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

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.

Download Full-text

RNA-Dependent RNA Polymerase 1 from Nicotiana tabacum Suppresses RNA Silencing and Enhances Viral Infection in Nicotiana benthamiana

The Plant Cell ◽

10.1105/tpc.109.072058 ◽

2010 ◽

Vol 22 (4) ◽

pp. 1358-1372 ◽

Cited By ~ 72

Author(s):

Xiao-Bao Ying ◽

Li Dong ◽

Hui Zhu ◽

Cheng-Guo Duan ◽

Quan-Sheng Du ◽

...

Keyword(s):

Nicotiana Tabacum ◽

Rna Polymerase ◽

Viral Infection ◽

Nicotiana Benthamiana ◽

Rna Silencing ◽

Rna Dependent Rna Polymerase ◽

Rna Polymerase 1

Download Full-text

Analysis of rice RNA-dependent RNA polymerase 1 (OsRDR1) in virus-mediated RNA silencing after particle bombardment

Journal of General Plant Pathology ◽

10.1007/s10327-010-0226-5 ◽

2010 ◽

Vol 76 (2) ◽

pp. 152-160 ◽

Cited By ~ 9

Author(s):

Hui Chen ◽

Atsushi Tamai ◽

Masashi Mori ◽

Masashi Ugaki ◽

Yoshikazu Tanaka ◽

...

Keyword(s):

Rna Polymerase ◽

Particle Bombardment ◽

Rna Silencing ◽

Rna Dependent Rna Polymerase ◽

Rna Polymerase 1

Download Full-text

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2115899118 ◽

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.

Download Full-text

Dynamics of a geminivirus-encoded pre-coat protein and host RNA-dependent RNA polymerase 1 in regulating symptom recovery in tobacco

Journal of Experimental Botany ◽

10.1093/jxb/ery043 ◽

2018 ◽

Vol 69 (8) ◽

pp. 2085-2102 ◽

Cited By ~ 13

Author(s):

Saumik Basu ◽

Nirbhay Kumar Kushwaha ◽

Ashish Kumar Singh ◽

Pranav Pankaj Sahu ◽

R Vinoth Kumar ◽

...

Keyword(s):

Coat Protein ◽

Rna Polymerase ◽

Rna Dependent Rna Polymerase ◽

Symptom Recovery ◽

Rna Polymerase 1

Download Full-text

Expressional and regulatory characterization of Arabidopsis RNA-dependent RNA polymerase 1

Planta ◽

10.1007/s00425-013-1863-7 ◽

2013 ◽

Vol 237 (6) ◽

pp. 1561-1569 ◽

Cited By ~ 10

Author(s):

Tao Xu ◽

Liang Zhang ◽

Jie Zhen ◽

Yunliu Fan ◽

Chunyi Zhang ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Dependent Rna Polymerase ◽

Rna Polymerase 1

Download Full-text

Use of Highly Conserved Motifs in Plant Virus RNA Polymerases as the Tags for Specific Detection of Carmovirus-Related RNA-Dependent RNA Polymerase Genes

Virology ◽

10.1006/viro.1995.1084 ◽

1995 ◽

Vol 207 (1) ◽

pp. 312-315 ◽

Cited By ~ 10

Author(s):

S.Yu. Morozov ◽

E.V. Ryabov ◽

R.-M. Leiser ◽

S.K. Zavriev

Keyword(s):

Rna Polymerase ◽

Plant Virus ◽

Rna Polymerases ◽

Specific Detection ◽

Rna Dependent Rna Polymerase ◽

Conserved Motifs ◽

Virus Rna

Download Full-text

Plant A20/AN1 proteins coordinate different immune responses including RNAi pathway for antiviral immunity

10.1101/622696 ◽

2019 ◽

Author(s):

Li Chang ◽

Ho-Hsiung Chang ◽

Yi-Shu Chiu ◽

Jui-Che Chang ◽

Duen-Wei Hsu ◽

...

Keyword(s):

Salicylic Acid ◽

Rna Polymerase ◽

Plant Stress ◽

Antiviral Immunity ◽

Rnai Pathway ◽

Functional Domain ◽

Rna Dependent Rna Polymerase ◽

Positive Role ◽

Stress Associated Protein ◽

Rna Polymerase 1

ABSTRACTSalicylic acid (SA)-mediated immunity plays important roles in combating virus in plants. Two plant stress associated protein (SAPs) containing dual A20/AN1 zinc-finger domain were found to play important roles in SA-mediated immunity; however, detailed mechanisms remain elusive. In this study, another orchid homolog gene of Pha13, Pha21, was analyzed. Pha21 confers antiviral immunity in both transgenic orchid and Arabidopsis overexpressing Pha21. Expression of Pha21 is early-induced by SA treatment, and is involved in the expression of the orchid homolog of the master regulator NPR1. Pha21 but not Pha13 is involved in the expression of key RNAi-related genes, Dicer-like nuclease 4 (DCL4) and Argonaut 1 (AGO1) in orchids. The involvement of SAPs in expression of orchid DCL4 and AGO1 is not limited to orchid, as AtSAP5 also plays essential role in the expression of Arabidopsis DCL4 and AGO1. However, unlike Pha13 and AtSAP5, Pha21 does not play positive role in the expression of orchid homolog gene of RNA-dependent RNA polymerase 1 (RdR1), an important gene in RNAi pathway. Pha21 can be found localized in the nucleus, and confers self-E3 ligase and ubiquitin binding activities. Functional domain analysis revealed that both A20 and AN1 domains of Pha21 are required for decreasing virus accumulation, and the AN1 domain plays a more important role in the expression of orchid DCL4. Collectively, our data suggests SA regulated SAPs play important roles in antiviral immunity and is involved in delicate regulation of key genes in RNAi-mediated pathway.IMPORTANCESalicylic acid (SA)-mediated antiviral immunity plays an important role to protect plants from virus infection; however, the detailed mechanisms remain elusive. We previously demonstrated that two plant A20/AN1 proteins, orchid Pha13 and Arabidopsis AtSAP5, function similarly and serve as an important hub to regulate SA-mediated antiviral immunity. In this study, we identified another orchid A20/AN1 protein, Pha21, which is involved in SA-mediated antiviral immunity. In contrast to Pha13 and AtSAP5, Pha21 plays minor negative roles in the expression of PhaRdR1 (orchid homolog of RNA-dependent RNA polymerase 1). However, Pha21 and AtSAP5, but not Pha13, are involved in the expression of important players in RNAi pathway, Dicer-like nuclease 4 (DCL4) and Argonaut 1 (AGO1), in orchid and Arabidopsis. Our data demonstrates that plant A20/AN1 proteins are conserved players in SA-mediated antiviral resistance among plants, and provide links between the A20/AN1 proteins and the RNAi pathway.

Download Full-text