019 ◽  
Author(s):  
Kaushik Panda ◽  
Andrea D. McCue ◽  
R. Keith Slotkin
Keyword(s):  
Dna Methylation ◽  
Transposable Elements ◽  
Rna Polymerase ◽  
Transcript Abundance ◽  
Gene Transcript ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Rna Polymerase Iv ◽  
Plant Life Cycle ◽  
And Function

AbstractThe plant-specific RNA Polymerase IV (Pol IV) transcribes heterochromatic regions, including many transposable elements, with the well-described role of generating 24 nucleotide (nt) small interfering RNAs (siRNAs). These siRNAs target DNA methylation back to transposable elements to reinforce the boundary between heterochromatin and euchromatin. In the male gametophytic phase of the plant life cycle, pollen, Pol IV switches to generating primarily 21-22 nt siRNAs, but the biogenesis and function of these siRNAs has been enigmatic. In contrast to being pollen-specific, we identified that Pol IV generates these 21-22 nt siRNAs in sporophytic tissues, likely from the same transcripts that are processed into the more abundant 24 nt siRNAs. The 21-22 nt forms are specifically generated by the combined activities of DICER proteins DCL2/DCL4 and can participate in RNA-directed DNA methylation. These 21-22 nt siRNAs are also loaded into ARGONAUTE1, which is known to function in post-transcriptional regulation. Like other plant siRNAs and microRNAs incorporated into AGO1, we find a signature of genic mRNA cleavage at the predicted target site of these siRNAs, suggesting that Pol IV-generated 21-22 nt siRNAs may function to regulate gene transcript abundance. Our data provides support for the existing model that in pollen Pol IV functions in gene regulation.

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