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 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’.

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 ◽  
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 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 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 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 Abundant expression of maternal siRNAs is a conserved feature of seed development

2020 ◽  
Vol 117 (26) ◽  
pp. 15305-15315 ◽  
Author(s):  
Jeffrey W. Grover ◽  
Diane Burgess ◽  
Timmy Kendall ◽  
Abdul Baten ◽  
Suresh Pokhrel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Seed Development ◽  
Small Rnas ◽  
Sequence Evolution ◽  
Small Interfering Rnas ◽  
Genomic Features ◽  
Pol Iv ◽  
Sirna Expression ◽  
Rapid Sequence ◽  
And Function

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). InBrassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression duringB. rapaseed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

scholarly journals SHH1, a Homeodomain Protein Required for DNA Methylation, As Well As RDR2, RDM4, and Chromatin Remodeling Factors, Associate with RNA Polymerase IV

PLoS Genetics ◽  
2011 ◽  
Vol 7 (7) ◽  
pp. e1002195 ◽  
Author(s):  
Julie A. Law ◽  
Ajay A. Vashisht ◽  
James A. Wohlschlegel ◽  
Steven E. Jacobsen
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Chromatin Remodeling ◽  
Homeodomain Protein ◽  
Rna Polymerase Iv

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

2019 ◽  
Vol 47 (17) ◽  
pp. 9024-9036 ◽  
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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