Demattei MV et al. (2011), Nuclear importation of Mariner transposases amo...

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PLoS One 2011 Jan 01;68:e23693. doi: 10.1371/journal.pone.0023693.

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Nuclear importation of Mariner transposases among eukaryotes: motif requirements and homo-protein interactions.

Demattei MV , Hedhili S , Sinzelle L , Bressac C , Casteret S , Moiré N , Cambefort J , Thomas X , Pollet N , Gantet P , Bigot Y .

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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.

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Species referenced: Xenopus
Genes referenced: rfk

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	Figure 1. Sequence features of MOS1, HIMAR1 and MCMAR1.(a) Alignment of the amino acid sequences of three MLE transposases. Basic residues, lysine (K), arginine (R) and histidine (H) are typed in red. Acidic residues, aspartate (D) and glutamate (E) are typed in blue. The C-terminal region with an acidic pI is boxed. Motifs corresponding to potential NLS in MLE sequences are boxed in grey and indicated above the sequence. The four putative monopartite and bipartite NLSs found in silico in MOS1 are referred to as M1, M2, M3 and M4. M1 is a putative bipartite NLS (RFK---KPPKR), whereas M2 is monopartite (KPPKR). Monopartite NLSs found in the HIMAR1 sequence are referred to as H1 and H2. HTH1 and HTH2 are located in the N-terminal domain. The regions corresponding to the Δ1 and the Δ3 segments are underlined. (b) Schematic representation of the transposase-GFP fusions used to explore the subcellular localization of MOS1, HIMAR1 and MCMAR1 in onion epidermal cells, drosophila cells, human cells and X. tropicalis cells. The amino acid at borders of each transposase segments are indicated in the right margin. (c) Schematic representation of the different truncated and mutant versions of MOS1-GFP fusion used in the study. The amino acid length of each transposase segment as well as the fluorescence results are indicated in the right panel. Mutated residues in each fusion are located with an “Ψ”.
	Figure 2. Localization of MOS1 in host cells.The GFP fluorescence patterns are analyzed in human HeLa cells, amphibian cells, insect cells, and onion epidermal cells transfected with plasmids expressing only GFP or a MOS1-FL GFP fusion. The left panels show GFP fluorescence, the middle panels show the nuclear genomic DNA staining by DAPI, the right panels correspond to merge pictures. For onion epidermal cells, the pattern of a GFP-SV40.NLS fusion was also verified since the localization of the MOS1-FL GFP was not homogenous in the nuclei. The scale bars correspond to 100 µm in HeLa cells, 200 µm in amphibian cells, 50 µm in insect cells, and 200 µm in plant cells.
	Figure 3. Localization of transposase Δ3 GFP fusions in host cells.The GFP fluorescence patterns are analyzed in human HeLa cells and amphibian cells transfected with plasmids expressing only MOS1-Δ3 GFP, HIMAR1-Δ3 GFP or MCMAR1-Δ3 GFP fusions. The left panels show GFP fluorescence, the middle panels show the nuclear genomic DNA stained by DAPI, the right panels correspond to merge pictures.
	Figure 4. Comparisons of fluorescence patterns between MOS1 Δ2-GFP and two variants.The GFP fluorescence patterns are analyzed in HeLa cells transfected with plasmids expressing only GFP or MOS1 Δ2-GFP as controls, MOS1 Δ4-GFP or MOS1 Δ2 R132A-GFP variants. The top panels show GFP fluorescence, the middle panels show the nuclear genomic DNA staining by DAPI, the bottom panels correspond to merge pictures. The observation of aggregates was already reported in the literature with the transposon protein MURB (about 26 kDa; [20]). This accessory protein is encoded by the plant transposon MuDR and assembles aggregates in the cytoplasm when it has an important expression rate.
	Figure 5. Localization of a highly expressed MOS1 in host cells.The GFP fluorescence patterns are analyzed in HeLa cells transfected with plasmids expressing only GFP or MOS1-FL GFP as controls, and MOS1-FLV2-GFP. In both MOS1-FLV2-GFP patterns, exposure times were 5-fold and 10-fold shorter than those obtained with GFP or MOS1-FL GFP. The top panels show GFP fluorescence, the middle panels show the nuclear genomic DNA staining by DAPI, and the bottom panels correspond to merge pictures.
	Figure 6. Molecular weight patterns of over-expressed MOS1 fusion in HeLa cells.Proteins extracted from HeLa cells over-expressing V5-MOS1 or MOS1V2-GFP were analyzed by immunoblotting after separation by polyacrylamide gel electrophoresis. V5-MOS1 and MOS1V2-GFP were repectively revealed by first hybridizing a mouse anti-V5 monoclonal antibody or a rabbit polyclonal anti-GFP a mouse a rabbit polyclonal anti-GFP. The filters were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG, followed by development using enhanced chemiluminescence.

References [+] :

Augé-Gouillou, Mariner Mos1 transposase dimerizes prior to ITR binding. 2005, Pubmed