Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) CFIm25 overexpression increases TAB2 and TBL1XR1 mRNA expression after STM infection, assessed by RT-qPCR analysis in control and CFIm25-OE macrophages at 2 and 6 hours postinfection with STM (n = 3). Data are presented as means ± SD (n = 3); *, P < 0.05; **, P < 0.01. (B) Sequence of primers used for RT-qPCR analysis of TAB2 and TBL1XR1 APA. (C) Schematic representation of the long/total TAB2 and TBL1XR1 transcript regions targeted for RT-qPCR to assess APA and mRNA expression.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Over Expression, Expressing, Infection, Quantitative RT-PCR, Control, Sequencing

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) Western blot analysis of CFIm25 protein in THP-1 macrophages six hours after infection with Salmonella Typhimurium (STM) compared to uninfected controls. A representative Western blot is shown on the left, and densitometric quantification of protein levels is shown on the right, normalized to GAPDH. (B-C) Flow cytometric quantification of ROS (B) and NO (C) production using DCFH-DA and DAF-FM, respectively, showing reduced microbicidal activity six hours postinfection relative to untreated macrophages. (D-E) Arginase activity, measured by urea production (D) and lactate levels (E), in macrophages six hours postinfection. All assays were compared with untreated macrophages (n = 3). Data are presented as means ± SD using three biological replicates; *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Western Blot, Infection, Activity Assay

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) Intracellular bacterial burden (CFU) in control (−) and CFIm25-overexpressing (CFIm25-OE, +) THP-1 macrophages at two and six hours postinfection. (B) Propidium iodide flow cytometric analysis of cell death in control and CFIm25-OE macrophages at two and six hours postinfection. Graphs show the percent of cells staining with propidium iodide; only dead cells take up the stain. (C) Quantification of ROS production using the DCFH-DA fluorescence assay. (D) Quantification of NO production using the DAF-FM fluorescence assay. (E) Antimicrobial activity of conditioned media from control and CFIm25-OE macrophages against STM, assessed by CFU recovery after incubation of bacteria with media. (F) Western blot analysis of CFIm25 and LL-37 protein levels in control and CFIm25-OE macrophages at two and six hours postinfection, (G) Quantitation of LL-37 levels by densitometry measurements and normalized to GAPDH. (H) Arginase activity measured by urea production in control and CFIm25-OE macrophages at two and six hours postinfection. (I) Lactate levels in control and CFIm25-OE macrophages at two and six hours postinfection were measured using a colorimetric lactate assay kit. (J) Flow cytometric analysis of M1 (CD80) and M2 (CD206) surface marker expression in control and CFIm25-OE macrophages at two and six hours postinfection. (K) Concentrations of the TNF-α, IL-12, TGF-β, and IL-10 cytokines in culture supernatants from control and CFIm25-OE macrophages at two h and six hours postinfection as measured by ELISA. Data are presented as means ± SD from three independent experiments (n = 3); *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Control, Staining, Fluorescence, Activity Assay, Incubation, Bacteria, Western Blot, Quantitation Assay, Lactate Assay, Marker, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A-B) RT-qPCR analysis of the log2 ratio of long/total TAB2 (A) and TBL1XR1 (B) mRNA isoforms in control and CFIm25-OE macrophages at two and six hours postinfection, normalized to GAPDH. (C) Western blot analysis of CFIm25, HLA-DR, CD 163, and the indicated immune signaling proteins (“P”, protein; “p”, phosphorylated form) in control and CFIm25-OE macrophages at two and six hours postinfection, with GAPDH as the loading control. (D) Quantification of changes of the indicated proteins in Western blots of samples from control and CFIm25-OE macrophages at two and six hours postinfection, with values normalized to GAPDH. (E) Representative Western blot showing nuclear and cytoplasmic distribution of NF-κB subunits in control and CFIm25-OE macrophages two and six hours postinfection, assessed by fractionation and immunoblotting. Data from all quantifications are presented as means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Quantitative RT-PCR, Control, Western Blot, Fractionation

Journal: bioRxiv

Article Title: The Alternative Polyadenylation Factor CFIm25 Orchestrates Macrophage Antibacterial Immunity by Amplifying TAB2-Mediated MAPK and NF-κB Signaling During Salmonella Infection

doi: 10.64898/2026.02.26.707986

Figure Lengend Snippet: (A) CFUs recovered at six hours postinfection from Intracellular STM in CFIm25-OE macrophages transfected with control siRNA, TAB2 siRNA, or TBL1XR1 siRNA. (B) Concentrations of TNF-α, IL-12, TGF-β, and IL-10 in culture supernatants from CFIm25-OE macrophages expressing control, TAB2, or TBL1XR1 siRNAs, harvested at six hours postinfection, and measured by ELISA. (C-F) ROS (C), NO (D), arginase activity (E), and lactate (F) levels at six hours postinfection in CFIm25-OE macrophages treated with control, TAB2, or TBL1XR1 siRNA. (G) Flow cytometric analysis of CD80 and CD206 expression at six hours postinfection in CFIm25-OE macrophages treated with control, TAB2, or TBL1XR1 siRNA. (H) Western blot analysis of the indicated NF-κB and MAPK signaling proteins at six hours postinfection in CFIm25-OE macrophages treated with control, TAB2, or TBL1XR1 siRNA, with densitometric quantification from three independent experiments (“P”, protein; “p”, phosphorylated form). Data are presented as means ± SD (n = 3); *, P < 0.05; **, P < 0.01.

Article Snippet: Antibodies used in this study included CFIm25 (Proteintech, 10322-1-AP), LL-37 (Santa Cruz, sc-166770), phosphorylated NF-κB P65 (Ser536) (Cell Signaling, 3033), NF-κB P65 (Cell Signaling, 8242), TAB2 (Cell Signaling, 3744), TBL1XR1 (Novus, NBP1-86996), phosphorylated STAT1 (Tyr701) (Cell Signaling, 9167), STAT1 (Cell Signaling, 14994), phosphorylated STAT3(Tyr705) (Cell Signaling, 9145), STAT3 (Cell Signaling, 4904), IκBα (Cell Signaling, 4814), phosphorylated P38(Thr180/Tyr182) (Cell Signaling, 4511), P38 (Cell Signaling, 9212), CD163 (Proteintech 68218-1-g), histone H3 (Cell Signaling, 9715), and GAPDH (Santa Cruz SC3233).

Techniques: Transfection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 1. A novel Nudt21 RNA is highly enriched in mouse testis and male germ cells. A) Schematic of male germ cell and somatic Nudt21 transcripts for mouse, showing that structural differences occur in their respective 30UTRs. DNA products generated by RT-PCR (A, B, C, E) and 3’RACE (D) from mouse testis or kidney total RNA are shown below the transcripts. Arrows indicate the position of the exon 1/intron 1 junction and translation initiation sites. B) Northern blot of total RNA isolated from mouse tissues and enriched adult male germ cells using RT-PCR product A as a probe (see A). Lanes: mouse testis (T), enriched adult male germ cells (gc), kidney (K), liver (L), brain (B), and heart (H). Ethidium bromide staining of RNA loading is shown below each lane. C) Northern analysis of mouse testis and kidney total RNA using a probe specific to Nudt21 exon 1 (product B, see A). Ethidium bromide staining of ribosomal RNAs is shown below. D) Hybridization of total RNA from mouse tissues with RT-PCR product E specific to the 30UTR of mouse somatic Nudt21 mRNA (see A). The blot used in the right panel of B was reprobed for somatic-specific 30UTR sequences and detected a ;4.5-kb mRNA in brain and other tissues, but not the 1.1-kb testis-enriched transcript.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Isolation, Staining, Hybridization

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 2. Nucleotide and coding sequences for mouse testis Nudt21. A) Composite cDNA sequence for mouse testis Nudt21. Primers used to amplify various RT-PCR and 3’RACE products are underlined. The translational initiation and stop codons (bold) as well as putative canonical and noncanonical polyadenylation signals (dashed boxes) and upstream CFIm binding sites (solid line boxes) in the 30UTR are indicated. B) Predicted coding sequences for NUDT21 from mouse testis and somatic human NUDT21 isoforms (hNUDT21–1, 2). See text for GenBank Accession numbers. Asterisks indicate identity with the predicted mouse testis amino acid sequence, while residues absent in the human splice variant hNUDT21–2 are shown with dashes.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Variant Assay

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 6. Distinct developmental variation of CFIm subunit mRNAs and proteins during male germ cell development. A) Total RNA from purified type A spermatogonia (lane A), type B spermatogonia (lane B), prepubertal pachytene spermatocytes (lane PP), pachytene spermatocytes (lane PS), round spermatids (lane RS), and residual bodies (RB) were hybridized with Nudt21 probe A and Cpsf6 probe F. The same blot was probed in each case. Ethidium bromide staining of RNA loading is shown below. B) NUDT21 and CPSF6 protein concentrations are similar in mitotic, meiotic, and spermiogenic germ cell populations. B, B type spermatogo- nia; PS, pachytene spermatocytes; RS, round spermatids; gc, enriched adult mouse male germ cells.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Purification, Staining

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 5. CFIm subunit proteins are elevated in mouse testis and male germ cells. A) Western analysis of NUDT21 (left panel) and CPSF6 (right panel) proteins in whole testis (T) and kidney (K). Equivalency of protein loading was confirmed by Ponceau-S staining (not shown). B) NUDT21 (left) and CPSF6 (right) proteins in nuclear extracts of enriched adult mouse spermatogenic cells (gc).

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Western Blot, Staining

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 8. CFIm transcripts are enriched in human testis. Northern blots of total RNA from different human tissues were probed for Nudt21 (upper panel; probe A) and Cpsf6 (lower panel; probe F) mRNAs. Ethidium bromide staining is shown below for each blot. Lanes: human liver (L), pancreas (P), heart (H), lung (Lu), muscle (M), kidney (K), spleen (S), testis (T), and small intestine (SI).

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Northern Blot, Staining

Journal: Biology of reproduction

Article Title: Pre-messenger RNA cleavage factor I (CFIm): potential role in alternative polyadenylation during spermatogenesis.

doi: 10.1095/biolreprod.107.064774

Figure Lengend Snippet: FIG. 9. ChIP analysis of NUDT21 in adult mouse germ cells and kidney. Formalde- hyde-cross-linked chromatin was precipi- tated with antibodies to NUDT21 (Anti- NUDT21) or using beads alone without primary antibody (No Ab Ctl). A) Following reversal of crosslinks, DNA precipitated from adult male germ cells was assayed for mouse Srebf2, Nr6a1, and Gabra6 genomic sequences by PCR. Specific bands generat- ed by the respective PCR reactions are shown in each case. Aliquots of input DNA (Input) were assayed as positive controls. B) NUDT21 ChIP analysis of proximal (Srebf2_v1) and distal (Srebf2) 30UTRs for the mouse Srebf2 gene in mouse sper- matogenic cell (Germ cells) and kidney chromatin.

Article Snippet: After blocking with 1% nonfat milk for 1 h at room temperature, the blots were incubated with antibody raised against NUDT21/ CPSF5 (1:1000; #BC001403; ProteinTech Group, Chicago, IL) or CPSF6 (1:1000; Abnova, Taipei City, Taiwan) overnight at 48C.

Techniques: Genomic Sequencing

Journal: Molecular and Cellular Biology

Article Title: ARS2 Regulates Nuclear Paraspeckle Formation through 3′-End Processing and Stability of NEAT1 Long Noncoding RNA

doi: 10.1128/MCB.00269-19

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: The antibodies used in this study are shown in . table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Antigen Type Source or reference ARS2 Rabbit Bethyl, Montgomery, TX CPSF6 Rabbit Bethyl, Montgomery, TX NUDT21 Rabbit Proteintech, Rosemont, IL FLAG Mouse Sigma-Aldrich, St. Louis, MO GAPDH Rabbit Abcam, Cambridge, UK α-Tubulin Rabbit Abcam, Cambridge, UK ZC3H18 Rabbit Sigma-Aldrich, St. Louis, MO DIG Mouse Abcam, Cambridge, UK SFPQ Mouse MBL, Aichi, Japan PHAX Mouse 21 CBP80 Mouse 21 Open in a separate window Antibodies used in this study

Techniques:

Journal: Nucleic Acids Research

Article Title: N4-acetylcytidine coordinates with NP1 and CPSF5 to facilitate alternative RNA processing during the replication of minute virus of canines

doi: 10.1093/nar/gkaf229

Figure Lengend Snippet: CPSF5 recruited by NP1 targets viral RNAs to further regulate the alternative RNA processing. ( A ) NP1 interacts with CPSF5, which was verified by performing an IP assay. Flag-tagged NP1 was transiently expressed in WRD cells and then purified by IP using anti-Flag antibodies and detected via MS (Table ) or western blotting. IgG was used as a negative control. ( B ) Southern blot assay showing MVC DNA replication in CPSF5 knockdown cells. Hirt DNA was extracted from MVC-infected cells with CPSF5 knockdown and subjected to Southern blot analysis at 48 h post-infection. ( C ) The expression of MVC protein was identified with CPSF5 knockdown. Western blot analysis of MVC protein expression by knocking down CPSF5 in WRD cells transfected with WT or mutant 3311m MVC infectious clone at 48 h; actin was used as a control. Relative intensity of VP2 versus actin was quantified using the ImageJ program. Data are means ± SDs ( n = 3). ***P ≤ 0.001, **P ≤ 0.01, unpaired Student’s t -test. ( D , E ) MVC RNA levels detected in CPSF5 knockdown cells. qRT-PCR was performed to determine the RNA levels from CPSF5 knockdown WRD cells transfected with infectious clone WT ( D ) or mutant 3311m ( E ) at 48 h by CPSF5, NP1, VP2 ORF primers, and GAPDH was used as a control. Data are means ± SEMs ( n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns: not significant, unpaired Student’s t -tests. ( F , G ) The relationship between CPSF5 and the ac4C-modified residue in regulating RNA processing was detected by RPA. RPA of total RNA extracted from CPSF5-knockdown WRD cells transfected with WT or 3311m mutant MVC infectious clone at 48 h was performed using a 3D-probe ( F ) and (pA)p-probe ( G ). ( H , I ) Ability of CPSF5 binding to the targeted MVC RNA based on formaldehyde-RIP-qRT-PCR. Flag-tagged CPSF5 was expressed in WRD cells, and formaldehyde-cross-linking cell lysates were subjected to IP with IgG or anti-Flag antibodies ( H ). WT and 3311m mutant infectious clones were used to transfect WRD cells in which CPSF5 was knocked down, then the cells were subjected to IP with anti-CPSF5 antibodies. qRT-PCR was performed to quantify MVC RNA ( I ). IgG was used as a negative control. Unpaired student’s t -tests were performed, and the data are presented as the means ± SEMs ( n = 3). ***P ≤ 0.001.

Article Snippet: Indicated proteins were detected using primary mouse monoclonal antibodies against beta-actin (sc47778, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (Cat. No. 60004-1-lg; Proteintech); rabbit polyclonal antibodies against CPSF5 (A4482, ABclonal), NAT10 (13365-1-AP, Proteintech), and Flag (F1804-1 MG, Sigma–Aldrich); anti-HA (66006-1-Ig, Proteintech) and anti-Histone H3 antibodies (Cat. No. GTX122148; GeneTex); and three rabbit polyclonal antibodies against MVC NP1, NS1, and VP2 that were previously generated [ ].

Techniques: Purification, Western Blot, Negative Control, Southern Blot, Knockdown, Infection, Expressing, Transfection, Mutagenesis, Control, Quantitative RT-PCR, Modification, Residue, Binding Assay, Clone Assay

Journal: Nucleic Acids Research

Article Title: N4-acetylcytidine coordinates with NP1 and CPSF5 to facilitate alternative RNA processing during the replication of minute virus of canines

doi: 10.1093/nar/gkaf229

Figure Lengend Snippet: MS data of NP1

Article Snippet: Indicated proteins were detected using primary mouse monoclonal antibodies against beta-actin (sc47778, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (Cat. No. 60004-1-lg; Proteintech); rabbit polyclonal antibodies against CPSF5 (A4482, ABclonal), NAT10 (13365-1-AP, Proteintech), and Flag (F1804-1 MG, Sigma–Aldrich); anti-HA (66006-1-Ig, Proteintech) and anti-Histone H3 antibodies (Cat. No. GTX122148; GeneTex); and three rabbit polyclonal antibodies against MVC NP1, NS1, and VP2 that were previously generated [ ].

Techniques:

Journal: Nucleic Acids Research

Article Title: N4-acetylcytidine coordinates with NP1 and CPSF5 to facilitate alternative RNA processing during the replication of minute virus of canines

doi: 10.1093/nar/gkaf229

Figure Lengend Snippet: CPSF5 mediated MVC RNA splicing is dependent on NP1 and ac4C modification at residue 3311. ( A , B ) The effect on CPSF5 binding to MVC RNA by increasing NP1 expression using formaldehyde-RIP-qRT-PCR. Vector or HA-NP1 and infectious clone WT or 3311m were co-transfected in WRD cells. Cell lysates cross-linked by formaldehyde were subjected to IP with IgG, anti-CPSF5 antibodies. qRT-PCR was performed to quantify the targeted MVC RNA. IgG was used as a negative control. Unpaired Student’s t -tests were performed, and the data are presented as means ± standard errors of the means ( n = 3). *** P ≤ 0.001, ns: not significant. ( C , D ) The effect on NP1 binding to MVC RNA by increasing CPSF5 expression using formaldehyde-RIP-qRT-PCR. Vector or Flag-CPSF5 and infectious clone WT or mutant 3311m were co-transfected in WRD cells. Cell lysates were subjected to IP with IgG, anti-NP1 antibodies. qRT-PCR was performed to quantify the targeted MVC RNA. IgG was used as a negative control. Unpaired Student’s t -tests were performed, and the data are presented as means ± standard errors of the means ( n = 3). ns: not significant. ( E ) Characterization of the cleavage and polyadenylation sites and downstream elements (DSEs) in (pA)p is shown at the top. The bottom panel shows a schematic representation of the RNA pulldown assay. The DNA template for T7 transcription consisted of three repeats of modified DSE. ( F , G ) Biotinylated RNA affinities of NP1 and CPSF5. HA-tagged NP1 or the vector was transiently expressed in WRD cells, and cell lysates were used to bind biotinylated RNA with or without the ac4C modification. In addition, purified NP1 or CPSF5 protein was mixed with RNA oligonucleotides with or without ac4C-modified RNA and analyzed via western blotting. ( H , I ) The binding analysis of CPSF5 or NP1 to oligo-RNA with or without the ac4C modification in the presence or absence of the NP1 ( H ) or CPSF5 ( I ) protein. ( J , K ) The binding analysis of NP1 and CPSF5 to ac4C-modified RNA. Equal amounts of purified CPSF5 ( J ) or NP1 ( K ) protein and increasing amounts of NP1 ( J ) or CPSF5 ( K ) protein were incubated with ac4C-modified RNA and analyzed via western blotting. Blots were detected using anti-CPSF5 or anti-NP1 antibodies.

Article Snippet: Indicated proteins were detected using primary mouse monoclonal antibodies against beta-actin (sc47778, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (Cat. No. 60004-1-lg; Proteintech); rabbit polyclonal antibodies against CPSF5 (A4482, ABclonal), NAT10 (13365-1-AP, Proteintech), and Flag (F1804-1 MG, Sigma–Aldrich); anti-HA (66006-1-Ig, Proteintech) and anti-Histone H3 antibodies (Cat. No. GTX122148; GeneTex); and three rabbit polyclonal antibodies against MVC NP1, NS1, and VP2 that were previously generated [ ].

Techniques: Modification, Residue, Binding Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation, Transfection, Negative Control, Mutagenesis, Purification, Western Blot, Incubation