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 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 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 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 Integrator terminates promoter-proximal Pol II to generate C. elegans piRNA precursors

2020 ◽  
Author(s):  
Toni Beltran ◽  
Elena Pahita ◽  
Subhanita Ghosh ◽  
Boris Lenhard ◽  
Peter Sarkies
Keyword(s):  
Catalytic Activity ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
List Type ◽  
Germline Development ◽  
Pol Ii ◽  
C Elegans

AbstractPiwi-interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C. elegans, piRNAs are transcribed from >15000 discrete genomic loci by RNA polymerase II, resulting in 28 nt short-capped piRNA precursors. Here we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. We show that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter-proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Our results draw new parallels between snRNA and piRNA biogenesis in nematodes, and provide evidence of a role for the Integrator complex as a terminator of promoter-proximal RNA polymerase II.Highlights- Integrator localises to sites of piRNA biogenesis in nematodes- Integrator cleaves nascent RNAs associated with promoter-proximal Pol II at piRNA loci to release short capped piRNA precursors from chromatin- Repression of Pol II elongation at the majority of piRNA loci is independent of Integrator

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 Regulation of expression of human RNA polymerase II-transcribed snRNA genes

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170073 ◽  
Author(s):  
Joana Guiro ◽  
Shona Murphy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Mrna Splicing ◽  
Regulation Of Expression ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Non Coding Rnas ◽  
Rna Genes

In addition to protein-coding genes, RNA polymerase II (pol II) transcribes numerous genes for non-coding RNAs, including the small-nuclear (sn)RNA genes. snRNAs are an important class of non-coding RNAs, several of which are involved in pre-mRNA splicing. The molecular mechanisms underlying expression of human pol II-transcribed snRNA genes are less well characterized than for protein-coding genes and there are important differences in expression of these two gene types. Here, we review the DNA features and proteins required for efficient transcription of snRNA genes and co-transcriptional 3′ end formation of the transcripts.

scholarly journals Association of the Influenza A Virus RNA-Dependent RNA Polymerase with Cellular RNA Polymerase II

2005 ◽  
Vol 79 (9) ◽  
pp. 5812-5818 ◽  
Author(s):  
Othmar G. Engelhardt ◽  
Matt Smith ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Influenza A ◽  
Large Subunit ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Virus Rna ◽  
Transcription Sites

ABSTRACT Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

scholarly journals Cyclin T1/CDK9 Interacts with Influenza A Virus Polymerase and Facilitates Its Association with Cellular RNA Polymerase II

2010 ◽  
Vol 84 (24) ◽  
pp. 12619-12627 ◽  
Author(s):  
Junjie Zhang ◽  
Gang Li ◽  
Xin Ye
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Influenza A ◽  
Viral Transcription ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Cyclin T1

ABSTRACT Influenza virus RNA-dependent RNA polymerase scavenges the 5′ cap from host pre-mRNA to prime viral transcription initiation. It is also well established that viral RNA-dependent RNA polymerase (vRNP) associates with cellular RNA polymerase II (Pol II), on which viral replication depends. Here we report that cyclin T1/CDK9 can interact with influenza virus polymerase and facilitate its association with cellular Pol II. The immunodepletion of cyclin T1/CDK9 totally abolished the association of vRNP with the C-terminal domain (CTD) Ser-2-phosphorylated form of RNA polymerase II. Further studies showed that overexpression of cyclin T1/CDK9 increased the transcription activity of vRNP, while knockdown of cyclin T1/CDK9 impaired viral replication. Our results suggest that cyclin T1/CDK9 serves as an adapter to mediate the interaction of vRNP and RNA Pol II and promote viral transcription.

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