DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

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.

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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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In-vitro inhibition of spring viremia of carp virus replication by RNA interference targeting the RNA-dependent RNA polymerase gene

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.10.008 ◽

2019 ◽

Vol 263 ◽

pp. 14-19 ◽

Cited By ~ 2

Author(s):

Alamira Marzouk Fouad ◽

Hatem Soliman ◽

Ebtsam S.H. Abdallah ◽

Sherif Ibrahim ◽

Mansour El-Matbouli ◽

...

Keyword(s):

Rna Interference ◽

Rna Polymerase ◽

Virus Replication ◽

Polymerase Gene ◽

Rna Dependent Rna Polymerase ◽

In Vitro Inhibition ◽

Rna Polymerase Gene

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Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA

Journal of Virology ◽

10.1128/jvi.72.11.8913-8920.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8913-8920 ◽

Cited By ~ 44

Author(s):

Kun Yao ◽

Vikram N. Vakharia

Keyword(s):

Rna Polymerase ◽

Reverse Genetics ◽

In Vitro Transcription ◽

Infectious Pancreatic Necrosis Virus ◽

Rna Dependent Rna Polymerase ◽

Rna Transcripts ◽

Recovered Virus ◽

Cloned Cdna ◽

Pancreatic Necrosis Virus

ABSTRACT We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of theBirnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3′-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3′ end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.

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