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

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.

Download Full-text

DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

Nucleic Acids Research ◽

10.1093/nar/gkaa160 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4230-4243 ◽

Cited By ~ 1

Author(s):

Marek Malicki ◽

Thomas Spaller ◽

Thomas Winckler ◽

Christian Hammann

Keyword(s):

Rna Interference ◽

Rna Polymerase ◽

Dictyostelium Discoideum ◽

Repeat Sequence ◽

Marker Genes ◽

Argonaute Protein ◽

Tyrosine Recombinase ◽

Rna Dependent Rna Polymerase ◽

Rna Transcripts ◽

Complementary Region

Abstract The Dictyostelium Intermediate Repeat Sequence 1 (DIRS-1) is the name-giving member of the DIRS order of tyrosine recombinase retrotransposons. In Dictyostelium discoideum, DIRS-1 is highly amplified and enriched in heterochromatic centromers of the D. discoideum genome. We show here that DIRS-1 it tightly controlled by the D. discoideum RNA interference machinery and is only mobilized in mutants lacking either the RNA dependent RNA polymerase RrpC or the Argonaute protein AgnA. DIRS retrotransposons contain an internal complementary region (ICR) that is thought to be required to reconstitute a full-length element from incomplete RNA transcripts. Using different versions of D. discoideum DIRS-1 equipped with retrotransposition marker genes, we show experimentally that the ICR is in fact essential to complete retrotransposition. We further show that DIRS-1 produces a mixture of single-stranded, mostly linear extrachromosomal cDNA intermediates. If this cDNA is isolated and transformed into D. discoideum cells, it can be used by DIRS-1 proteins to complete productive retrotransposition. This work provides the first experimental evidence to propose a general retrotransposition mechanism of the class of DIRS like tyrosine recombinase retrotransposons.

Download Full-text

The IDR-containing protein PID-2 affects Z granules and is required for piRNA-induced silencing in the embryo

10.1101/2020.04.14.040584 ◽

2020 ◽

Author(s):

Maria Placentino ◽

António Miguel de Jesus Domingues ◽

Jan Schreier ◽

Sabrina Dietz ◽

Svenja Hellmann ◽

...

Keyword(s):

Gene Regulation ◽

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Small Rna ◽

Rna Silencing ◽

Rna Dependent Rna Polymerase ◽

Prolyl Aminopeptidase ◽

C Elegans

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.

Download Full-text

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.

Download Full-text