scholarly journals An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation

Nature Plants ◽  
2021 ◽  
Author(s):  
Meredith J. Sigman ◽  
Kaushik Panda ◽  
Rachel Kirchner ◽  
Lauren L. McLain ◽  
Hayden Payne ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Argonaute Protein ◽  
Unanswered Question ◽  
Small Interfering Rnas ◽  
Pol V ◽  
Target Locus ◽  
Target Sites ◽  
Foreign Dna ◽  
Epigenetic Repression

AbstractIn mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

scholarly journals An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

2021 ◽  
Author(s):  
Meredith J Sigman ◽  
Kaushik Panda ◽  
Rachel Kirchner ◽  
Lauren L McLain ◽  
Hayden Payne ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Argonaute Protein ◽  
Unanswered Question ◽  
Small Interfering Rnas ◽  
Pol V ◽  
Target Locus ◽  
Target Sites ◽  
Foreign Dna ◽  
Epigenetic Repression

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

scholarly journals The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice

2021 ◽  
Vol 118 (30) ◽  
pp. e2100709118
Author(s):  
Kezhi Zheng ◽  
Lili Wang ◽  
Longjun Zeng ◽  
Dachao Xu ◽  
Zhongxin Guo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Histone Modifications ◽  
Noncoding Rna ◽  
De Novo ◽  
Noncoding Rnas ◽  
Reproductive Development ◽  
Small Interfering Rnas ◽  
Pol V ◽  
Specific Manner

RNA-directed DNA methylation (RdDM) functions in de novo methylation in CG, CHG, and CHH contexts. Here, we performed map-based cloning of OsNRPE1, which encodes the largest subunit of RNA polymerase V (Pol V), a key regulator of gene silencing and reproductive development in rice. We found that rice Pol V is required for CHH methylation on RdDM loci by transcribing long noncoding RNAs. Pol V influences the accumulation of 24-nucleotide small interfering RNAs (24-nt siRNAs) in a locus-specific manner. Biosynthesis of 24-nt siRNAs on loci with high CHH methylation levels and low CG and CHG methylation levels tends to depend on Pol V. In contrast, low methylation levels in the CHH context and high methylation levels in CG and CHG contexts predisposes 24-nt siRNA accumulation to be independent of Pol V. H3K9me1 and H3K9me2 tend to be enriched on Pol V–independent 24-nt siRNA loci, whereas various active histone modifications are enriched on Pol V–dependent 24-nt siRNA loci. DNA methylation is required for 24-nt siRNAs biosynthesis on Pol V–dependent loci but not on Pol V–independent loci. Our results reveal the function of rice Pol V for long noncoding RNA production, DNA methylation, 24-nt siRNA accumulation, and reproductive development.

scholarly journals The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis

2019 ◽  
Vol 47 (17) ◽  
pp. 9037-9052 ◽  
Author(s):  
Laura Ferrafiat ◽  
David Pflieger ◽  
Jasleen Singh ◽  
Michael Thieme ◽  
Marcel Böhrer ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Surveillance Systems ◽  
Missense Mutations ◽  
Small Interfering Rnas ◽  
Mutant Genotype ◽  
Pol Iv ◽  
N Terminus

Abstract RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV’s largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.

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.

scholarly journals DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

2015 ◽  
Vol 112 (3) ◽  
pp. 911-916 ◽  
Author(s):  
Xuehua Zhong ◽  
Christopher J. Hale ◽  
Minh Nguyen ◽  
Israel Ausin ◽  
Martin Groth ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Noncoding Rna ◽  
Dna Methyltransferase ◽  
Transcript Abundance ◽  
Transcriptional Elongation ◽  
Rna Transcripts ◽  
Pol V ◽  
Genome Defense ◽  
Eukaryotic Organisms

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation.

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

2020 ◽  
Vol 375 (1795) ◽  
pp. 20190417 ◽  
Author(s):  
Kaushik Panda ◽  
Andrea D. McCue ◽  
R. Keith Slotkin
Keyword(s):  
Dna Methylation ◽  
Gene Regulation ◽  
Rna Polymerase ◽  
Transcript Abundance ◽  
Gene Transcript ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Rna Polymerase Iv ◽  
Plant Life Cycle ◽  
And Function

The plant-specific RNA Polymerase IV (Pol IV) transcribes heterochromatic regions, including many transposable elements (TEs), with the well-described role of generating 24 nucleotide (nt) small interfering RNAs (siRNAs). These siRNAs target DNA methylation back to TEs 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 have 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 (AGO1), which is known to function in post-transcriptional gene 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 provide support for the existing model that in pollen Pol IV functions in gene regulation. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

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