Genetically tagged TRE5-A retrotransposons reveal high amplification rates and authentic target site preference in the Dictyostelium discoideum genome

Siol, Oliver; Spaller, Thomas; Schiefner, Jana; Winckler, Thomas

doi:10.1093/nar/gkr261

Cited by 6 publications

(17 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The “HindIII arm” was generated by amplification of nucleotides 1–1166 of the agnC gene including its authentic translation start codon. The pGEM-agnC-GA plasmid was linearized and transformed into D. discoideum AX2 or DH1 cells and transformants were selected in HL5 medium (Formedium, Hunstanton, UK) containing 6 μg/ml blasticidin (Life Technologies, Carlsbad, USA) [ 37 ]. From such clones genomic DNA was isolated and screened by PCR for insertion of the GA cassette at the targeted locus using one primer specific for the blasticidin resistance gene and a second primer that hybridized outside of the DNA sequences covered by the HindIII arm.…”

Section: Methodsmentioning

confidence: 99%

A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein

Schmith¹,

Spaller²,

Gaube³

et al. 2015

Mobile DNA

View full text Add to dashboard Cite

BackgroundIn the compact and haploid genome of Dictyostelium discoideum control of transposon activity is of particular importance to maintain viability. The non-long terminal repeat retrotransposon TRE5-A amplifies continuously in D. discoideum cells even though it produces considerable amounts of minus-strand (antisense) RNA in the presence of an active RNA interference machinery. Removal of the host-encoded C-module-binding factor (CbfA) from D. discoideum cells resulted in a more than 90 % reduction of both plus- and minus-strand RNA of TRE5-A and a strong decrease of the retrotransposition activity of the cellular TRE5-A population. Transcriptome analysis revealed an approximately 230-fold overexpression of the gene coding for the Argonaute-like protein AgnC in a CbfA-depleted mutant.ResultsThe D. discoideum genome contains orthologs of RNA-dependent RNA polymerases, Dicer-like proteins, and Argonaute proteins that are supposed to represent RNA interference pathways. We analyzed available mutants in these genes for altered expression of TRE5-A. We found that the retrotransposon was overexpressed in mutants lacking the Argonaute proteins AgnC and AgnE. Because the agnC gene is barely expressed in wild-type cells, probably due to repression by CbfA, we employed a new method of promoter-swapping to overexpress agnC in a CbfA-independent manner. In these strains we established an in vivo retrotransposition assay that determines the retrotransposition frequency of the cellular TRE5-A population. We observed that both the TRE5-A steady-state RNA level and retrotransposition rate dropped to less than 10 % of wild-type in the agnC overexpressor strains.ConclusionsThe data suggest that TRE5-A amplification is controlled by a distinct pathway of the Dictyostelium RNA interference machinery that does not require RNA-dependent RNA polymerases but involves AgnC. This control is at least partially overcome by the activity of CbfA, a factor derived from the retrotransposon’s host. This unusual regulation of mobile element activity most likely had a profound effect on genome evolution in D. discoideum.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-015-0045-5) contains supplementary material, which is available to authorized users.

show abstract

Section: Methodsmentioning

confidence: 99%

A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein

Schmith¹,

Spaller²,

Gaube³

et al. 2015

Mobile DNA

View full text Add to dashboard Cite

show abstract

“…However, similar to Ty3 elements, mutations of the box B promoter that interfere with binding of TFIIIC abolish the targeting of TRE5-A to the tRNA target gene [ 133 ]. TRE5-A insertion profiling demonstrated that TRE5-A can also integrate at the Pol III-transcribed ribosomal 5S gene which is located on a multi-copy extrachromosomal DNA element harboring the rRNA genes [ 134 , 135 ]. Unlike TRE5, TRE3 has a broader range of insertion that is 40–150 bp downstream of tRNA genes in the same transcription orientation (Fig.…”

Section: Tes Target Rna Pol III Transcribed Genes In Dictyomentioning

confidence: 99%

Retrotransposon targeting to RNA polymerase III-transcribed genes

2018

View full text Add to dashboard Cite

Retrotransposons are genetic elements that are similar in structure and life cycle to retroviruses by replicating via an RNA intermediate and inserting into a host genome. The Saccharomyces cerevisiae (S. cerevisiae) Ty1–5 elements are long terminal repeat (LTR) retrotransposons that are members of the Ty1-copia (Pseudoviridae) or Ty3-gypsy (Metaviridae) families. Four of the five S. cerevisiae Ty elements are inserted into the genome upstream of RNA Polymerase (Pol) III-transcribed genes such as transfer RNA (tRNA) genes. This particular genomic locus provides a safe environment for Ty element insertion without disruption of the host genome and is a targeting strategy used by retrotransposons that insert into compact genomes of hosts such as S. cerevisiae and the social amoeba Dictyostelium. The mechanism by which Ty1 targeting is achieved has been recently solved due to the discovery of an interaction between Ty1 Integrase (IN) and RNA Pol III subunits. We describe the methods used to identify the Ty1-IN interaction with Pol III and the Ty1 targeting consequences if the interaction is perturbed. The details of Ty1 targeting are just beginning to emerge and many unexplored areas remain including consideration of the 3-dimensional shape of genome. We present a variety of other retrotransposon families that insert adjacent to Pol III-transcribed genes and the mechanism by which the host machinery has been hijacked to accomplish this targeting strategy. Finally, we discuss why retrotransposons selected Pol III-transcribed genes as a target during evolution and how retrotransposons have shaped genome architecture.

show abstract

“…By using a pre-defined integration site, the assay can analyze the structural requirements of genomic loci to be recognized by TRE5-A. The right part outlines the use of defined, genetically tagged TRE5-A retrotransposons 17 to determine the target site specificity of TRE5-A retrotransposons. Considering that TRE5-A bsr elements mimic the natural integration preference of endogenous TRE5-As, this assay can identify natural integration sites and determine the rate of off-site integrations.…”

Section: Genetically Tagged Tre5-as: Defining the Retrotransposon Strmentioning

confidence: 99%

“…This prediction would be supported by results showing that B boxes occur on the extrachromosomal DNA elements that carry the D. discoideum ribosomal tRNA genes. 17 Thus, it will be interesting to see if TRE5-A bsr elements will integrate at loci not associated with tRNA genes and whether these loci are off-site targets or previously unrecognized functional B boxes occupied by the TFIIIC/TFIIIB complex. If we predict that every authentic TRE5-A bsr integration difficult to estimate how frequently tRNA gene-specific integration events have occurred.…”

Section: Trna Gene-targeted Retrotransposition: Avoidingmentioning

confidence: 99%

See 1 more Smart Citation

Dictyostelium transfer RNA gene-targeting retrotransposons

Winckler

Schiefner

Spaller

et al. 2011

Mobile Genetic Elements

View full text Add to dashboard Cite

The model species of social amoebae, Dictyostelium discoideum, has a compact genome consisting of about two thirds protein-coding regions, with intergenic regions that are rarely larger than 1,000 bp. We hypothesize that the haploid state of D. discoideum cells provides defense against the amplification of mobile elements whose transposition activities would otherwise lead to the accumulation of heterozygous, potentially lethal mutations in diploid populations. We further speculate that complex transposon clusters found on D. discoideum chromosomes do not a priori result from integration preferences of these transposons, but that the clusters instead result from negative selection against cells harboring insertional mutations in genes. D. discoideum cells contain a fraction of retrotransposons that are found in the close vicinity of tRNA genes. Growing evidence suggests that these retrotransposons use active recognition mechanisms to determine suitable integration sites. However, the question remains whether these retrotransposons also cause insertional mutagenesis of genes, resulting in their enrichment at tRNA genes, which are relatively safe sites in euchromatic regions. Recently developed in vivo retrotransposition assays will allow a detailed, genome-wide analysis of de novo integration events in the D. discoideum genome.

show abstract

Genetically tagged TRE5-A retrotransposons reveal high amplification rates and authentic target site preference in the Dictyostelium discoideum genome

Cited by 6 publications

References 53 publications

A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein

A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein

Retrotransposon targeting to RNA polymerase III-transcribed genes

Dictyostelium transfer RNA gene-targeting retrotransposons

Contact Info

Product

Resources

About