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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.

Transposable elements amplify in genomes as selfish DNA elements and challenge host fitness because their intrinsic integration steps during mobilization can compromise genome integrity. In gene-dense genomes, transposable elements are notably under selection to avoid insertional mutagenesis of host protein-coding genes. We describe an example of convergent evolution in the distantly related amoebozoan Dictyostelium discoideum and the yeast Saccharomyces cerevisiae, in which the D. discoideum retrotransposon DGLT-A and the yeast Ty3 element developed different mechanisms to facilitate position-specific integration at similar sites upstream of tRNA genes. Transcription of tRNA genes by RNA polymerase III requires the transcription factor complexes TFIIIB and TFIIIC. Whereas Ty3 recognizes tRNA genes mainly through interactions of its integrase with TFIIIB subunits, the DGLT-A-encoded ribonuclease H contacts TFIIIC subunit Tfc4 at an interface that covers tetratricopeptide repeats (TPRs) 7 and 8. A major function of this interface is to connect TFIIIC subcomplexes τA and τB and to facilitate TFIIIB assembly. During the initiation of tRNA gene transcription τB is displaced from τA, which transiently exposes the TPR 7/8 surface of Tfc4 on τA. We propose that the DGLT-A intasome uses this binding site to obtain access to genomic DNA for integration during tRNA gene transcription.

The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-Absr) integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-Absr targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III) transcriptome. We determined that both natural TRE5-A and cloned TRE5-Absr retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element.

Background In gene-dense genomes, mobile elements are confronted with highly selective pressure to amplify without causing excessive damage to the host. The targeting of tRNA genes as potentially safe integration sites has been developed by retrotransposons in various organisms such as the social amoeba Dictyostelium discoideum and the yeast Saccharomyces cerevisiae. In D. discoideum, tRNA gene-targeting retrotransposons have expanded to approximately 3 % of the genome. Recently obtained genome sequences of species representing the evolutionary history of social amoebae enabled us to determine whether the targeting of tRNA genes is a generally successful strategy for mobile elements to colonize compact genomes. Results During the evolution of dictyostelids, different retrotransposon types independently developed the targeting of tRNA genes at least six times. DGLT-A elements are long terminal repeat (LTR) retrotransposons that display integration preferences ~15 bp upstream of tRNA gene-coding regions reminiscent of the yeast Ty3 element. Skipper elements are chromoviruses that have developed two subgroups: one has canonical chromo domains that may favor integration in centromeric regions, whereas the other has diverged chromo domains and is found ~100 bp downstream of tRNA genes. The integration of D. discoideum non-LTR retrotransposons ~50 bp upstream (TRE5 elements) and ~100 bp downstream (TRE3 elements) of tRNA genes, respectively, likely emerged at the root of dictyostelid evolution. We identified two novel non-LTR retrotransposons unrelated to TREs: one with a TRE5-like integration behavior and the other with preference ~4 bp upstream of tRNA genes. Conclusions Dictyostelid retrotransposons demonstrate convergent evolution of tRNA gene targeting as a probable means to colonize the compact genomes of their hosts without being excessively mutagenic. However, high copy numbers of tRNA gene-associated retrotransposons, such as those observed in D. discoideum, are an exception, suggesting that the targeting of tRNA genes does not necessarily favor the amplification of position-specific integrating elements to high copy numbers under the repressive conditions that prevail in most host cells. Electronic supplementary material The online version of this article (doi:10.1186/s13100-016-0073-9) contains supplementary material, which is available to authorized users.

Background In 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. Results The 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. Conclusions The 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 material The online version of this article (doi:10.1186/s13100-015-0045-5) contains supplementary material, which is available to authorized users.

C-module-binding factor A (CbfA) is a jumonji-type transcription regulator that is important for maintaining the expression and mobility of the retrotransposable element TRE5-A in the social amoeba Dictyostelium discoideum . CbfA-deficient cells have lost TRE5-A retrotransposition, are impaired in the ability to feed on bacteria, and do not enter multicellular development because of a block in cell aggregation. In this study, we performed Illumina RNA-seq of growing CbfA mutant cells to obtain a list of CbfA-regulated genes. We demonstrate that the carboxy-terminal domain of CbfA alone is sufficient to mediate most CbfA-dependent gene expression. The carboxy-terminal domain of CbfA from the distantly related social amoeba Polysphondylium pallidum restored the expression of CbfA-dependent genes in the D. discoideum CbfA mutant, indicating a deep conservation in the gene regulatory function of this domain in the dictyostelid clade. The CbfA-like protein CbfB displays ∼25% sequence identity with CbfA in the amino-terminal region, which contains a JmjC domain and two zinc finger regions and is thought to mediate chromatin-remodeling activity. In contrast to CbfA proteins, where the carboxy-terminal domains are strictly conserved in all dictyostelids, CbfB proteins have completely unrelated carboxy-terminal domains. Outside the dictyostelid clade, CbfA-like proteins with the CbfA-archetypical JmjC/zinc finger arrangement and individual carboxy-terminal domains are prominent in filamentous fungi but are not found in yeasts, plants, and metazoans. Our data suggest that two functional regions of the CbfA-like proteins evolved at different rates to allow the occurrence of species-specific adaptation processes during genome evolution.

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.

Retrotransposons contribute significantly to the evolution of eukaryotic genomes. They replicate by producing DNA copies of their own RNA, which are integrated at new locations in the host cell genome. In the gene-dense genome of the social amoeba Dictyostelium discoideum, retrotransposon TRE5-A avoids insertional mutagenesis by targeting the transcription factor (TF) IIIC/IIIB complex and integrating ∼ 50 bp upstream of tRNA genes. We generated synthetic TRE5-A retrotransposons (TRE5-A(bsr)) that were tagged with a selection marker that conferred resistance to blasticidin after a complete retrotransposition cycle. We found that the TRE5-A(bsr) elements were efficiently mobilized in trans by proteins expressed from the endogenous TRE5-A population found in D. discoideum cells. ORF1 protein translated from TRE5-A(bsr) elements significantly enhanced retrotransposition. We observed that the 5' untranslated region of TRE5-A could be replaced by an unrelated promoter, whereas the 3' untranslated region of TRE5-A was essential for retrotransposition. A predicted secondary structure in the RNA of the 3' untranslated region of TRE5-A may be involved in the retrotransposition process. The TRE5-A(bsr) elements were capable of identifying authentic integration targets in vivo, including formerly unnoticed, putative binding sites for TFIIIC on the extrachromosomal DNA element that carries the ribosomal RNA genes.