14 research outputs found
Nuclear Importation of Mariner Transposases among Eukaryotes: Motif Requirements and Homo-Protein Interactions
Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm
Regulation of mariner transposition: the peculiar case of Mos1.
BACKGROUND: Mariner elements represent the most successful family of autonomous DNA transposons, being present in various plant and animal genomes, including humans. The introduction and co-evolution of mariners within host genomes imply a strict regulation of the transposon activity. Biochemical data accumulated during the past decade have led to a convergent picture of the transposition cycle of mariner elements, suggesting that mariner transposition does not rely on host-specific factors. This model does not account for differences of transposition efficiency in human cells between mariners. We thus wondered whether apparent similarities in transposition cycle could hide differences in the intrinsic parameters that control mariner transposition. PRINCIPAL FINDINGS: We find that Mos1 transposase concentrations in excess to the Mos1 ends prevent the paired-end complex assembly. However, we observe that Mos1 transposition is not impaired by transposase high concentration, dismissing the idea that transposase over production plays an obligatory role in the down-regulation of mariner transposition. Our main finding is that the paired-end complex is formed in a cooperative way, regardless of the transposase concentration. We also show that an element framed by two identical ITRs (Inverted Terminal Repeats) is more efficient in driving transposition than an element framed by two different ITRs (i.e. the natural Mos1 copy), the latter being more sensitive to transposase concentration variations. Finally, we show that the current Mos1 ITRs correspond to the ancestral ones. CONCLUSIONS: We provide new insights on intrinsic properties supporting the self-regulation of the Mos1 element. These properties (transposase specific activity, aggregation, ITR sequences, transposase concentration/transposon copy number ratio...) could have played a role in the dynamics of host-genomes invasion by Mos1, accounting (at least in part) for the current low copy number of Mos1 within host genomes
Physical properties of DNA components affecting the transposition efficiency of the mariner Mos1 element
International audiencePrevious studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1 mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active
pBC-3T3 transposition rates.
<p><i>Top panels</i>: Transposition rates were assayed with various amounts of pBC-3T3 (1.6 to 16 nM) and three MOS1 concentrations: 10 nM (grey bars), 100 nM (checkerboard bars), 1 µM (hatched bars). Each bar is the mean (+/− SD) of at least five independent assays. Kruskal-Wallis and post hoc tests were used to monitor the significance of the differences. ns: no statistic differences. (*) p<0.05. (**) p<0.005. <i>Bottom panel</i>: Transposition rates (from the top panel) are plotted as a function of the amount of pBC-3T3 used in the assay, for each transposase concentration. 10 nM MOS1: black line, 100 nM: gray line, 1 µM: dotted line.</p
5′ ITR sequences of the <i>Mos1</i>-tribe elements.
<p>The accession numbers are given on the right. The host species is given in brackets. Ds: <i>Drosophila simulans</i>; Md: <i>Musca domestica</i>; Zt: <i>Zaprionus tuberculatus</i>; Dm: <i>Drosophila mauritiana</i>; Dse: <i>Drosophila sechelia</i>; Dt: <i>Drosophila tsacasi</i>; Bg: <i>Blattella germanica.</i> The differences between the consensus and the element are in underlined lower cases letters. The Mos1 sequence and accession number are in bold.</p
pBC-5T3 transposition rates.
<p><i>Top panels</i>: Transposition rates were assayed with various amounts of pBC-5T3 (3.2 to 16 nM) and three MOS1 concentrations: 10 nM (grey bars), 100 nM (checkerboard bars), 1 µM (hatched bars). Each bar is the mean (+/− SD) of at least five independent assays. Kruskal-Wallis and post hoc tests were used to monitor the significance of the differences. ns: no statistic differences. (*) p<0.05. (**) p<0.005. <i>Bottom panel</i>: Transposition rates (from the top panel) are plotted as a function of the amount of pBC-5T3 used in the assay, for each transposase concentration. 10 nM MOS1: black line, 100 nM: gray line, 1 µM: dotted line.</p
The PEC contains two ITRs and two transposases. A.
<p>Sequence of the double-strand oligonucleotide (short-PC: short pre-cleaved ITR) used in EMSAs. TS: transferred strand; NTS: non-transferred strand. The 3′ITR is shown in bold, with a 3-bases overhang in 3′ of the TS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043365#pone.0043365-Dawson1" target="_blank">[14]</a>. Inner sequence: narrow letters. The long-PC oligonucleotide (long pre-cleaved ITR) has the same sequence, with a longer inner sequence. An asterisk marks the position of the <sup>32</sup>P labeling. <b>B.</b> Short/long transposase analyses. EMSAs were performed with 250 nM of short-PC (short pre-cleaved) labeled ITR (as a probe), and 250 nM of purified MBP-MOS1. Lane 1: probe alone, Lane 2: complexes assembly without factor-Xa treatment. Lanes 3 to 5, complexes were subjected to factor-Xa cleavage (1H, 2H and 5H respectively) before electrophoresis. The MOS1 dimer in the PEC was assayed here. We have taken advantage of the fact that the MBP-MOS1 fusion protein contains a cleavage site for factor-Xa between MBP and MOS1. If the PEC contains MBP-MOS1 dimer, then a three-band pattern is expected after cleavage of factor-Xa: one band containing uncleaved MBP-MOS1 (in native PEC, as seen at T = 0), one band containing one cleaved and one uncleaved MOS1 in the complex, and one band containing two cleaved MOS1s in the complex. SEC2 disappears as a result of the factor-Xa cleavage, since it contains two MBP-MOS1 molecules that are converted into MOS1 molecules by the release of the MBP moiety, giving bands with faster mobility in electrophoresis. The proteins present in the various PECs are drawn on the right. <b>C.</b> Short/long ITR analyses. EMSAs were performed with 250 nM of purified MBP-MOS1 and 250 nM of short/long ITR combinations (as indicated). The number of ITRs in the PEC is assayed here. The ITRs present in the complexes are drawn on the right. Short-PC: short pre-cleaved ITR. Long-PC: long pre-cleaved ITR. S*: labeled short-PC. L*: labeled long-PC.</p
ITR sequence analyses.
<p>Sequences of the <i>Mos1-</i>tribe ITRs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043365#pone-0043365-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043365#pone-0043365-t002" target="_blank">2</a>) were aligned to obtain 5′ and 3′ITRs logos and consensus. The majority rule was used to define variable positions, which are 1, 2, 3, 14, 18, and 26 in the 5′ITR and 1, 2, 3, and 14 in the 3′ITR. Doing so, the 24 conserved positions in <i>Mos1</i> ITRs are conserved in the consensus ITRs (black letters). When compared to each other, 5′ and 3′ consensus ITRs contain three clear differences at positions 1, 18, and 26, which are the same as that found in <i>Mos1</i> ITRs (blue letters). Position 16 (purple letter) in the 5′ consensus remains ambiguous, being either a G (as in the 5′ <i>Mos1</i> ITR) or a T (as in the 3′ <i>Mos1</i> ITR).</p
<i>Mariner</i> transposition cycle.
<p>A representative <i>mariner</i> element is depicted at the top of the figure, with its main components, <i>i.e.</i> the transposase coding sequence (in grey), the inverted terminal repeats (5′ITR and 3′ITR, in orange), and the TA dinucleotide flanking the element (landmark for transposition). According to published data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043365#pone.0043365-Carpentier1" target="_blank">[7] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043365#pone.0043365-Richardson1" target="_blank">[18]</a>, <i>Mos1</i> transposition consists of five main steps: (<b>1</b>) dimerization of MOS1 proteins, the <i>Mos1</i> transposase (green circle) for subsequent ITR binding, thus forming SEC2 (Single-end complex 2). (<b>2</b>) Synaptic complex assembly is obtained by the addition of the second ITR to SEC2, thus forming the PEC (Paired-end complex). (<b>3</b>) DNA strands are then cleaved by the transposase, promoting the excision. Once the PIC (Pre-integration complex) has been produced, the capture of the target DNA occurs (<b>4</b>), followed by the integration of the element into a TA target dinucleotide (<b>5</b>). The results presented in this study argue for generalized this model to all <i>mariner</i> elements.</p