ADAR1 restricts LINE-1 retrotransposition

Abstract

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.

Figure 1.

Results of the dual tag affinity purification procedure. Whole cell lysates from 293T cells transfected with either pADAR1-p150-V5 or pV5 empty vector and the proviral NL4-3 genome were subjected to immunoprecipitation (IP) with the NiNTA Magnetic Beads followed by several washes and elution of His-tagged native protein complex from the beads. The eluted protein complex was then subjected to a second step of IP using the anti-V5-tag magnetic beads followed by several washes with NP40 buffer. The resulting beads were resuspended in SDS loading buffer and the proteins were separated by SDS-PAGE, visualized by Sypro–Ruby staining and subjected to LC-MS/MS. The single asterisk indicates the p150 isoform of ADAR1-V5, the double asterisk corresponds to the ADAR1-V5 protein of about 110 kDa produced by translation machinery from a second methionine at position 296, as previously reported (69). Both isoforms migrate as doublets of bands possibly due to post-translation modification as originally observed (18). All the 14 non-ribosomal proteins identified as putative ADAR1-interacting factors are indicated.

Figure 2.

Validation of the results of the nano-LC MS/MS data by co-immunoprecipitation (Co-IP) analysis. Lysates of 293T cells co-transfected with pADAR1-p150-V5 or pV5 empty vector and the proviral NL4-3 genome were subjected to IP with anti-V5-tag magnetic beads (IP V5) followed by Western blot (WB) analysis with anti-V5, anti-NONO, anti-PSF, anti-hnRNP L and anti-NCL, anti-HSPA1A and anti-PABPC1 antibodies (left panel). Total cell lysates were mock-treated or RNase (A+V1)-treated prior to IP. WB analysis of 20 μg of cell lysate inputs (input) is shown. Complete RNA digestion after RNase-treatment of the cell extract (lysate) prior IP was confirmed by loading a fraction of the treated and untreated cell extract onto 1% agarose/formaldehyde gel followed by electrophoresis and ethidium bromide staining (right panel). The 28S and 18S rRNA are indicated.

Figure 3.

Validation of the results of the nano-LC MS/MS data by reciprocal IP. Lysate of 293T cells was subjected to IP using either IgGs or anti-NONO or anti-PSF or anti- hnRNP L or anti-NCL or anti-HSPA1A or anti-PABPC1 antibodies followed by WB analysis using specific antibodies. WB analysis of 20 μg of cell lysate inputs (input) is shown.

Figure 4.

ADAR1 regulates L1 retrotransposition. (A) HeLa cells were transiently transfected with specific anti-ADAR1 shRNA plasmids or controls (empty vector or scramble shRNA plasmid). WB analysis using anti-ADAR1 and anti-GAPDH antibodies of total cell extract prepared from the transfected cells confirms the specific knock-down of ADAR1 protein 72 h after transfection. (B) Schematic representation of the pYX014 cassette and the rational of L1 retrotransposition assay, as previously described (36). The Fluc indicator cassette is cloned into the L1 3′UTR in antisense orientation relative to L1 transcription. This cassette has its own promoter (P2) and the Fluc coding sequence is interrupted by a gamma globin intron. An Rluc cassette, containing its own promoter (P3), is incorporated into the backbone of the plasmid and allows measurement of the transfection efficiency. The Fluc gene can by expressed only when the L1 transcript (L1 pre-mRNA) is spliced (L1 mRNA), reverse transcribed and inserted into genomic DNA. (C) Representative Fluc retrotransposition assay results: HeLa cells previously transfected with anti-ADAR1 shRNA plasmids or control plasmids (empty vector or scramble shRNA plasmid) were then transfected with the luciferase assay vector pYX014. Four days after pYX014 transfection and puromycin selection, cells were lysed for luminescence analysis. The X-axis indicates the construct name. The Y-axis indicates the L1 activity measured as the ratio of the Fluc/Rluc values as previously reported (36). Data are reported as the mean ± SD from three independent experiments. Asterisks indicate statistically significant differences from the scr shRNA sample (P-values were calculated by two-tailed t-test and are indicated above each histogram, *P < 0.05). (D) Representative G418 retrotransposition assay results: HeLa cells previously transfected with anti-ADAR1 shRNA plasmids or control plasmids (empty vector or scramble shRNA plasmid) were further transfected with the pJM101/L1.3 cassette. Seventy-two hours later, cells were grown in media supplemented with G418 and after ∼10 days of G418 selection, the remaining cells were fixed and then stained with crystal violet to facilitate the visualization and allow the counting of the colonies formed for individual retrotransposition events. The X-axis in the graph indicates the constructs name. The Y-axis indicates the number of G418-resistant foci per cell culture dish. Data are reported as the mean ± SD from three technical replicates of a single representative experiment. Asterisks indicate statistically significant differences from the scr shRNA sample (P-values were calculated by two-tailed t-test and are indicated above each histogram, *P < 0.05). The experiment was conducted five times (biological replicates) with similar results. (E) Representative T25 flasks with crystal violet-stained G418-resitant HeLa colonies of the experiments described in (D) are shown. (F) Representative T25 flasks with crystal violet-stained G418-resitant HeLa colonies of experiments performed as described in (D) by substituting the pJM101/L1.3 cassette with the pcDNA3.1 plasmid are shown.

Figure 5.

Inhibition of LINE-1 retrotransposition by ADAR1 over-expression. (A) Representative Fluc retrotransposition assay results: Hela cells were co-transfected with a constant amount of the pYX014 retrotransposition cassette and a decreasing amount of pADAR1-p150-V5 plasmid ranging from a 1:1 to 25:1 ratio. Four days post-transfection, cells were lysed for both luminescence analysis to measure L1 activity (top panel) and for WB analysis (bottom panel) with anti-V5 and anti-GAPDH antibodies. In the top panel, the X-axis indicates the construct name and the plasmids ratio. The Y-axis indicates the L1 activity measured as the ratio of the Fluc/Rluc values as previously reported (36). Data are reported as the mean ± SD from three independent experiments. (B) HeLa cells were co-transfected with the pYX014 retrotransposition cassette together with a 5:1 ratio of either pADAR1-p150-V5 plasmid or pADAR1-Dcat plasmid expressing a mutant of ADAR1 lacking the deaminase domain (bottom panel) or pV5 empty vector. Four days post-transfection, cells were lysed for both luminescence analysis to measure L1 activity (top panel) and for WB analysis (middle panel) with anti-V5, anti- eIF-2α and anti- P- eIF-2α antibodies. As positive control, HeLa cells were treated with 1 μM sodium arsenite for 48 h previously reported to induce eIF-2α phoshorylation (middle panel). The ratio P- eIF-2α /total eIF-2α is indicated. In the top panel, the X-axis indicates the construct name. The Y-axis indicates the L1 activity measured as the ratio of the Fluc/Rluc values as previously reported (36). Data are reported as the mean ± SD from four independent experiments. Asterisks indicate statistically significant differences from the pV5 sample (P-values were calculated by two-tailed t-test and are indicated above each histogram, **P < 0.005).

Figure 6.

ADAR1 binds the basal L1 RNP complex without affecting the accumulation of its components. (A) Lysates of 293T cells transfected with either pV5-ADAR1-p150 or pV5 empty vector together with pES2TE1 cassette were subjected to IP with anti-V5-tag magnetic beads (IP V5) followed by WB analysis with anti-V5 and anti-T7 tag antibodies. WB analysis of 7 μg of cell lysate inputs (input) is shown. (B) Total RNA isolated from a fraction of the immunocomplexes (IP V5) obtained in (A) and total RNA isolated from the co-transfected 293T cells with pV5-ADAR1-p150 plasmid together with pES2TE1 cassette (input) were subjected to RT-PCR analysis using specific primers to amplify fragments of the ectopically expressed L1 RNA and actin mRNA. (C) Results of the RT-qPCR experiments: HeLa cells previously transfected with anti-ADAR1 shRNAs or scr shRNA plasmids were further transfected with the pES2TE1 cassette. Five days post-transfection the ectopically expressed L1 RNA level was determined. The X-axis indicates the construct name. The Y-axis indicates the relative level of L1 RNA generated from the pES2TE1 cassette and normalized for the hygromycin mRNA level generated from the same cassette. The ratio L1 RNA/HYG mRNA of the scr shRNA sample is assigned as 1. Data are reported as the mean ± SD from three independent experiments. (D) ORF1p protein expression: total cell extract prepared from the HeLa cells transfected as described in (C) were analyzed by WB using the anti-T7 and the anti-GAPDH antibodies. One WB analysis representative of three independent experiments is shown.