Retrotransposon Domestication and Control in Dictyostelium discoideum

Abstract

Transposable elements, identified in all eukaryotes, are mobile genetic units that can change their genomic position. Transposons usually employ an excision and reintegration mechanism, by which they change position, but not copy number. In contrast, retrotransposons amplify via RNA intermediates, increasing their genomic copy number. Hence, they represent a particular threat to the structural and informational integrity of the invaded genome. The social amoeba Dictyostelium discoideum, model organism of the evolutionary Amoebozoa supergroup, features a haploid, gene-dense genome that offers limited space for damage-free transposition. Several of its contemporary retrotransposons display intrinsic integration preferences, for example by inserting next to transfer RNA genes or other retroelements. Likely, any retrotransposons that invaded the genome of the amoeba in a non-directed manner were lost during evolution, as this would result in decreased fitness of the organism. Thus, the positional preference of the Dictyostelium retroelements might represent a domestication of the selfish elements. Likewise, the reduced danger of such domesticated transposable elements led to their accumulation, and they represent about 10% of the current genome of D. discoideum. To prevent the uncontrolled spreading of retrotransposons, the amoeba employs control mechanisms including RNA interference and heterochromatization. Here, we review TRE5-A, DIRS-1 and Skipper-1, as representatives of the three retrotransposon classes in D. discoideum, which make up 5.7% of the Dictyostelium genome. We compile open questions with respect to their mobility and cellular regulation, and suggest strategies, how these questions might be addressed experimentally.

Keywords: DIRS-1; RNA interference; Skipper-1; TRE5-A; retrovirus.

Figure 1

Possible fates of transposable elements. Shown is a cell (gray) with a nucleus (yellow), in which a transposable element (TE; blue) invaded. Depending on the TE features and its control by cellular elements, different scenarios can be envisaged. Lack of cellular control of the TE will lead to its significant accumulation, posing additional burden to the host (left). Random integration (center) by lack of TE site preference is prone to hit essential genes. Compared to a cellularly controlled TE that additionally was domesticated by displaying a safe integration site preference (right), the former two scenarios would result in cells with decreased fitness. This is expected to eventually lead to their loss during evolution (indicated by dashed lines). A combination of the first two scenarios (not shown) is thought to result in a particularly disfavored cell.

Figure 2

Retrotransposons encoded in the D. discoideum genome and strategies for their investigation. (A) Shown are the structures of the consensus elements with open reading frames (ORFs) in blue, drawn to scale (see bottom). The non-LTR retrotransposon family is separated into two subgroups, namely TRE5 and TRE3 (for 5′ and 3′ tRNA gene targeted retroelement, respectively), based on their integration preferences upstream or downstream of tRNA genes. Typical for all TRE elements is the presence of two ORFs, where the second one encodes for an apurinic/apyrimidinic endonuclease (APE), a reverse transcriptase (RT), and a zinc-finger domain (ZF). The bordering short untranslated regions (UTR) are indicated, which contain in the TRE5 subfamily distinct modules A–C. The LTR retrotransposons represent Ty3/gypsy-like elements that are surrounded by directed long terminal repeats (LTR, boxed triangles). DGLT-A has a single ORF encoding for a group-specific antigen (GAG) protein with a zinc finger-like signature followed by RT, ribonuclease H (RH), and integrase (INT) domains. Skipper-1 contains two ORFs. ORF1 codes for a GAG protein with a defined zinc finger-like motif. ORF2 codes for a protease (PRO), RT, RH, INT. Additionally, ORF2 of Skipper-1 contains a chromo domain (CHD) at the C-terminus. Note that all copies of DGLT-A and Skipper-2 in the genome of D. discoideum are incomplete, contrary to other dictyostelids (Spaller et al., 2016). DIRS-1 is the founding member of the class of tyrosine recombinase (YR) retrotransposons. Its three ORFs are surrounded by inverted LTRs. ORF1 encodes a putative GAG protein, ORF2 overlaps with ORF3 and encodes RT, RH and methyl transferase (MT) domains, while ORF3 contains the YR. (B) Schematic representation of the retrotransposition assay with a genetically tagged transposable element TE^mbsrI (mbsrI: minus-strand blasticidin S resistance (bsr) gene disrupted by an inverse intron), as applied successfully in the investigation of Ty1 from Saccharomyces cerevisiae, mammalian LINEs and TRE5-A from D. discoideum (Boeke et al., ; Esnault et al., ; Ostertag et al., ; Siol et al., 2011). Shown is the orientation of the resistance cassette (referred to as “master element” when disrupted by an intron) with respect to the TE sequence, in which the cassette is embedded. A15P denotes the actin15 promoter and terminator sequence (term.) is shown. Upon co-transformation with a marker plasmid, e.g., pISAR, which confers G418 resistance to D. discoideum. Transformants with stably integrated plasmids are selected with G418. The master element is not able to generate blasticidin S (BS) resistant clones, because the respective gene is inactivated by an intron (from the S17 gene, 74 bp in size). After a complete retrotransposition cycle, however, the intron is spliced out, resulting in TE^mbsr (minus-strand bsr gene), referred to as “copy element” upon loosing the intron in the resistance gene sequence. This enables expression of a functional bsr messenger RNA (mRNA), thereby conferring BS resistance (BS^R). BS resistant clones thus are cells, in which at least one full retrotransposition cycle of the TE under investigation has been performed. Primers P1 and P2 can be used to confirm the splicing of the intron by PCR, and the size of the PCR product is indicative for the master element or the copy element. (C) Mapping of novel integration sites using tagged TEs. To address integration sites, genomic DNA (gDNA) is digested using restriction enzymes (RE) that cut outside the TE. Upon circularization of resulting fragments by ligation and RE digestion inside the TE sequence, linear fragments are obtained. These can be analyzed by PCR employing primers P1 and P2, or directly subjected to next generation sequencing.