siRNA biogenesis, DNA methylation and target locus silencing are distinct and separable Pol IV sub-functions differentially dependent on the largest subunit's CTD

ABSTRACTPlant nuclear multisubunit RNA polymerase IV plays a key role in the RNA-directed DNA methylation (RdDM) pathway for transcriptional silencing of transposons, viruses and specific genes by synthesizing precursors of 24 nt siRNAs that guide the process. The Pol IV largest subunit, NRPD1 is derived from the Pol II largest subunit but has a unique carboxy-terminal domain (CTD) of unknown function. We show that the NRPD1 CTD is critical for transcriptional silencing of target loci and for producing 24 nt siRNAs at high levels. However, the CTD is surprisingly dispensable for near wild-type levels of Pol IV-dependent genomic cytosine methylation. These results suggest that low levels of 24 nt siRNAs, produced at only 20-30% of wild-type levels, are sufficient for full RNA-directed DNA methylation, yet insufficient for silencing, suggesting additional roles for siRNAs beyond DNA methylation. Moreover, at a subset of target loci, neither siRNA levels nor cytosine methylation are impaired upon deletion of the CTD, yet silencing is lost. Collectively, the non-linear relationships between siRNA levels, cytosine methylation and silencing suggest the existence of additional mechanisms of silencing dependent on Pol IV transcription and mediated by the CTD, such as promoter occlusion to inhibit the activities of other polymerases.

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

eLife ◽

10.7554/elife.09591 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 120

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.

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The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation

Nucleic Acids Research ◽

10.1093/nar/gkz615 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9024-9036 ◽

Cited By ~ 4

Author(s):

Jered M Wendte ◽

Jeremy R Haag ◽

Olga M Pontes ◽

Jasleen Singh ◽

Sara Metcalf ◽

...

Keyword(s):

Dna Methylation ◽

Catalytic Activity ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cytosine Methylation ◽

Transcriptional Gene Silencing ◽

Rna Dependent Rna Polymerase ◽

Pol Ii ◽

Pol Iv ◽

Non Coding Rnas

Abstract In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

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

10.1101/679795 ◽

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.

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

10.1101/2021.09.06.459145 ◽

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.

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RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays

PLoS Genetics ◽

10.1371/journal.pgen.1009326 ◽

2021 ◽

Vol 17 (6) ◽

pp. e1009326

Author(s):

Wei Guo ◽

Dafang Wang ◽

Damon Lisch

Keyword(s):

Dna Methylation ◽

Transposable Elements ◽

Histone H3 ◽

Transcriptional Silencing ◽

Epigenetic Silencing ◽

Terminal Inverted Repeat ◽

Wild Type ◽

Rna Dependent Rna Polymerase ◽

Plant Genomes ◽

Transposon Silencing

In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Mediator of Paramutation 1 (Mop1), a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.

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

Journal of Virology ◽

10.1128/jvi.00656-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7529-7540 ◽

Cited By ~ 26

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.

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The CCR4‐NOT complex component NOT1 regulates RNA‐directed DNA methylation and transcriptional silencing by facilitating Pol IV‐dependent siRNA production

The Plant Journal ◽

10.1111/tpj.14818 ◽

2020 ◽

Vol 103 (4) ◽

pp. 1503-1515

Author(s):

Hao‐Ran Zhou ◽

Rong‐Nan Lin ◽

Huan‐Wei Huang ◽

Lin Li ◽

Tao Cai ◽

...

Keyword(s):

Dna Methylation ◽

Transcriptional Silencing ◽

Complex Component ◽

Pol Iv

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

10.1101/2021.03.24.436774 ◽

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.

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RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.

10.1101/2021.01.08.425849 ◽

2021 ◽

Author(s):

Damon Lisch ◽

Wei Guo ◽

Dafang Wang

Keyword(s):

Dna Methylation ◽

Heat Stress ◽

Transposable Elements ◽

Transcriptional Silencing ◽

Epigenetic Silencing ◽

Terminal Inverted Repeat ◽

Wild Type ◽

Rna Dependent Rna Polymerase ◽

Plant Genomes ◽

Transposon Silencing

In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Modifer of Paramutation 1 (Mop1 ), a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via H3K9 and H3K27 dimethylation, even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by H3K29 trimethylation, a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.

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

10.1101/832634 ◽

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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