Journal: Nucleic Acids Research

Article Title: Alternative polyadenylation links RNA processing to iron metabolism in human erythropoiesis

doi: 10.1093/nar/gkag218

Figure Lengend Snippet: CPSF6 may regulate the APA during erythropoiesis. (A) Heatmap showing mRNA expression of 19 core APA regulators during erythropoiesis and basophilopoiesis. Regulators are grouped into the cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CSTF), cleavage factor Ⅰ (CFⅠ), and cleavage factor Ⅱ (CFⅡ) complexes, as well as individual (Single) proteins. CPM, counts per million. Wilcoxon rank sum test by Seurat Find All Markers , adjusted by BH. **** P < 0.0001. (B) CPSF6 mRNA expression relative to (A) . CPM, counts per million. (C) Representative western blots showing CPSF6 protein levels in erythroblasts induced from human UCB-derived CD34⁺ HSPCs and cultured for 4, 8, 11, 13, and 15 days. Normalized CPSF6/β-actin fold changes are indicated. (D) Density distribution of the UGUA motif near the most proximal and most distal PASs of differential APA-genes, respectively. The left panel shows motif distributions for the APA-genes from APA patterns 1–3 and patterns 4–7, respectively; the right panel shows motif distributions for the 3′UTR continuously shortening and continuously lengthening APA-genes from . The Kolmogorov–Smirnov test was used to compare the UGUA distribution between the most proximal and most distal PASs. (E) Schematic diagram of PAS selection by CPSF6.

Article Snippet: To prepare the RNA immunoprecipitation (RIP) reaction, 50 μL of protein A/G magnetic beads (Thermo Fisher, Cat # 10004D) were mixed with 4 μg of anti-CPSF6 antibody (Bethyl, Cat # A301-356A) and rotated at 4°C for 6 h. Harvested K562 cells were lysed in 500 μL lysis buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 0.5 mM EDTA pH 8.0, 10 mM KCl, 1 mM MgCl 2 , 0.5% NP-40, 10% glycerol, 1.5 mM DTT), with protease inhibitor cocktail (Beyotime, Cat # P1008), 10 U/mL RNase inhibitor (Beyotime, Cat # RE135-01).

Techniques: Expressing, Western Blot, Derivative Assay, Cell Culture, Selection

Journal: Nucleic Acids Research

Article Title: Alternative polyadenylation links RNA processing to iron metabolism in human erythropoiesis

doi: 10.1093/nar/gkag218

Figure Lengend Snippet: CPSF6 is essential for human erythropoiesis. (A) Schematic diagram of experimental design. Human UCB-derived CD34 + HSPCs were differentiated into erythroid cells in vitro , and infected with short hairpin RNA (shRNA) lentivirus on day 4 to knock down CPSF6. Cells on day 11 were subjected to a cell assay. (B) Western blot showing CPSF6 and β-actin protein in untreated, control-shRNA, or CPSF6-shRNA transduced erythroblasts cultured on day 11. (C and D) Colony-forming ability of cells cultured on days 7 and 14, which contain mixed populations of cells that include BFU-E and CFU-E. (C) shows the morphology of BFU-E and CFU-E colonies. (D) shows quantification of BFU-E and CFU-E colonies. ( E and F ) Representative flow cytometry plots of erythroblasts on day 11 and the percentage of CD235a + /CD71 + erythroblasts with CPSF6 knockdown. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To prepare the RNA immunoprecipitation (RIP) reaction, 50 μL of protein A/G magnetic beads (Thermo Fisher, Cat # 10004D) were mixed with 4 μg of anti-CPSF6 antibody (Bethyl, Cat # A301-356A) and rotated at 4°C for 6 h. Harvested K562 cells were lysed in 500 μL lysis buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 0.5 mM EDTA pH 8.0, 10 mM KCl, 1 mM MgCl 2 , 0.5% NP-40, 10% glycerol, 1.5 mM DTT), with protease inhibitor cocktail (Beyotime, Cat # P1008), 10 U/mL RNase inhibitor (Beyotime, Cat # RE135-01).

Techniques: Derivative Assay, In Vitro, Infection, shRNA, Knockdown, Western Blot, Control, Cell Culture, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Alternative polyadenylation links RNA processing to iron metabolism in human erythropoiesis

doi: 10.1093/nar/gkag218

Figure Lengend Snippet: CPSF6 knockdown alters 3′UTR-APA. (A) Schematic diagram of experimental design. Human UCB-derived CD34 + HSPCs were induced to erythroid cells in vitro , and infected with shRNA lentivirus to knock down CPSF6. Cells on day 11 (D11) and 13 (D13) were subjected to bulk RNA-seq and 3′-seq. Schematic read distributions from 3′-seq and RNA-seq illustrating 3′UTR APA changes are shown on the right. (B) Cumulative distribution of the pPUI in differentiated cells with control and CPSF6 knockdown. Every shCPSF6 sample was compared to its control by the Kruskal–Wallis test, and the P values were all less than 2.2e-16. The pie chart shows the number and proportion of differentially shortening and lengthening APA-genes. (C) Volcano plot of differential APA-genes of CPSF6 knockdown samples compared to control samples identified by bulk RNA-seq at D11. (D) Venn diagram showing genes with differential APA identified in normal erythropoiesis (scRNA-seq) and in CPSF6 knockdown cells (bulk RNA-seq and 3′-seq at days 11 and 13). See . ( E and F ) Genome browser tracks of 3′-seq and RT-qPCR validation of representative iron metabolism-related differential APA genes after CPSF6 knockdown. (G) GSEA plot of APA-gene pPUI in 3′-seq datasets on day 11.

Article Snippet: To prepare the RNA immunoprecipitation (RIP) reaction, 50 μL of protein A/G magnetic beads (Thermo Fisher, Cat # 10004D) were mixed with 4 μg of anti-CPSF6 antibody (Bethyl, Cat # A301-356A) and rotated at 4°C for 6 h. Harvested K562 cells were lysed in 500 μL lysis buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 0.5 mM EDTA pH 8.0, 10 mM KCl, 1 mM MgCl 2 , 0.5% NP-40, 10% glycerol, 1.5 mM DTT), with protease inhibitor cocktail (Beyotime, Cat # P1008), 10 U/mL RNase inhibitor (Beyotime, Cat # RE135-01).

Techniques: Knockdown, Derivative Assay, In Vitro, Infection, shRNA, RNA Sequencing, Control, Quantitative RT-PCR, Biomarker Discovery

Journal: Nucleic Acids Research

Article Title: Alternative polyadenylation links RNA processing to iron metabolism in human erythropoiesis

doi: 10.1093/nar/gkag218

Figure Lengend Snippet: CPSF6 mediates FAM210B APA to support iron homeostasis during erythropoiesis. (A-B) Detection of intracellular Fe 2+ and Fe 3+ content on day 11. (C) Western blot analysis of FAM210B protein expression in CPSF6-knockdown cells compared with shCtrl on day 11. (D) Schematic representation of FAM210B long transcript (LT) and short transcript (ST). The amino acid (aa) positions are annotated in the CDS region. (E) Representative fluorescence images of the GFP protein expression with 3′UTR of FAM210B -LT and 3′UTR of FAM210B -ST in the HEK293FT cell line. (F-H) Western blot quality control of CPSF6 RNA immunoprecipitation (RIP) in K562 cells (F) , (G-H) RT-qPCR analysis of RIP enrichment for FAM210B LT and ST. (I) The representative image of cell pellets from cord blood–derived CD34 + HSCPCs induced toward erythroid differentiation for 8 days in vitro . Experimental groups included shCtrl, shCPSF6, and shCPSF6 + ST (FAM210B-ST overexpression upon CPSF6 knockdown). (J) Validation of manipulation efficiency by RT-qPCR. mRNA levels of CPSF6 (left) and reconstituted FAM210B isoforms (right) were measured (mean ± SD, n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( K and L ) Representative flow cytometry plots of erythroblasts (L) and quantification of CD235a + CD71 + erythroblasts (mean ± SD, n = 3) (M) . * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To prepare the RNA immunoprecipitation (RIP) reaction, 50 μL of protein A/G magnetic beads (Thermo Fisher, Cat # 10004D) were mixed with 4 μg of anti-CPSF6 antibody (Bethyl, Cat # A301-356A) and rotated at 4°C for 6 h. Harvested K562 cells were lysed in 500 μL lysis buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 0.5 mM EDTA pH 8.0, 10 mM KCl, 1 mM MgCl 2 , 0.5% NP-40, 10% glycerol, 1.5 mM DTT), with protease inhibitor cocktail (Beyotime, Cat # P1008), 10 U/mL RNase inhibitor (Beyotime, Cat # RE135-01).

Techniques: Western Blot, Expressing, Knockdown, Fluorescence, Control, RNA Immunoprecipitation, Quantitative RT-PCR, Derivative Assay, In Vitro, Over Expression, Biomarker Discovery, Flow Cytometry

Journal: iScience

Article Title: SNW1 promotes lymphatic metastasis in bladder cancer by modulating SRPK1 splicing

doi: 10.1016/j.isci.2026.114811

Figure Lengend Snippet: SNW1 interacts with NUDT21/CPSF6 to regulate SRPK1 splicing (A) Proteins interacting with SNW1 were immunoprecipitated with anti-SNW1 antibody and identified by LC‒MS/MS. Table lists the top 10 SNW1-associated proteins in T24 cells. (B) Pearson correlation analysis showing associations between SNW1 and NUDT21, and SNW1 and CPSF6, in the TCGA cohort. (C and D) CoIP validation of interactions between endogenous SNW1, NUDT21, and CPSF6. (E and F) Western blot analysis of SRPK1 protein following NUDT21 (E) or CPSF6 (F) knockdown. (G–J) RT-qPCR analysis of SRPK1-L (G, I) and SRPK1-S (H, J) transcript levels. Data are presented as mean ± SD, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (one-way ANOVA). (K–N) Transwell assays show reduced migration and invasion of T24 (K, L) and UM-UC-3 (M, N) cells after NUDT21 or CPSF6 knockdown. (O and P) RIP assays with anti-NUDT21 (O) or anti-CPSF6 (P) antibodies showing reduced SRPK1 pre-mRNA enrichment in SNW1 KD cells relative to Control KD cells. Data are presented as mean ± SD, ∗∗∗ p < 0.001 (one-way ANOVA).

Article Snippet: Primary antibodies against SNW1 (259261-AP), NUDT21 (10322-1-AP), SRPK1 (14073-1-AP), CPSF6 (15489-1-AP) were from Proteintech (Wuhan, China).

Techniques: Immunoprecipitation, Biomarker Discovery, Western Blot, Knockdown, Quantitative RT-PCR, Migration, Control

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: Cpsf6 is downregulated during silica-induced pulmonary fibrogenesis. A The C57BL/6 mice were sacrificed on days 7, 14, and 28 after intratracheal instillation of silica suspended in saline. Histological changes in lung tissues were observed by hematoxylin and eosin (H&E) staining. B Masson staining was performed to measure fibrotic lesions on day 28. C The hydroxyproline content of the lung tissues was used to assess the degree of collagen deposition ( n = 3), with * P < 0.05. D Representative images of immunohistochemical staining of α-SMA in lung sections. E - F The mRNA and protein levels of myofibroblast markers (Collagen I and α-SMA) in lung tissues ( n = 3), with * P < 0.05, ** P < 0.01. G - H Cpsf6 mRNA and protein expression in murine lung tissues ( n = 3), with ** P < 0.01. I - K The expression of Cpsf6, Collagen I, and α-SMA was assessed via qRT-PCR and western blot analysis in mouse primary lung fibroblasts ( n = 3), with ** P < 0.01

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Saline, Staining, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: Knockdown of Cpsf6 exacerbates pulmonary fibrosis induced by silica in vivo. A Strategy for Cpsf6 knockdown in silica-induced pulmonary fibrosis mouse model. B Cpsf6 expression was measured by qRT-PCR in the treated mouse lung tissues for the indicated groups ( n = 3), with ** P < 0.01 vs. the control group. C H&E staining, Masson staining, and IHC staining for α-SMA were conducted to assess the severity of pulmonary fibrosis. D The silencing of Cpsf6 increased the hydroxyproline levels in the lungs of mice subjected to SiO 2 treatment ( n = 3), with * P < 0.05, ** P < 0.01 vs. the control group. E The protein expression levels of Cpsf6, Collagen I, and α-SMA in murine lung tissues were measured via western blot analysis. GAPDH served as an internal loading control. F qPCR analysis of mRNA levels of Collagen I and α-SMA in lung tissue ( n = 3), with * P < 0.05, ** P < 0.01. G Cpsf6, Collagen I, and α-SMA expression levels were detected by western blot analysis in mouse primary lung fibroblasts

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Knockdown, In Vivo, Expressing, Quantitative RT-PCR, Control, Staining, Immunohistochemistry, Western Blot

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: Overexpression of Cpsf6 alleviates silica-induced pulmonary fibrosis. A Strategy for Cpsf6 overexpression in silica-induced pulmonary fibrosis mouse model. B The mRNA level of Cpsf6 was detected by qPCR in lung tissues after treatment with AAV-Cpsf6 ( n = 3), with * P < 0.05 and ** P < 0.01 vs. the control group, and ## P < 0.01 vs. the SiO 2 + AAV-control group. C H&E staining, Masson staining, and IHC staining for α-SMA were performed to evaluate the extent of pulmonary fibrosis. D Quantification of hydroxyproline in lung tissues ( n = 3), with ** P < 0.01. E The protein expression levels of Cpsf6, Collagen I, and α-SMA in murine lung tissues. GAPDH served as an internal loading control. F The mRNA levels of Collagen I and α-SMA in lung tissue ( n = 3), with ** P < 0.01 vs. the control group, and # P < 0.05 vs. the SiO 2 + AAV-control group. G Western blot analysis of the protein expression of Cpsf6, Collagen I, and α-SMA in mouse primary lung fibroblasts for the indicated groups

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Control, Staining, Immunohistochemistry, Expressing, Western Blot

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: CPSF6 is downregulated in active pulmonary fibroblasts induced by TGF-β1. A Expression of α-SMA and Collagen I in MRC-5 was assessed by Western blot after 48 h of treatment with the indicated doses of TGF-β1 (0–5 ng/mL). GAPDH served as an internal loading control. B - C immunofluorescence staining of α-SMA (red) and Collagen I (green) in MRC-5 fibroblasts following exposure to TGF-β1. Nuclei were counterstained with DAPI (blue). Scale bar = 125 μm. D Quantification of relative cell viability assessed by MTT assay in MRC-5 fibroblasts treated with different concentrations of TGF-β1 ( n = 3). * p < 0.05 vs. control. E EdU incorporation assay illustrating proliferative activity of MRC-5 cells with or without TGF-β1 stimulation, with green representing EdU and blue indicating nuclei. Scale bar = 125 μm. F Heatmap showing dysregulated genes (red, upregulated genes; blue, downregulated genes) identified by DEGs. G qRT-PCR analysis of CPSF6 expression in MRC-5 cells ( n = 3), with * P < 0.05, ** P < 0.01. H Western blot analysis of CPSF6 protein levels in MRC-5 fibroblasts following TGF-β1 exposure at the indicated concentrations. GAPDH was used as a reference control. I The expression of α-SMA (red) and Collagen I (green) in mouse primary lung fibroblasts was assessed via immunofluorescence staining to show fibroblast activation. Scale bar = 125 μm. J Immunofluorescence staining validating CPSF6 (red) downregulation in TGF-β1-treated mouse primary lung fibroblasts. Nuclei are stained with DAPI (blue). Scale bar = 125 μm. K CPSF6 protein expression in primary lung fibroblasts

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining, MTT Assay, Activity Assay, Quantitative RT-PCR, Activation Assay

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: CPSF6 plays a key role in fibroblast-myofibroblast transition (FMT). A The efficiency of CPSF6 knockdown in MRC-5 cells was assessed by qRT-PCR ( n = 3), with ** P < 0.01. B Western blot analysis of the protein expression levels of CPSF6, Collagen I, and α-SMA in MRC-5 cells for the indicated groups ( n = 3). C Immunofluorescence staining of Collagen I and α-SMA in MRC-5 cells for the indicated groups. Red represents α-SMA staining; green represents Collagen I staining; blue represents nuclear DNA staining by DAPI. Scale bar = 125 μm. D - E EdU and MTT assays evaluated the proliferative capacity of MRC-5 cells ( n = 3), with ** P < 0.01. Scale bar = 125 μm. F MRC-5 cells’ contraction was measured using the collagen gel assay. G The mRNA levels of CPSF6 in cells treated with CPSF6 plasmid and TGF-β1, as indicated for 48 h, were determined by RT-qPCR. H Western blot analysis of Cpsf6, Collagen I, and α-SMA in MRC-5 cells treated with CPSF6 plasmid and TGF-β1 as indicated for 48 h. I - J MTT and EdU assays evaluated the proliferative capacity of MRC-5 cells ( n = 3), with * P < 0.05 vs. the control group, and # P < 0.05 vs. the TGF-β1 + NC group. Scale bar = 125 μm. K Immunofluorescence staining of α-SMA and Collagen I in MRC-5 cells. Red represents α-SMA staining; green represents Collagen I staining; blue represents nuclear DNA staining by DAPI. Scale bar = 125 μm

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Staining, Plasmid Preparation, Control

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: RUNX1 is a downstream functional target of CPSF6. A Western blot analysis of RUNX1 protein levels in MRC-5 cells treated with CPSF6 siRNA. B Immunofluorescence staining of RUNX1 in MRC-5 cells. Green represents RUNX1 staining; blue represents nuclear DNA staining by DAPI. Scale bar = 125 μm. C Western blot analysis of the protein expression of RUNX1 in MRC-5 cells for the indicated groups. D Western blot and densitometric analysis of RUNX1, Collagen I, and α-SMA in MRC-5 cells transfected with RUNX1 siRNA or control siRNA then treated with 5ng/ml TGF-β1 for 48 h ( n = 3), with * P < 0.05, ** P < 0.01 vs. the control group, and ## P < 0.01 vs. TGF-β1 + control siRNA group. E Immunofluorescence staining of α-SMA (red) and Collagen I (green) in MRC-5 cells for the indicated groups. Blue represents nuclear DNA staining by DAPI. Scale bar = 125 μm. F - G MTT assay and EdU staining were used to assess cell proliferation in MRC-5 cells ( n = 3), with * P < 0.05 vs. the control group, and # P < 0.05 vs. the TGF-β1 + control siRNA group. Scale bar = 125 μm. H Contraction assay of the collagen gel complex in the presence or absence of RUNX1 siRNA

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Functional Assay, Western Blot, Immunofluorescence, Staining, Expressing, Transfection, Control, MTT Assay, Contraction Assay

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: RUNX1 mediates the CPSF6 function during fibroblast activation. A Western blot analysis of Collagen I and α-SMA in MRC-5 cells transfected with RUNX1 siRNA. B The expression of α-SMA (red) and Collagen I (green) was assessed via immunofluorescence staining to show fibroblast activation. Scale bar = 125 μm. C - D EdU and MTT assays were performed to evaluate cell proliferative ability in MRC-5 cells ( n = 3), * P < 0.05, ** P < 0.01. Scale bar = 125 μm. E Fibroblast contraction was determined. F Western blot analysis displayed Collagen I and α-SMA levels in MRC-5 cells treated with CPSF6 plasmid and RUNX1 plasmid. G MRC-5 cells were stained with a primary antibody against the myofibroblast marker α-SMA (red) and Collagen I (green). Nuclei were stained with DAPI. Scale bars correspond to 125 μm. H MTT assay was performed to evaluate cell proliferation ability in MRC-5 cells ( n = 3), with ** P < 0.01. I Contraction assay of the collagen gel complex treated with TGF-β1 and CPSF6 or RUNX1 plasmid

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Activation Assay, Western Blot, Transfection, Expressing, Immunofluorescence, Staining, Plasmid Preparation, Marker, MTT Assay, Contraction Assay

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: CPSF6 knockdown induces 3'UTR shortening of RUNX1 to regulate its expression. A - B The qRT-PCR determined the relative levels of two transcripts of RUNX1 and CPSF6 in MRC-5 cells treated with CPSF6 siRNA or CPSF6 plasmid. C Agarose gel electrophoresis demonstrates 3'RACE amplification of RUNX1 using RNAs extracted from MRC-5 cells treated with CPSF6 siRNA and cntrol group. D The abundance of two transcripts of RUNX1 was assessed by qRT-PCR in MRC-5 cells treated with Actinomycin D (2µM) at the indicated time points. E Long/short 3'UTR RUNX1 activity via luciferase assay ( n = 3), ** P < 0.01. F Schematic illustration exhibiting the overlapping of the target miRNAs of long-3'UTR RUNX1 inform predicted by Target Scan, miRDB, miRWalk, and GSE32538 . G The expression of miRNAs was detected by qRT-PCR in MRC-5 cells. H - I Luciferase activities of Luc-long-3'UTR or Luc-short-3'UTR in MRC-5 cells co-transfected with miR-30a-5p mimic, miR-30a-5p inhibitor, or N.C. were determined by a luciferase reporter assay ( n = 3), ** P < 0.01. J RIP assays for long-3'UTR RUNX1 mRNA and miR-30a-5p levels in MRC-5 cells were detected by qRT-PCR analysis ( n = 3), ** P < 0.01. K RUNX1 expression levels were detected by western blot analysis. L Immunofluorescence staining of RUNX1 in MRC-5 cells for the indicated groups. Green represents RUNX1 staining; blue represents nuclear DNA staining by DAPI. Scale bar = 125 μm

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Activity Assay, Luciferase, Transfection, Reporter Assay, Western Blot, Immunofluorescence, Staining

Journal: Respiratory Research

Article Title: CPSF6-mediated alternative polyadenylation of RUNX1 to regulate silica-induced pulmonary fibrosis progression

doi: 10.1186/s12931-026-03507-7

Figure Lengend Snippet: Schematic illustration delineates the CPSF6-mediated mechanism implicated in the pathogenesis of pulmonary fibrosis. The image was generated utilizing Biorender.

Article Snippet: Adeno-associated virus serotype 9 (AAV9) vectors encoding short hairpin RNA targeting CPSF6 (sh-CPSF6) or CPSF6 were constructed by Gene Co., Ltd. (Shanghai, China).

Techniques: Generated

Journal: eLife

Article Title: Decoding the biogenesis of HIV-induced CPSF6 puncta and their fusion with nuclear speckles

doi: 10.7554/eLife.103725

Figure Lengend Snippet: Spinning disk confocal images of HEK293 SRRM2 HaloTag cells acquired 9 hr after VSV-G/HIV-1ΔEnvIN HA LAI (BRU) (MOI 10) infection occurred in the presence of Nevirapine (10 µM). Cells were previously transduced for 24 hr with CPSF6-mNeonGreen lentiviral vector (MOI 0.5) to detect CPSF6 (visualized in green). SRRM2 MLOs were detected using the TMR-HaloTag Ligand (in red) and the nuclei were stained with Hoechst (blue). Acquisitions were performed continuously for 27 min, which allowed us to see the independent formation of CPSF6 puncta, their migration towards the SRRM2 MLOs and the final fusion of CPSF6 puncta and SRRM2 MLOs.

Article Snippet: Antibody , Rabbit monoclonal anti-CPSF6 , Novus Biologicals , #NBP1-85676 , IF 1:400 WB 1:500.

Techniques:

Journal: eLife

Article Title: Decoding the biogenesis of HIV-induced CPSF6 puncta and their fusion with nuclear speckles

doi: 10.7554/eLife.103725

Figure Lengend Snippet: Spinning disk confocal images of HEK293 SRRM2 HaloTag cells acquired 9 hr after VSV-G/HIV-1ΔEnvIN HA LAI (BRU) (MOI 10) infection occurred in the presence of Nevirapine (10 µM). Cells were previously transduced for 24 hr with CPSF6-mNeonGreen lentiviral vector (MOI 0.5) to detect CPSF6 (visualized in green). SRRM2 MLOs were detected using the TMR-HaloTag Ligand (in red) and the nuclei were stained with Hoechst (blue). Acquisitions were performed continuously for 27 min, which allowed us to see the independent formation of CPSF6 puncta, their migration towards the SRRM2 MLOs and the final fusion of CPSF6 puncta and SRRM2 MLOs.

Article Snippet: Antibody , Rabbit monoclonal anti-CPSF6 , Novus Biologicals , #NBP1-85676 , IF 1:400 WB 1:500.

Techniques:

Journal: mBio

Article Title: HIV-1 infection regulates gene expression by altering alternative polyadenylation correlated with CPSF6 and CPSF5 redistribution

doi: 10.1128/mbio.02865-25

Figure Lengend Snippet: HIV-1 infection induces changes in APA. ( A ) To induce the translocation of CPSF6 to nuclear speckles, human A549 cells were challenged with HIV-1-GFP at an MOI of 2 for 24 h. Infection was assessed as the percentage of GFP-positive cells by flow cytometry. The results of three independent experiments with standard deviation are shown. Significance was determined using an unpaired t -test; *** P < 0.001. ( B ) Cells were subsequently fixed, permeabilized, and immunolabeled using specific antibodies against SC35 (red, marker for nuclear speckles) and CPSF6 (green). Nuclei were stained with DAPI (blue). ( C ) The average (and standard deviation) percentage of cells containing CPSF6 in nuclear speckles (SC-35 positive compartments) from three independent experiments, as determined by visual examination of 200 cells, is shown. Significance was determined using an unpaired t -test; *** P < 0.001. ( D ) Total RNA from three infected and three mock-infected samples was sequenced, and gene expression is displayed in a Volcano plot. The x-axis shows log 2 fold changes in gene expression, with positive indicating upregulation and negative indicating downregulation. The y-axis shows the statistical significance, expressed as −Log 10 P. ( E ) Gene ontology analysis of all genes upregulated and downregulated by HIV-1 infection. The number of genes in each pathway is expressed as the size of the sphere, and the color represents the P -adjusted value. ( F ) Volcano plot showing polyadenylation signal (PAS) changes. The x-axis shows compositional fold change, which represents the 3’ UTR length for each transcript. A positive compositional fold change indicates the presence of a longer 3’UTR (red), and a negative compositional fold change indicates the presence of a shorter 3’UTR (green). The y-axis shows the statistical significance of the result, conveyed as the −Log 10 P. CPSF6, cleavage and polyadenylation specificity factor subunit 6; DAPI, 4’6-diamino-phenylindole; GFP, green fluorescent protein; MOI, multiplicity of infection; PAS, polyadenylation signal; UTR, untranslated region.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8; Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Infection, Translocation Assay, Flow Cytometry, Standard Deviation, Immunolabeling, Marker, Staining, Gene Expression

Journal: mBio

Article Title: HIV-1 infection regulates gene expression by altering alternative polyadenylation correlated with CPSF6 and CPSF5 redistribution

doi: 10.1128/mbio.02865-25

Figure Lengend Snippet: HIV-1 infection induces APA changes in human A549 cells that depend on the interaction between the viral capsid and CPSF6. ( A–C ) Human A549 cells were challenged with HIV-1-GFP ( A ), HIV-1-N74D-GFP ( B ), or HIV-1-A77V-GFP ( C ), using an MOI of 2 for 48 h. Cells were harvested, and total RNA was extracted. Total RNA from three infected and three mock-infected samples was sequenced to analyze changes in APA using PAC-seq. Scatterplots were generated from PolyAMiner analysis of PAC-seq results, plotted as the PolyAIndex (PAI) of HIV-infected cells relative to mock-infected cells. Negative PAI denotes 3’UTR shortening, whereas positive PAI denotes 3’UTR lengthening. Significance is defined as PAI<−0.5 or PAI>0.5, with an adjusted P -value < 0.05. ( D–F ) Genome browser images of IRF7 ( D ), VMA21 ( E ), and TBC1D12 ( F ) with PAC-seq reads shown. Scale is set to 2 Kb, and the y-axis shows read values. The position of the annotated polyadenylation signal (PAS) is shown in red. APA, alternative polyadenylation; IRF7, interferon responsive factor 7; MOI, multiplicity of infection; PAC-seq, Poly(A)-Click-Sequencing; TBC1D12, TBC1 domain family member 12; UTR, untranslated region; VMA21, vacuolar membrane protein 21.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8; Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Infection, Generated, Sequencing, Membrane

Journal: mBio

Article Title: HIV-1 infection regulates gene expression by altering alternative polyadenylation correlated with CPSF6 and CPSF5 redistribution

doi: 10.1128/mbio.02865-25

Figure Lengend Snippet: HIV-1 infection induces APA changes in human primary CD4 + T cells that depend on the interaction between the viral capsid and CPSF6. Human primary CD4 + T cells were challenged with HIV-1-GFP ( A ), HIV-1-N74D-GFP ( B ), or HIV-1-A77V-GFP ( C ) using an MOI of 2 for 48 h. After 48 h, cells were sorted to enrich for GFP-positive cells. Subsequently, GFP-positive cells were grown until a total of 96 h of HIV-1 infection was reached. Cells were harvested, and total RNA was extracted. Total RNA from three infected and three mock-infected samples was sequenced to analyze changes in APA using PAC-seq. Scatterplots were generated using PolyAMiner analysis of PAC-seq results, plotted as the PolyAIndex (PAI) of HIV-infected cells relative to mock-infected cells. A negative PAI denotes 3’UTR shortening in infected cells, whereas a positive PAI denotes 3’UTR lengthening. Significance was defined as PAI <−0.5 or PAI>0.5, with an adjusted P -value of <0.05. MOI, multiplicity of infection; PAC-seq, Poly(A)-Click-sequencing; UTR, untranslated region.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8; Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Infection, Generated, Sequencing

Journal: mBio

Article Title: HIV-1 infection regulates gene expression by altering alternative polyadenylation correlated with CPSF6 and CPSF5 redistribution

doi: 10.1128/mbio.02865-25

Figure Lengend Snippet: Loss of CPSF6 expression globally shortens the 3’UTR of cellular mRNAs. ( A ) A549 WT, NT#H1, CPSF6-KO#B4, CPSF6-KO#B7, and CPSF6-KO#C8 cells were lysed, and proteins were analyzed by Nu-PAGE, followed by western blot using anti-CPSF6, anti-CPSF5, anti-CPSF7, and anti-GAPDH antibodies. Experiments were repeated at least three times, and a representative image is shown. Graphs show the average densitometry quantification of three replicates with standard deviation. Significance was determined using ANOVA multiple comparisons tests; *** P < 0.001; ns, not significant. ( B ) Total RNA from CPSF6-KO and WT A549 cells was prepared, and polyadenylated transcripts were identified by PAC-seq, followed by APA analysis using PolyAMiner. ( C ) A549 WT, NT#H1, CPSF6-KO#B4, CPSF6-KO#B7, and CPSF6-KO#C8 cells were infected with increasing amounts of HIV-1-GFP for 24 or 48 h. Infection was assessed as the percentage of GFP-positive cells by flow cytometry. ( D ) WT and CPSF6-KO#B7 A549 cells were infected with HIV-1-GFP at an MOI of 2 for 48 h. Cells were fixed, permeabilized, and stained using the following antibodies: (i) anti-CPSF5 (red) and anti-CPSF6 (green); (ii) anti-SC-35 (red) and anti-CPSF6 (green); (iii) anti-LEDGF/p75 (red) and anti-CPSF6 (green); and (iv) anti-LEDGF/p75 (red) and anti-SC35 (green). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. ( E ) Percentage of A549 cells containing CPSF6, CPSF5, or CPSF7 in nuclear speckles (condensates) upon HIV-1 infection (average of three independent experiments with standard deviation). Cells containing CPSF6, CPSF5, or CPSF7 in nuclear speckles were determined by visual examination of 200 cells. Significance was determined using unpaired t -test; *** P < 0.001; ns, not significant. APA, alternative polyadenylation; CPSF5, cleavage and polyadenylation specificity factor subunit 5; CPSF6, cleavage and polyadenylation specificity factor subunit 6; CPSF7, cleavage and polyadenylation specificity factor subunit 7; DAPI, 4’,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KO, knockout; LEDGF/p75, lens epithelium-derived growth factor; NT, non-targeting; PAC-Seq, Poly(A)-Click-Sequencing; UTR, untranslated region; WT, wild-type; hpi, hours post-infection.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8; Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Expressing, Western Blot, Standard Deviation, Infection, Flow Cytometry, Staining, Knock-Out, Derivative Assay, Sequencing

Journal: mBio

Article Title: HIV-1 infection regulates gene expression by altering alternative polyadenylation correlated with CPSF6 and CPSF5 redistribution

doi: 10.1128/mbio.02865-25

Figure Lengend Snippet: HIV-1 infection mimics the CPSF6-KO phenotype. ( A ) CPSF6-KO and control (WT and NT#H1) A549 cells were analyzed by western blot using anti-SLFN5 and anti-CPSF6 antibodies. Anti-GAPDH antibodies were used as a loading control. ( B ) A549 cells were challenged with HIV-1-GFP or HIV-1-Luc using an MOI of 2 for 48 h. Subsequently, cells were lysed, and extracts were analyzed by western blot using anti-SLFN5, anti-CPSF6, and anti-CPSF5. Virus presence was assessed using anti-p24 antibodies, and anti-GAPDH antibodies were used as a protein loading control. ( C ) A549 cells were challenged with three different HIV-1-A77V-GFP preparations using an MOI of 2 for 48 h. Cells were lysed and analyzed by western blot using anti-SLFN5 and anti-CPSF5. Virus presence was assessed using anti-p24 antibodies, and anti-GAPDH antibodies were used as a protein loading control. ( A–C ) All experiments were repeated at least three times, and a representative image is shown. Graphs show the average densitometry quantification of at least three replicates with standard deviation. Significance was determined using ANOVA multiple comparisons tests; *** P < 0.001; ns, not significant. CPSF5, cleavage and polyadenylation specificity factor subunit 5; CPSF6, cleavage and polyadenylation specificity factor subunit 6; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; KO, knockout; Luc, luciferase; MOI, multiplicity of infection; NT, non-targeting; p24, viral capsid; SLFN5, Schlafen family member 5; WT, wild-type.

Article Snippet: We used mouse monoclonal antibodies targeting the following proteins: SC35 (clone SC-35; Cat# ab11826, Abcam), CPSF5 (clone 3F8; Cat# H00011051-M12, Novus Biologicals), CPSF7 (clone A-9; Cat# sc-393880, Santa Cruz), CPSF6 (clone F-3; Cat# sc-376228, Santa Cruz), ISG15 (clone F-9; Cat# sc-166755, Santa Cruz), and HIV-1 p24 (clone 183-H12-5C; Cat# ARP-3537, NIH AIDS Reagent Program).

Techniques: Infection, Control, Western Blot, Virus, Standard Deviation, Knock-Out, Luciferase