Fig 2: ZCCHC3 decreased LINE-1 insertions. (A) Diagrams of Taqman real-time PCR to detect novel LINE-1 integrates. The real-time PCR was performed with two primers and one probe across the intron to detect the integrated novel LINE-1. (B) Overexpression of ZCCHC3 decreased novel LINE-1 integrates. HEK293T cells were transfected with pWA367 and ZCCHC3 DNA. Four days post-transfection, genomic DNA was extracted and analyzed for Taqman real-time PCR (top panel). Expression of the Flag-ZCCHC3 was analyzed by immunoblot, and Tubulin was detected as an internal control (bottom panel). (C) Knockdown of ZCCHC3 increased novel LINE-1 integrates. HEK293T cells were transfected with pWA367 together with the siRNA targeting ZCCHC3. Four days post-transfection, genomic DNA was extracted and analyzed for Taqman real-time PCR (top panel). Expression of the ZCCHC3 was analyzed by immunoblot, and Tubulin was detected as an internal control (bottom panel). Data are representative of at least three independent experiments, values are expressed as means ± SD, and representative blots and images are shown.

Fig 3: The zinc-finger domain is essential for ZCCHC3-mediated LINE-1 restriction. (A) The illustration of ZCCHC3 truncation. (B) ZCCHC3 dc does not inhibit LINE-1. HEK293T cells were transfected with pWA367 and either empty vector, Flag-ZCCHC3 or Flag-ZCCHC3-dc. 4 days post-transfection, cell lysates were analyzed for luminescence. (C) ZCCHC3 dc does not diminish LINE-1 RNA and ORF1p. Two days post-transfection, total RNA was extracted and LINE-1 RNA was normalized by the GAPDH and analyzed for Taqman real-time PCR (left panel). Expression of the LINE-1-ORF1p and Flag-ZCCHC3 were analyzed by immunoblot, and Tubulin was detected as an internal control (right panel). (D) ZCCHC3 dc does not interact with LINE-1 ORF1p. HEK293T cells were transfected with Myc-ORF1p and Flag-ZCCHC3 or Flag-ZCCHC3-dc. The cells were collected and lysed 48 h post-transfection. Equal amounts of supernatant were incubated with Myc antibody(mouse), Myc-ORF1p, and Flag-ZCCHC3 or Flag-ZCCHC3-dc and were analyzed by Western blots using antibodies against Myc and Flag(rabbit). (E) ZCCHC3 dc does not bind to LINE-1 RNA. HEK293T cells were co-transfected with pWA367 DNA and ZCCHC3-WT or ZCCHC3-dC DNA. Forty-eight hours later, an equal amount of cell lysis was used to detect LINE-1 RNA by RNA-IP. LINE-1 RNA pulled down by ZCCHC3 was quantified by RT–qPCR. (F) ZCCHC3 dc is not located with LINE-1 ORF1p. Hela cells were transfected with an empty vector or vector encoding Flag-ZCCHC3 or Flag-ZCCHC3-dc and Myc-ORF1p. Forty-eight hours post-transfection, Myc-ORF1p and Flag ZCCHC3 or Flag-ZCCHC3-dc were detected by indirect immunofluorescence staining. Data are representative of at least three independent experiments, values are expressed as means ± SD, and representative blots and images are shown.

Fig 4: ZCCHC3 interacts with LINE-1 in the cytoplasm. (A) ZCCHC3 is co-located with LINE-1 ORF1p. Hela cells were transfected with pWA367 plasmid and Flag-ZCCHC3 DNA. Forty-eight hours post-transfection, LINE-1 ORF1p and Flag ZCCHC3 were detected by indirect immunofluorescence staining. (B) ZCCHC3 interacts with LINE-1 ORF1p. HEK293T cells were transfected with pWA367 plasmid and Flag-ZCCHC3 DNA. The cells were collected and lysed 48 h post-transfection. Equal amounts of supernatant were incubated with Flag antibody or lgG (mouse), LINE-1 ORF1p, and Flag-ZCCHC3 were analyzed by Western blots using antibodies against ORF1p and Flag (rabbit). (C) The interaction between ZCCHC3 and LINE-1 ORF1p is RNA-dependent. HEK293T cells were transfected with Flag-ZCCHC3 and Myc-ORF1p DNA. Forty-eight post-transfection, the cells were collected and lysed, and then treated with RNase at 37°C for 30 min, equal amounts of supernatant were incubated with Flag or Myc antibody (mouse) and then analyzed by Western blots using antibodies against Myc or Flag (rabbit). (D) ZCCHC3 binds to LINE-1 RNA. HEK293T cells were co-transfected with pWA367 DNA and ZCCHC3 DNA. Forty-eight hours later, an equal amount of cell lysis was used to detect LINE-1 RNA by RNA-IP. LINE-1 RNA pulled down by ZCCHC3 was quantified by RT–qPCR. These experiments were repeated three times and representative blots and images are shown.

Fig 5: ZCCHC3 diminished LINE-1 RNA and ORF1p. (A) Overexpression of ZCCHC3 decreases LINE-1 RNA and ORF1p. HEK293T cells were transfected with pWA367 and ZCCHC3 DNA. Total RNA was extracted for Taqman real-time PCR analysis, the RNA level was normalized to GAPDH (left panel). Expression of the LINE-1 ORF1p and Flag-ZCCHC3 were analyzed by immunoblot, and Tubulin was detected as an internal control (right panel). (B) Knockdown of ZCCHC3 increases LINE-1 RNA and ORF1p level. HEK 293T cells were transfected with pWA367 together with the siRNA targeting ZCCHC3. Total RNA was extracted for Taqman real-time PCR analysis, the RNA level was normalized to GAPDH (left panel). Expression of the LINE-1 ORF1p and ZCCHC3 were analyzed by immunoblot, and Tubulin was detected as an internal control (right panel). (C) ZCCHC3 does not affect the activity of the LINE-1 5′UTR promoter. HEK293T cells were transfected with the L1-FL reporter plasmid and ZCCHC3 DNA. Cell lysates were analyzed for luminescence (left panel). Expression of the ZCCHC3 was analyzed by immunoblot, and Tubulin was detected as an internal control (left panel). (D) The detection of RNA stability. HEK293 cells were co-transfected with pWA367 and either ZCCHC3, MOV10 DNA, or pcDNA3.0. Twenty-four hours post-transfection, the cells were treated with actinomycin D, and then detected LINE-1 RNA levels at various times as indicated by Taqman real-time PCR, the RNA level was normalized to GAPDH (left panel). Expression of the ZCCHC3 and MOV10 were analyzed by immunoblot, and Tubulin was detected as an internal control (left panel). Data are representative of at least three independent experiments, values are expressed as means ± SD, and representative blots and images are shown.