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-Absr) that were tagged with a selection marker that conferred resistance to blasticidin after a complete retrotransposition cycle. We found that the TRE5-Absr 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-Absr 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-Absr 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.

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.

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.