Spreading of RNA Targeting and DNA Methylation in RNA Silencing Requires Transcription of the Target Gene and a Putative RNA-Dependent RNA Polymerase

AbstractIn Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG-1-bound 21U RNAs trigger silencing mechanisms mediated by RNA-dependent RNA polymerase (RdRP)-synthetized 22G RNAs. This silencing can become PRG-1-independent, and heritable over many generations. This state is named RNAe. It is unknown how and when RNAe is established, and how it is maintained. We show that maternally provided 21U RNAs can be sufficient to trigger RNAe in embryos. Additionally, we identify the IDR-containing protein PID-2, as a factor required to establish and maintain RNAe. PID-2 interacts with two novel, partially redundant, eTudor domain proteins, PID-4 and PID-5. Additionally, PID-5 has a domain related to the X-prolyl aminopeptidase protein APP-1, and binds APP-1, implicating N-terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in the C. elegans small RNA silencing network.

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

Nucleic Acids Research ◽

10.1093/nar/gkz615 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9024-9036 ◽

Cited By ~ 4

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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Investigation of Host Range of and Host Defense against a Mitochondrially Replicating Mitovirus

Journal of Virology ◽

10.1128/jvi.01503-18 ◽

2019 ◽

Vol 93 (6) ◽

Cited By ~ 14

Author(s):

Sabitree Shahi ◽

Ana Eusebio-Cope ◽

Hideki Kondo ◽

Bradley I. Hillman ◽

Nobuhiro Suzuki

Keyword(s):

Rna Polymerase ◽

Host Range ◽

Protoplast Fusion ◽

Rna Silencing ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Rna Dependent Rna Polymerase ◽

Content Type ◽

Host Interactions ◽

Chestnut Blight Fungus

ABSTRACT Mitoviruses (genus Mitovirus, family Narnaviridae) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.2 to 4.4 kb with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase. Cryphonectria parasitica mitovirus 1 (CpMV1) from U.S. strain NB631 of the chestnut blight fungus, Cryphonectria parasitica, was the first virus identified as a mitochondrially replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single-conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriaceae, Cryphonectria radicalis and Valsa ceratosperma, also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae. PCR-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and -deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, which is consistent with its mitochondrial replication. Collectively, the results of study provide a foundation to further explore the biology of mitoviruses. IMPORTANCE Capsidless mitoviruses, which are ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.2 to 4.4 kb, encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria parasitica mitovirus 1 (CpMV1), which was isolated from strain NB631 of the chestnut blight fungus (Cryphonectria parasitica), a model filamentous fungus for studying virus-host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica and different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and -competent strains showed clearly that the virus was unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus-host interactions.

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

10.1101/2021.08.31.458351 ◽

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.

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A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation

10.1101/2021.09.06.459145 ◽

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.

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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

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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

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The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

Nucleic Acids Research ◽

10.1093/nar/gkt1337 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3330-3345 ◽

Cited By ~ 9

Author(s):

Stephan Wiegand ◽

Doreen Meier ◽

Carsten Seehafer ◽

Marek Malicki ◽

Patrick Hofmann ◽

...

Keyword(s):

Rna Polymerase ◽

Dictyostelium Discoideum ◽

Rna Silencing ◽

Repeat Sequence ◽

Transcriptional Level ◽

Asymmetric Distribution ◽

Messenger Rnas ◽

Long Terminal Repeats ◽

Rna Dependent Rna Polymerase ◽

Antisense Orientation

Abstract Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC– strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC– strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5′ and 3′ directions.

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