Fig. 6

PML-I shuttles to the cytoplasm to suppress L1 by promoting the degradation of ORF1p. (A) PML mutants were generated encompassing the CDE (aa positions 596–882) and tagged with both an SV40 NLS and the PML-I NES at the N-terminus. Localizations of the mutants are shown in Supplementary Figure S14A. The mutants lack the C-terminal RBCC domain of PML. (B) Loss of PML elevates L1 activity which can be reversed with the addback of PML-I and the NES-PML mutant (n = 3). Resultant plates from the assay were stained with 0.5% crystal violet and quantified. Data was analysed by a repeated measures one-way ANOVA and then a Tukey's multiple comparisons test between groups. Asterisks indicate the following: ****P< 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. (C) Transfection of the human L1 retrotransposition vector led to cytoplasmic PML puncta forming. PML co-localized partly with TREX1 in the cytoplasm after L1 retroelements were active. (D) The nucleocytoplasmic shuttling of PML-I is CRM1-dependent. U2OS cells expressing GFP-PML-I were transfected with empty vector or L1 expression vector (L1.3, JM101.1) treated for 3 h with or without CRM1 inhibitor Leptomycin B (10 ng/ml). Cytoplasmic puncta of GFP-PML-I are indicated by the white the arrows. Scale bars represent 10 μm for images in (C) and (D). (E) U2OS cells (WT and PML KO) were transfected with an empty vector (-) or the L1 element with T7-tagged ORF1p. Cells were then treated with different concentrations of ubiquitin-proteasome inhibitor MG132 for 10 h and T7-ORF1p protein levels were examined.