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

Abstract

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.

Keywords: Argonaute; Dictyostelium; RNAi; Retrotransposition; siRNA.

Fig. 1

Expression of mobile elements in the CbfA underexpressing mutant JH.D. Expression data are derived from a previous RNA-seq analysis [28]. RPKM values (reads per kb of mRNA and standardized to 1 million reads) were obtained for the indicated mobile elements and calculated as ratio of JH.D versus AX2 and are shown as “fold change” of expression, meaning that values >1 represent overexpression of genes in JH.D. Values are means from three independent cultures ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, relative to control AX2 cells (Student’s t-test). Black bars indicate non-LTR retrotransposons, grey bars indicate LTR retrotransposons (including the tyrosine recombinase retrotransposon DIRS-1), and white bars indicate putative DNA transposons

Fig. 2

Expression of RNAi components in CbfA mutant JH.D cells. Expression of Dicer-like proteins (drnA, drnB), RdRPs (rrpA-C) and Argonaute genes (agnA-E) was analyzed by RNA-seq (gray bars, n = 3) in wild-type AX2 and CbfA-mutant JH.D cells from three independent cultures [28]. These three RNA samples, and RNA from three additional independent cultures, were analyzed by qRT-PCR (black bars, n = 6). Data are shown as fold change of expression with values >1 meaning that expression in the mutant cells was higher than in the control cells. Values are means ± SD from three and six independent cultures, respectively. **p < 0.01, ***p < 0.001, relative to wild-type AX2 cells (Student’s t-test)

Fig. 3

CbfA controls AgnC expression. a Scheme of the CbfA protein. The 1000-amino acid protein can be roughly divided into an amino-terminal part that may have chromatin-remodeling activity and a carboxy-terminal part that may facilitate DNA-binding [28, 39]. JmjC: carboxy-terminal jumonji domain", ZF: zinc finger-like motif; NRD: asparagine-rich domain; CTD: carboxy-terminal domain. The CbfA proteins expressed in mutant JH.D comprised either the full length protein (amino acids 2–1000) or the CbfA-CTD (amino acids 724–1000). b Expression of CbfA in JH.D cells. CbfA mutant JH.D cells were transformed with plasmids allowing for the expression of either untagged, full-length CbfA [21] or the GFP-tagged, carboxy-terminal domain of CbfA (CbfA-CTD) [28]. Shown is a western blot of whole-cell extracts prepared from the indicated strains. CbfA was visualized with the monoclonal antibody 7 F3 that detects CbfA-CTD. Numbers to the left indicate the sizes of the protein standards in kDa. c Complementation of the agnC overexpression phenotype in JH.D cells. Expression of agnC in the indicated strains was determined by qRT-PCR. Expression levels in JH.D cells and JH.D transformants were compared to AX2 wild-type cells and are expressed as “fold change” of expression, meaning that values >1 represent overexpression of genes in the JH.D strains and a value of 1 would indicate complete reversion of the overexpression in JH.D cells. Values are means from six independent cultures ± SD. **p < 0.01, relative to control AX2 cells (Student’s t-test)

Fig. 4

Expression of TRE5-A in knockout mutants of RNAi components. Expression of TRE5-A ORF1 was analyzed by qRT-PCR in the indicated knockout mutants of Argonaute genes and the Dicer-like protein DrnB. Phenotype reversion in agnC and agnE knockouts was accomplished using TAP-tagged agnC and agnE overexpressed in the respective mutants (agnC ⁻[agnC ^OE] and agnE ⁻[ agnE ^OE]). TRE5-A expression in JH.D cells is shown for comparison. Expression levels were compared to AX2 wild-type cells and are expressed as fold change of expression, meaning that values >1 represent overexpression of genes in the mutants. Data represent means from three independent cultures ± SD. *p < 0.05, **p < 0.01 relative to AX2 cells (Student’s t-test)

Fig. 5

TRE5-A expression in agnC ^GA mutants. a Construction of agnC “gene activation” mutants. The agnC locus on chromosome 2 is indicated by nucleotide positions. The gene activation cassette consisted of a hybrid actin6/actin15 promoter (arrows indicate transcription direction). The BamHI arm contained a 1200 bp DNA fragment covering the complete coding sequence of gene DDB_G0271884, including 273 bp of upstream sequence. The HindIII arm contained 1200 bp of agnC coding sequence, including the original translation start site. After double-recombination of the agnC ^GA vector with genomic DNA, the expression of agnC was driven by the act15 promoter, whereas expression of the neighboring gene DDB_G0271884 was unaffected. b Semi-quantitative RT-PCR analysis of RNA from AX2, JH.D, and three independent agnC ^GA mutants demonstrating overexpression of agnC, normal expression of the neighboring gene DDB_G0271884 and gpdA (loading control), and silencing of TRE5-A (ORF1 and ORF2 sequences). NTC: no template control. c Quantitative RT-PCR of TRE5-A (ORF1) expression on RNA from JH.D and three agnC ^GA mutants. Expression levels were compared to AX2 cells and are expressed as fold change of expression, meaning that values <1 represent lower levels of TRE5-A in the mutants relative to wild-type AX2 cells. Data represent means from four independent cultures of the indicated strains ± SD

Fig. 6

Outline of the TRE trap retrotransposition assay. The TRE ^trap gene is a modified version of the pyr56 gene, which codes for the D. discoideum UMP synthase. The pyr56 gene contains an intron (dashed line; SD: splice donor site, SA: splice acceptor site) into shich a tRNA gene is inserted as bait for the integration of TRE5-A. The TRE ^trap gene is transformed into the ura⁻ strain DH1 that has a complete deletion of the pyr56 gene. The TRE ^trap gene converts transformants to ura⁺ because the intron is functionally spliced. If an element of the endogenous TRE5-A population targets the tRNA gene in the TRE ^trap gene for integration, the TRE ^trap gene is disrupted even if the integration actually occurs in the intronic sequence. The resulting ura⁻ cells gain resistance to the drug 5-fluoroorotic acid (5-FOA) and grow out clonally if uracil is added to the medium [18]

Fig. 7

Retrotransposition of the TRE5-A population in agnC overexpressing strains. a Semi-quantitative RT-PCR analysis of RNA from DH1 and three independent DH1[agnC ^GA] mutants. Note that agnC was overexpressed comapred to DH1. Expression of the neighboring gene DDB_G0271884 (compare Figure 5) was not affected, whereas expression of TRE5-A ORF1 was reduced in the agnC ^GA mutants. Expression of the house-keeping gene gpdA is shown as control. NTC: no template control. b DH1 and DH1[agnC ^GA] cells were transformed with either the empty TRE ^trap gene (i.e., no tRNA gene inserted in the intron) or the TRE ^trap gene containing a Val ^UAC tRNA gene. In this TRE trap assay, one clone of DH1 transformants and two independent clones of DH1[agnC ^GA] cells (clones #1 and #2) were analyzed for TRE5-A retrotransposition. For each strain, five plates containing 10⁷ cells each were prepared. Cells were cultured in FM medium containing 5-FOA and uracil until clones appeared. Clones were counted and results are presented as retrotransposition frequency ± SD. ***p < 0.001 (Student’s t-test). This assay was reproduced once with similar results