scholarly journals Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

2021 ◽  
Author(s):  
Akihito Fukudome ◽  
Jasleen Singh ◽  
Vibhor Mishra ◽  
Eswar Reddem ◽  
Francisco Martinez-Marquez ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Rna Polymerases ◽  
Rna Recognition Motif ◽  
Rna Dependent Rna Polymerase ◽  
Guide Rna ◽  
Pol Iv ◽  
Dna Strands ◽  
Dna Strand ◽  
Dna Template

AbstractRNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3’ ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNA’s 3’ end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3’ end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3’ end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand.SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3’ end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

scholarly journals Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

2021 ◽  
Vol 118 (51) ◽  
pp. e2115899118
Author(s):  
Akihito Fukudome ◽  
Jasleen Singh ◽  
Vibhor Mishra ◽  
Eswar Reddem ◽  
Francisco Martinez-Marquez ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerases ◽  
Rna Recognition Motif ◽  
Rna Dependent Rna Polymerase ◽  
Guide Rna ◽  
Pol Iv ◽  
Dna Strands ◽  
Tightly Coupled ◽  
Rna Polymerase Iv ◽  
Dna Strand

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV–RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3′ ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA’s 3′ end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3′ end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3′ end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

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.

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 Oxazinomycin arrests RNA polymerase at the polythymidine sequences

2019 ◽  
Vol 47 (19) ◽  
pp. 10296-10312 ◽  
Author(s):  
Ranjit K Prajapati ◽  
Petja Rosenqvist ◽  
Kaisa Palmu ◽  
Janne J Mäkinen ◽  
Anssi M Malinen ◽  
...  
Keyword(s):  
Oxygen Atom ◽  
Rna Polymerase ◽  
Inhibitory Effect ◽  
Trojan Horse ◽  
Rna Polymerases ◽  
Streptomyces Hygroscopicus ◽  
Rna Cleavage ◽  
Sequence Context ◽  
Good Substrate ◽  
Dna Template

Abstract Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.

scholarly journals RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Lydia J. R. Hunter ◽  
Samuel F. Brockington ◽  
Alex M. Murphy ◽  
Adrienne E. Pate ◽  
Kristina Gruden ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Rna Polymerase ◽  
Rna Polymerases ◽  
Rna Dependent Rna Polymerase ◽  
Rna Polymerase 1

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 Assembly of a double-stranded RNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 docks with NUCLEAR RNA POLYMERASE IV at the clamp domain

2020 ◽  
Author(s):  
Vibhor Mishra ◽  
Jasleen Singh ◽  
Akihito Fukudome ◽  
Feng Wang ◽  
Yixiang Zhang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mrna Translation ◽  
Transcriptional Gene Silencing ◽  
Exit Channel ◽  
Rna Dependent Rna Polymerase ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Rapid Conversion ◽  
Nuclear Rna Polymerase

AbstractIn plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24 nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 co-immunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that RDR2 stably associates with Pol IV’s largest catalytic subunit, NRPD1 at three sites, all within the clamp module. The clamp is a ubiquitous feature of DNA-dependent RNA polymerases that opens to allow DNA template entry and closes to encase the DNA-RNA hybrid adjacent to the RNA exit channel. The clamp also provides binding sites for polymerase-specific subunits or regulatory proteins, thus RDR2 binding to the Pol IV clamp is consistent with this theme. Within RDR2, the site of interaction with NRPD1 is very near the catalytic center. The locations of the NRPD1-RDR2 contact sites suggest a model in which transcripts emanating from Pol IV’s RNA exit channel align with the template cleft of RDR2, facilitating rapid conversion of terminated Pol IV transcripts into double-stranded RNAs.Significance StatementShort interfering RNAs (siRNAs) play important roles in gene regulation by inhibiting mRNA translation into proteins or by guiding chromatin modifications that inhibit gene transcription. In plants, transcriptional gene silencing is guided by siRNAs derived from double-stranded (ds) RNAs generated by coupling the activities of DNA-dependent NUCLEAR RNA POLYMERASE IV and RNA-DEPENDENT RNA POLYMERASE 2. We show that the physical basis for Pol IV-RDR2 coupling is RDR2 binding to the clamp domain of Pol IV’s largest subunit. The positions of the protein docking sites suggest that nascent Pol IV transcripts are generated in close proximity to RDR2’s catalytic site, enabling rapid conversion of Pol IV transcripts into dsRNAs.

scholarly journals Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV

2021 ◽  
Vol 118 (13) ◽  
pp. e2019276118
Author(s):  
Vibhor Mishra ◽  
Jasleen Singh ◽  
Feng Wang ◽  
Yixiang Zhang ◽  
Akihito Fukudome ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Active Site ◽  
Rna Dependent Rna Polymerase ◽  
Dna Viruses ◽  
Selfish Genetic Elements ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Tightly Coupled ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV’s 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3′ ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

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.

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