An active murine transposon family pair: retrotransposition of "master" MusD copies and ETn trans-mobilization

Abstract

The ETn (Early Transposon) elements are among the most active murine mobile sequences, being responsible for a series of mutations by insertion in vivo. Yet they are noncoding, and it had long been suspected that ETn are mobilized in trans by coding-competent elements, most probably from the closely related MusD family of LTR-retrotransposons. A genome-wide in silico search for coding-competent MusD elements identified a total of nine such copies, which we cloned and marked to test their transpositional activity, using an ex vivo assay in heterologous cells. Three copies were found to be autonomous for transposition, with each gag, pro, and pol MusD gene absolutely required for mobility. These active MusD copies specifically trigger retrotransposition of marked ETn elements with high efficiency, by complementation in trans. Characterization of the structures of de novo transposed MusD and ETn marked elements, as well as of their integration sites, disclosed canonical retroviral-like retrotransposition, with 6-bp target site duplications common to both elements. These results highlight the parasitic molecular strategies that are used by the ETn elements for their mobility, and unambiguously identify their "master genes."

Figure 1.

Structure of the related MusD and ETn elements and rationale of the assay for retrotransposition. (A) Genomic organization of MusD, ETn type II and I elements. The LTRs (dark gray box) with a U3-R-U5 organization border three ORFs homologous to the retroviral gag, pro, and pol genes (light gray boxes). The transcription start site is marked with an arrow, and the signals necessary for retroviral reverse transcription are indicated: (PBS) primer binding site; (PPT) poly purine tract. ETn elements are closely related to MusD despite the replacement of the retroviral genes by a noncoding sequence (hatched box). ETn I elements differ from ETn II only in the LTRs and 5′-UTR region (squared boxes). (B) Schematic representation of a MusD/ETn element under the control of the CMV promoter and marked with the neo^TNF reporter gene (light gray), in which the neo gene placed in backward orientation with its own promoter (Pr) is rendered inactive by the presence of a forward intron (top), which should be spliced out in the transposition RNA intermediate (middle), thus resulting in an active neo gene in the de novo MusD/ETn insertion (bottom). (C) Experimental procedure for detection of MusD/ETn retrotransposition. Cells were transfected either with a neo^TNF-marked defective MusD/ETn together with an MusD expression vector, or with a full-length neo^TNF-marked MusD alone. Cells were amplified for a week, and retrotransposition events were detected upon G418 selection.

Figure 2.

Assay for MusD gene products and retrotransposition. (A) Structure of the MusD expression vector and neo^TNF-marked reporter. (B) Western blot analysis of the Gag-related gene products from the nine coding-competent MusD copies (see Table 1 for copy and accession nos). Lysates of cells transfected with a control plasmid (pCMVβ; mock) or the expression vectors for the coding-competent MusD copies were electrophoresed in a denaturing polyacrylamide gel (SDS-PAGE), blotted, and hybridized with a rabbit anti-serum directed against the Gag polyprotein. The apparent molecular masses of the major bands are indicated. (C) Assay for retrotransposition of the defective neo^TNF-marked MusD reporter upon transcomplementation by the coding-competent MusD copies. Retrotransposition was assayed in heterologous human cells upon cotransfection of the reporter with a control plasmid (pCMVβ) (data not shown) or expression vectors for the selected MusD elements. Results of the G418 selections are illustrated with images of the plates after the G418^R foci have been fixed and stained. The retrotransposition frequencies, as derived from the number of foci per seeded cells, are given for each construct as the mean of two to four independent experiments, carried out with at least 3 × 10⁶ cells, with standard errors indicated.

Figure 3.

Assay for autonomous MusD retrotransposition and characterization of the de novo insertions. (A) Each of the three MusD elements encoding functional proteins, as determined in the assay in trans, was marked with the neo^TNF indicator downstream from the pol ORF as schematized and assayed for its autonomous retrotransposition. Images of the plates and retrotransposition frequencies are given as in Figure 2 (two to three independent experiments, with standard errors indicated). (B) Structure and chromosomal localization of four transposed MusD copies. The complete characterization of transposed MusD copies and insertion sites was performed using G418^R clones from MusD-1 and MusD-6 marked elements assayed for transposition in HeLa cells. The structures of the marked MusD expression vector, of the intermediate MusD transcript, and of the de novo insertion (with reconstituted LTRs and target site duplication, TSD) are schematized, together with the corresponding empty target site. The sequences of four insertions are given below, with the [TG... CA] LTR borders and flanking DNA sequences indicated; target-site duplications (grayed) are found in all four cases, associated with complete LTRs. The GenBank Accession no. of each insertion site is given together with its chromosomal localization (with the R bands in white and the G bands in dark gray).

Figure 4.

MusD gene products required for retrotransposition. (A) Assay for MusD retrotransposition was performed as in Figure 3, with either the wild-type neo^TNF marked MusD-6 (WT) or the same element rendered defective for gag or pro (via an in-frame deletion in the corresponding gene so as not to alter translation of the downstream ORFs) or pol (via an out-of-frame deletion). The retrotransposition frequencies obtained for each construct are indicated. (B) Western blot analysis for cleavage of the MusD Gag polyprotein. Lysates of cells transfected with a control plasmid (mock) or the MusD constructs in A were electrophoresed (SDS-PAGE), blotted, and hybridized with a rabbit anti-serum directed against the Gag polyprotein. The apparent molecular mass for the major bands of the wild-type MusD is indicated.

Figure 5.

MusD-mediated retrotransposition of the ETn elements. (A) Assay for retrotransposition of neo^TNF-marked ETn elements (schematized on the left with their copy number indicated) (see also Table 1) upon transcomplementation with the autonomous MusD-6 copy (+MusD) or a control plasmid (none). Images of the plates and retrotransposition frequencies are given as in Figure 2 (two to three independent experiments, with standard errors indicated). Additional experiments included assays with a neo^TNF-marked defective IAP element (marked IAP-IL3) as well as complementation in trans with a functional IAP vector (RP23-92L23; +IAP) (Dewannieux et al. 2004). (B) Four ETn II insertions (originating from the ETn IIβ-1 element) are illustrated as in Figure 3, with the GenBank accession no. and chromosomal localization of each insertion site indicated.