Interplay between RNASEH2 and MOV10 controls LINE-1 retrotransposition

Figures

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  Figure 1.. Interaction between RNASEH2 and MOV10 in an RNA-dependent manner. (A) Immunoblot analysis of RNASEH2 subunits in both cell lysates and eluates in co-immunoprecipitation experiments. Corresponding expression constructs were co-transfected into HeLa cells. At 48 h post-transfection, the cells were harvested for the co-immunoprecipitation assays. Total cell lysates were treated with or without RNase A prior to the immunoprecipitation procedure. GAPDH served as the loading control, and molecular weight standards are indicated in kDa. (B) Immunoblot analysis of endogenous MOV10 and RNASEH2A followed by co-immunoprecipitation with either anti-IgG or anti-MOV10 antibody. (C) Immunoblot analysis results showing levels of endogenous MOV10 and RNASEH2A in nuclear and cytosol fractions. (D) Western blot analysis of total cell extracts prepared from shRNA-mediated knockdown HeLa cells using anti-MOV10, anti-RNASEH2A and anti-GAPDH antibody. The knockdown efficiency of either endogenous MOV10 or RNASEH2A was confirmed. GAPDH served as the loading control. (E) Immunoblot analysis showing levels of RNASEH2 components in either control or MOV10-deficient HeLa cells following…

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  Figure 2.. Suppression of L1 mobility by interplay between RNASEH2 and MOV10. (A) A schematic drawing of the L1 construct and an overview of the L1 retrotransposition assay. The L1-neoTET expression cassette is a complete retrocompetent L1 element that encodes L1 ORF1p and L1 ORF2p driven by a CMV promoter. The L1 construct carries a retrotransposition indicator cassette near its 3′ UTR. The cassette contains the neomycin phosphotransferase gene (neor) interrupted by a tetrahymena self-splicing intron (36) in an anti-sense orientation relative to the transcriptional orientation of the L1 element. The intron is spliced out of the full-length L1 RNA transcript. The spliced L1 RNA is reverse-transcribed, and the resulting cDNA is integrated into the genome. Retrotransposition of the resulting RNA leads to expression of the indicator gene, conferring G418-resistance to host cells. (B) L1 assays were carried out following co-transfection of the indicated expression vectors with the L1-neoTET expression cassette at a ratio of 1:1 into HeLa cells. The cells were subjected to selection for up to 14 days in the presence of G418 (1 mg/ml). Following G418 selection, G418-resistant foci were stained…

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  Figure 3.. Association of RNASEH2 with L1 RNAs in MOV10-dependent manner. (A) Immunofluorescent confocal microscopy (including Z-stacks) showing the subcellular distribution of L1-derived RNAs, MOV10 and RNASEH2A. HeLa cells were transfected with pAD3TE1, a plasmid expressing a nuclear localized MS2-GFP fusion protein. These microscopy results revealed L1 RNA accumulation in both cytoplasmic and nuclear foci by exploiting the 24 MS2-binding sites in the pAD3TE1-derived L1 RNA. For detection of MOV10 and RNASEH2A, either anti-MOV10 antibody or anti-RNASEH2A antibody was used as a primary antibody, respectively. Green and red lines indicate corresponding points in the orthogonal planes, showing localization of the label within the pictured cell. The scale bar represents 10 μm. (B) RNA immunoprecipitation carried out in HeLa cells co-transfected with either N-terminally HA-tagged MOV10 or N-terminally HA-tagged MOV10K530A and the L1-luc cassette (pYX017). At 48 h post-transfection, whole cell extracts were subjected to RNA immunoprecipitation using anti-HA antibody. The RT-qPCR was carried out using primers specific to spliced Fluc cassette. The relative abundances of the immunoprecipitated…

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  Figure 4.. Accumulation of L1-derived RNA-DNA hybrids in MOV10- and RNASEH2A-deficient cells. (A) Immunofluorescence analysis of RNA-DNA hybrids using S9.6 antibody showing the control, MOV10- and RNASEH2A-depleted cells, respectively. A merge of the two channels is shown with the nucleus stained with DAPI. The scale bar represents 10 μm. (B) A schematic comparison of DRIP-qPCR and DRIP-RT-qPCR procedure. (C) Results of DRIP‐qPCR following immunoprecipitation using the S9.6 antibody upon 36 h post-transfection with pYX017 (L1-luc). DRIP‐qPCR was performed using primers specific to the spliced Fluc region in transfected HeLa cells. The relative abundance of RNA-DNA hybrids immunoprecipitated is represented as fold change over the input material. The data were normalized to MDM2 levels. The data are represented as mean ± SD values from three independent experiments analyzed by two-tailed Student’s t-test with p-values indicated. (D) DRIP-RT-qPCR performed in HeLa cells transfected with pYX017 (L1-luc). Samples were harvested at 24 h post-transfection and subjected to immunoprecipitation using the S9.6 antibody. The resulting RNAs in the immunoprecipitates were quantified by RT-qPCR using…

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  Figure 5.. Contribution of MOV10 as an interaction mediator between L1 ORF1p and RNASEH2 for L1 suppression. (A) (Upper panel) A schematic drawing of MOV10 variants. (Lower panel) L1 retrotransposition assays using L1-luc cassette performed in HeLa cells co-transfected with corresponding MOV10 variants and pYX017 at a ratio of 1:1. Treatment of d4T (50 μM) served as a negative control. Data are presented as the mean ± SD of three independent experiments and were analyzed with two-tailed Student’s t-test with the p-values indicated. (B) Immunoblot results showing the levels of MOV10 variants in both cell lysates and eluates followed by co-immunoprecipitation. Both L1 ORF1p expression vector and the corresponding MOV10 variants were co-transfected into HeLa cells. At 48 h post-transfection, the cells were harvested for co-immunoprecipitation assays using anti-FLAG antibody. (C–E) Western blot analyses showing levels of RNASEH2 components in HeLa cells co-transfected with indicated combinations of expression plasmids following co-immunoprecipitation with anti-HA antibody. GAPDH served as the loading control, and molecular weight standards are indicated in kDa.

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  Figure 6.. A causal relationship between L1 restriction and RA-related gene expressions. (A) L1 retrotransposition assays using L1-neoTET cassette performed in synovial SW982 cells co-transfected with corresponding expression vectors and the L1-neoTET expression cassette at a ratio of 1:1. The transfected cells were subjected to selection for 10 ∼ 12 days with G418 (1 mg/ml). Following selection, G418-resistant foci were stained with crystal violet solution. Representative culture dishes for each condition are shown. Quantitation of the L1 assays was plotted. The Y-axis depicts the number of G418-resistant foci per 40,000 cells. Data are shown as the mean ± SD from an experiment with three replicates and were analyzed by two-tailed Student’s t-test with the p-values indicated. (B–D) Levels of inflammatory cytokines and MMP-3 activity. In parallel to L1 assay shown in (A), cell culture supernatants were harvested at 72 h post-transfection and analyzed by enzyme-linked immunosorbent assay (ELISA) to detect (B) IL-6 and (C) TNF-α levels in SW982 cells, and fluorometric immunocapture assays were performed to detect (D) MMP-3 activity. Data are presented as the mean ± SD of three independent…