Journal: iScience

Article Title: Schlafen-5 inhibits LINE-1 retrotransposition

doi: 10.1016/j.isci.2023.107968

Figure Lengend Snippet: SLFN5 inhibits LINE-1 retrotransposition (A) Schematic of the CMV-L1-neo RT reporter and an overview of the LINE-1 retrotransposition assay. Following transcription from the 5′ UTR promoter of LINE-1, the intron in the neomycin resistance gene is removed. The intronless mRNA is then reverse transcribed into cDNA, which can produce a functional neomycin resistance mRNA. (B) Overexpressing SLFN5 inhibited LINE-1 mobilization. HeLa cells were transfected with 1000 ng CMV-L1-neo RT DNA and 250, 500, and 1000 ng SLFN5 plasmids, followed by retrotransposition assay. SLFN5 expression was monitored by Western blot using an anti-Flag probe. The colony numbers represent LINE-1 mobilization activity. Colonies were visualized with crystal violet staining. The data from three independent experiments are summarized in the bar graph. (C) Depletion of SLFN5 enhances LINE-1 mobility. The SLFN5 knockout HeLa cell line (SLFN5-KO-HeLa) and control HeLa cells (sgControl-HeLa) were transfected with 1000 ng CMV-L1-neo RT DNA followed by treatment with or without IFNα/β, and then a retrotransposition assay was performed. Endogenous SLFN5 was detected by an anti-SLFN5 probe. The data from three independent experiments are summarized in the bar graph. Throughout the figure, statistical significance was determined by unpaired two-tailed Student’s t test. ∗p < 0.05; ∗∗p < 0.01.

Article Snippet: The LINE-1 promoter reporter plasmid L1-FL, which contains a 670-nt LINE-1 5′-UTR sequence in the pGL3-Basic vector (Promega), was described previously.

Techniques: Functional Assay, Transfection, Expressing, Western Blot, Activity Assay, Staining, Knock-Out, Two Tailed Test

Journal: iScience

Article Title: Schlafen-5 inhibits LINE-1 retrotransposition

doi: 10.1016/j.isci.2023.107968

Figure Lengend Snippet: SLFN5 specifically inhibits LINE-1 (A) Schematics of the MusD-neo TNF and IAP-neo TNF reporter. (B) G418 resistance retrotransposition assays. HeLa cells were transfected with 1000 ng of CMV-L1-neo RT , MusD-neo TNF , IAP-neo TNF or pcDNA3.1 DNA with 250, 500, and 1000 ng SLFN5 DNA, respectively, and then performed a retrotransposition assay (left panel). SLFN5 expression was monitored by Western blot using an anti-Flag probe (right panel). The colony numbers represent LINE-1 retrotransposition activity. Colonies were visualized with crystal violet staining. The data from three independent experiments are summarized in the bar graph.

Article Snippet: The LINE-1 promoter reporter plasmid L1-FL, which contains a 670-nt LINE-1 5′-UTR sequence in the pGL3-Basic vector (Promega), was described previously.

Techniques: Transfection, Expressing, Western Blot, Activity Assay, Staining

Journal: iScience

Article Title: Schlafen-5 inhibits LINE-1 retrotransposition

doi: 10.1016/j.isci.2023.107968

Figure Lengend Snippet: Helicase motifs are required for SLFN5 to inhibit LINE-1 retrotransposition (A) Upper panel: Scheme of the domain architecture of the wild-type and mutant SLFN5 variants tested. The numbers indicate the amino acid deletions in SLFN5 mutants. The positions of the mutated amino acids Lys and Asp in the Walker A and Walker B motifs are also indicated. Lower panel: The inhibitory activity of SLFN5 and its mutants. HeLa cells were transfected with CMV-L1-neo RT DNA and SLFN5 or SLFN5 mutant plasmids, respectively, followed by retrotransposition assays. SLFN5 and its mutant expression were monitored by Western blotting using an anti-Flag probe. The colony numbers represent LINE-1 mobilization activity. Colonies were visualized with crystal violet staining. The data from three independent experiments are summarized in the bar graph. (B) Detection of the inhibitory activity of SLFN5, SLFN5 (K584R), SLFN5 (D649A) and SLFN5 (K584R/D649A) on LINE-1 using a retrotransposition assay. The data from three independent experiments are summarized in the bar graph. Throughout the figure, statistical significance was determined by unpaired two-tailed Student’s t test. n.s. not significant, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The LINE-1 promoter reporter plasmid L1-FL, which contains a 670-nt LINE-1 5′-UTR sequence in the pGL3-Basic vector (Promega), was described previously.

Techniques: Mutagenesis, Activity Assay, Transfection, Expressing, Western Blot, Staining, Two Tailed Test

Journal: iScience

Article Title: Schlafen-5 inhibits LINE-1 retrotransposition

doi: 10.1016/j.isci.2023.107968

Figure Lengend Snippet: SLFN5 diminished LINE-1 cDNA levels but did not affect its reverse transcriptase activity (A) SLFN5 can diminish LINE-1 cDNA. Upper panel(left): Illustration of the primer locations used for amplifying CMV-L1-neo RT cDNA. Upper panel (right): PCR products of the plasmid DNA and reverse transcribed cDNA of CMV-L1-neo RT by the designed primers. The lower panel (left): HeLa cells were cotransfected with 1000 ng CMV-L1-neo RT DNA with 0, 500 and 1000 ng SLFN5-expressing plasmids, and then LINE-1 cDNA was detected by qPCR. The lower panel (right): HeLa cells were cotransfected with 1000 ng CMV-L1-neo RT DNA with 500 ng SLFN5, SLFN5 (K584R), SLFN5 (D649A) and SLFN5 (K584R/D649A) DNA, and then LINE-1 cDNA was detected by qPCR. The data from three independent experiments are summarized in the bar graph. The expression of SLFN5 was detected by Western blot. (B) DRIP-qPCR was performed in SLFN5-transfected HeLa cells and control cells. The relative abundance of the DNA-RNA hybrid is represented as the fold change over the control. The sample with RNase H treatment was set as a negative control, in which the RNA strands of the RNA-DNA hybrids were specifically removed and therefore could not be immunoprecipitated by the S9.6 antibody. (C) SLFN5 did not affect the reverse transcriptase activity of LINE-1 ORF2p. HeLa cells were transfected with 1000 ng CMV-L1-neo RT DNA with 500 ng SLFN5, and 48 h later, the cells were collected to detect reverse transcriptase activity by LEAP assay. The reverse transcribed cDNA was quantified by qPCR (left panel) and visualized by conventional PCR (bottom right panel), and the data from three independent experiments are summarized in the bar graph. Throughout the figure, statistical significance was determined by unpaired two-tailed Student’s t test. ∗p < 0.05; ∗∗p < 0.01; n.s., not significant.

Article Snippet: The LINE-1 promoter reporter plasmid L1-FL, which contains a 670-nt LINE-1 5′-UTR sequence in the pGL3-Basic vector (Promega), was described previously.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Western Blot, Transfection, Negative Control, Immunoprecipitation, Two Tailed Test

Journal: iScience

Article Title: Schlafen-5 inhibits LINE-1 retrotransposition

doi: 10.1016/j.isci.2023.107968

Figure Lengend Snippet: SLFN5 has no effect on the transcription and translation of LINE-1 (A) Exogenous expressing SLFN5 did not decrease LINE-1 RNA level. HeLa cells were transfected 1000 ng CMV-L1-neo RT DNA with 250, 500 and 1000 ng of SLFN5 plasmids respectively. 48 h later, the cells were collected for total RNA extraction, and performed RT-qPCR. (B) SLFN5 knockout did not affect LINE-1 RNA level. SLFN5-KO-HeLa cell line, and control HeLa cell line were transfected with 1000 ng CMV-L1-neo RT DNA, and then total cellular RNA was extracted for RT–qPCR (left panel). 1x10 6 of SLFN5-KO-HeLa cells and control cells were harvested and total RNA was extracted to detect endogenous LINE-1 RNA expression by RT–qPCR (right panel). SLFN5 expression was monitored by Western blot using an anti-Flag probe. The RT–qPCR data from three independent experiments are summarized in the bar graph. (C) SLFN5 did not inhibit the 5′UTR promoter activity of LINE-1. HEK293 cells were cotransfected with 1000 ng L1-FL DNA together with 250, 500 and 1000 ng of SLFN5 plasmids. The firefly luciferase activity was measured to represent the activity of the LINE-1 5′-UTR promoter. pGL3-basic contains the firefly luciferase gene that lacks a promoter at its 5′ end. Firefly luciferase activity from this vector was measured to reflect the basal expression of the firefly luciferase gene. SLFN5 expression was monitored by Western blot using an anti-Flag probe. The luciferase activity assay from three independent experiments is summarized in the bar graph. (D) SLFN5 expression did not affect LINE-1 RNA translation. HeLa cells were transfected with CMV-L1-neo RT DNA with 250, 500 and 1000 ng of SLFN5 plasmids. The cell lysates were used to detect SLFN5 (anti-Flag), ORF1p (anti-ORF1p) and β-actin (anti-β-actin) expression using Western blotting. Throughout the figure, statistical significance was determined by unpaired two-tailed Student’s t test. n.s., not significant.

Article Snippet: The LINE-1 promoter reporter plasmid L1-FL, which contains a 670-nt LINE-1 5′-UTR sequence in the pGL3-Basic vector (Promega), was described previously.

Techniques: Expressing, Transfection, RNA Extraction, Quantitative RT-PCR, Knock-Out, RNA Expression, Western Blot, Activity Assay, Luciferase, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: Schlafen-5 inhibits LINE-1 retrotransposition

doi: 10.1016/j.isci.2023.107968

Figure Lengend Snippet: SLFN5 decreased nuclear LINE-1 RNA level (A) SLFN5 blocks LINE-1 RNA entering nucleus. HeLa cells were cotransfected with CMV-L1-neo RT and different amounts of SLFN5 DNA. Forty-eight hours later, cells were collected for extracting whole cell and nuclear RNA to quantify the LINE-1 RNA level by RT–qPCR. The expression of SLFN5 was detected by Western blot. (B) The effects of SLFN5 and its mutants on LINE-1 RNA levels in the nucleus (left panel). The expression of SLFN5 was detected by Western blot. p84 protein and β-actin was used as a nuclear and cytoplasmic marker (right panel). (C) The effects of SLFN5 and its mutants on LINE-1 RNA levels in the cytoplasm. (D) Detection of the effects of SLFN5 on the ORF1p and ORF2p levels in the nucleus and cytoplasm by Western blot. p84 protein and β-actin was used as a nuclear and cytoplasmic marker. (E) SLFN5 changed the distribution of LINE-1 RNA in cells. Upper panel: Confocal microscopy showed subcellular localization of LINE-1 RNA (red) and SLFN5 (green). Lower panel: Quantification of subcellular localization of LINE-1 RNA was performed on 15 individual cells for each sample by measuring the fluorescence intensities of nuclear and cytoplasmic subcompartments per cell. The scale bar represents 10 µm. The data from three independent experiments are summarized in the bar graph. Throughout the figure, statistical significance was determined by unpaired two-tailed Student’s t test. ∗p < 0.05; ∗∗p < 0.01. n.s., not significant.

Article Snippet: The LINE-1 promoter reporter plasmid L1-FL, which contains a 670-nt LINE-1 5′-UTR sequence in the pGL3-Basic vector (Promega), was described previously.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Marker, Confocal Microscopy, Fluorescence, Two Tailed Test

Journal: iScience

Article Title: Schlafen-5 inhibits LINE-1 retrotransposition

doi: 10.1016/j.isci.2023.107968

Figure Lengend Snippet: SLFN5 binds to LINE-1 RNA and blocks the recruitment of ORF1p required for LINE-1 RNP (A) SLFN5 affects the formation of LINE-1 RNP. HeLa cells were cotransfected with 1000 ng CMV-L1-neo RT DNA and 500 ng SLFN5, SLFN5 (K584R), SLFN5 (D649A) or SLFN5 (K584R/D649A) DNA. Forty-eight hours later, equal amounts of cell lysates were used to isolate LINE-1 RNP by ultracentrifugation. The LINE-1 RNP samples were quantified by detecting LINE-1 ORF1p using the Odyssey infrared imaging system. (B) SLFN5 and its mutants bind to LINE-1 RNA. HeLa cells were cotransfected with CMV-L1-neo RT DNA and SLFN5, SLFN5 (K584R), SLFN5 (D649A) and SLFN5 (K584R/D649A) DNA. Forty-eight hours later, an equal amount of cell lysis was used to detect LINE-1 RNA and SLFN5 by RNA IP. LINE-1 RNA pulled down by ORF1p was quantified by RT–qPCR. The expression of SLFN5 and ORF1p was detected by Western blot using anti-Flag and anti-ORF1p antibodies, respectively. (C) The distribution of LINE-1 RNA and ORF1p was analyzed by 5%–50% discontinuous sucrose gradient ultracentrifugation. Each fraction collected was analyzed by RT–PCR and Western blot. The numbers under the graph represent different fractions. (D) Detection of the interaction between LINE-1 RNA and SLFN5 mutants by RIP. Equal amounts of cell lysates were immunoprecipitated with anti-Myc, and the levels of LINE-1 and GAPDH RNA were determined by qRT–PCR. IgG served as the negative control in the RIP. The data from three independent experiments are summarized in the bar graph. The expression of SLFN5 and its mutants was detected by Western blot. Throughout the figure, statistical significance was determined by unpaired two-tailed Student’s t test. ∗p < 0.05; ∗∗p < 0.01; ns, not significant.

Article Snippet: The LINE-1 promoter reporter plasmid L1-FL, which contains a 670-nt LINE-1 5′-UTR sequence in the pGL3-Basic vector (Promega), was described previously.

Techniques: Imaging, Lysis, Quantitative RT-PCR, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Two Tailed Test