Journal: bioRxiv

Article Title: mRNA poly(A)-tail length is a battleground for coronavirus–host competition

doi: 10.1101/2025.10.09.680815

Figure Lengend Snippet: (A) Outline of infection time course and fractionation. (B) Assessment of subcellular fractionation. Shown are immunoblots of whole-cell lysates (WCL), cytoplasmic lysates (Cyto), and nuclear lysates (Nuc) of mock and infected cells (11 hpi) probed for a cytoplasmic marker (PABPC1) as well as a nuclear marker (Histone H3). (C) Assessment of subcellular fractionation by processing of Xbp1 mRNA. Shown is an agarose gel of RT-PCR products for Xbp1 mRNA in WCLs, cytoplasmic lysates, and nuclear lysates of mock or MHV-infected cells. (D) Outline of the rapid capture of DMVs adopted from organelle IP – (top), as well as metabolic labeling for capturing the nascent mRNAs (bottom). (E) Assessment of DMV IP. Shown are immunoblots of cytoplasmic inputs and GFP IPs of cells infected with MHV-A59 or MHV-Δ2-GFP3 probed for viral protein nsp9 and various cytoplasmic markers. (F) Tail-length distributions of bulk and nascent mitochondrial mRNAs. Plotted are CDFs of poly(A)-tail lengths of total cytoplasmic and nascent mitochondrial mRNAs (input and 5-EU, respectively).

Article Snippet: The primary antibodies were used at the following dilutions: PABPC1 (Cell Signaling Technology, #4992S, 1:1000), Histone H3 (Cell Signaling Technology, #9715S, 1:5000), nsp9 (MHV; Thermo Fisher, #200-301-A56, 1:1000), LC3 (Cell Signaling Technology, #2775S, 1:1000), CALR (Cell Signaling Technology, #12238S, 1:1000), CTSB (Cell Signaling Technology, #31718S, 1:1000), Golgin-97 (Cell Signaling Technology, #13192S, 1:1000), CSDE1 (Abcam, #ab201688, 1:1000), Vinculin (Cell Signaling Technology, #13901S, 1:5000), GFP (Cell Signaling Technology, #2956S, 1:2000), and Puromycin (MilliporeSigma, #MABE343, 1:1000).

Techniques: Infection, Fractionation, Western Blot, Marker, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Labeling

Journal: bioRxiv

Article Title: mRNA poly(A)-tail length is a battleground for coronavirus–host competition

doi: 10.1101/2025.10.09.680815

Figure Lengend Snippet: (A) Narrow tail-length distribution of viral mRNAs compared to host mRNAs. Plotted are tail-length distributions of viral mRNAs (at 3 and 9 hpi) and host mRNAs (at 3 and 9 hpi) upon infection. (B) Narrow tail-length distribution of MHV mRNAs compared to host mRNAs. Shown for cytoplasmic mRNAs of virus or each host gene passing the expression cutoff are standard deviations for tail lengths in MHV-infected cells (at 9 hpi) plotted as a function of median tail length. (C) Narrow distribution of SARS-CoV-2 mRNAs compared to host mRNAs at 6 hpi; otherwise, as in (B). (D) AF3 prediction of multimers between virus-associated RBPs and PABPC1. Plotted are PEAK scores for an AF3 screen of multimers of virus-associated mRNA-binding proteins and either full-length PABPC1 or its individual domains. (E) The PAE plot generated from the AF3 model of the multimer between CSDE1 and RRM1-RRM2 domain of PABPC1. (F) Schematic and the 3D model of AF3-predicted complex between CSDE1 and RRM1-RRM2 domain of PABPC1. The poly(A) tail in the 3D model (right) is inserted from the superimposed structures of AF3 model and PDB: 4F02. (G) Biochemical support for the AF3 model of the complex between CSDE1 and RRM1-RRM2 domain of PABPC1. Shown are size-exclusion chromatograms of the minimal components of the complex: Residues 2-186 of CSDE1 comprising CSD1 and CSD2, residues 2-191 of PABPC1 comprising RRM1 and RRM2 of PABPC1, and oligo(A) 10 . The CSDE1 mut harbors mutations (V136R and Y138R) predicted to disrupt the interface between CSDE1 and the RRM1-RRM2 domain of PABPC1.

Article Snippet: The primary antibodies were used at the following dilutions: PABPC1 (Cell Signaling Technology, #4992S, 1:1000), Histone H3 (Cell Signaling Technology, #9715S, 1:5000), nsp9 (MHV; Thermo Fisher, #200-301-A56, 1:1000), LC3 (Cell Signaling Technology, #2775S, 1:1000), CALR (Cell Signaling Technology, #12238S, 1:1000), CTSB (Cell Signaling Technology, #31718S, 1:1000), Golgin-97 (Cell Signaling Technology, #13192S, 1:1000), CSDE1 (Abcam, #ab201688, 1:1000), Vinculin (Cell Signaling Technology, #13901S, 1:5000), GFP (Cell Signaling Technology, #2956S, 1:2000), and Puromycin (MilliporeSigma, #MABE343, 1:1000).

Techniques: Infection, Virus, Expressing, Binding Assay, Generated

Journal: bioRxiv

Article Title: mRNA poly(A)-tail length is a battleground for coronavirus–host competition

doi: 10.1101/2025.10.09.680815

Figure Lengend Snippet: (A) (A) Replot of RAP-MS data for SARS-CoV-2 mRNAs . Plotted are changes in abundance of RBPs pulled down with probes targeting SARS-CoV-2 genomic mRNA compared to proteins pulled down with probes targeting RNA component of mitochondrial RNA processing endoribonuclease (RMRP). (B) Identification of CSDE1 as a pro-viral factor. Plotted are the ranked score of CSDE1 and other proteins found in CRISPR screens designed to identify host dependencies of SARS-CoV-2 infection , . (C) Formation of a ternary complex involving full-length CSDE1, PABPC1, and the poly(A) tail. Shown are size-exclusion chromatograms of the full-length components of the CSDE1–PABPC1–oligo(A) 30 complex. (D) Preferential binding of CSDE1 to poly(A)-bound PABPC1. Shown are size-exclusion chromatograms of CSDE1 and PABPC1 in the absence or presence of the oligo(A) 30 . (E) Stoichiometry of the ternary complex involving CSDE1, PABPC1, and oligo(A) 30 . Plotted are mass photometry profiles of the full-length components of the CSDE1–PABPC1–oligo(A) 30 complex. (F) Predicted binding of CSD2 to the RRM1 domain of PABPC paralogs. Plotted are PEAK scores for a ColabFold screen of multimers of CSD2 of CSDE1 and all RRM domains in the human proteome. (G) Biochemical support for the AF3 model of the complex between CSDE1 and RRM1-RRM2 domain of PABPC1. Shown are size-exclusion chromatograms for absorbance at 260 nm, which enables detection of the RNA, for the experiment shown in .

Article Snippet: The primary antibodies were used at the following dilutions: PABPC1 (Cell Signaling Technology, #4992S, 1:1000), Histone H3 (Cell Signaling Technology, #9715S, 1:5000), nsp9 (MHV; Thermo Fisher, #200-301-A56, 1:1000), LC3 (Cell Signaling Technology, #2775S, 1:1000), CALR (Cell Signaling Technology, #12238S, 1:1000), CTSB (Cell Signaling Technology, #31718S, 1:1000), Golgin-97 (Cell Signaling Technology, #13192S, 1:1000), CSDE1 (Abcam, #ab201688, 1:1000), Vinculin (Cell Signaling Technology, #13901S, 1:5000), GFP (Cell Signaling Technology, #2956S, 1:2000), and Puromycin (MilliporeSigma, #MABE343, 1:1000).

Techniques: CRISPR, Infection, Binding Assay

Journal: bioRxiv

Article Title: mRNA poly(A)-tail length is a battleground for coronavirus–host competition

doi: 10.1101/2025.10.09.680815

Figure Lengend Snippet: (A) Repression of deadenylation by CSD1–2. Shown are time courses of in vitro deadenylation of a UCUAAAU(A)□□ substrate by the full CCR4–NOT complex. Reactions were performed in the presence of either no added protein (–), PABPC1 only, CSDE1 residues 2–186 (containing CSD1 and CSD2) only in either wild-type (WT, top panel) or mutant (mut, bottom panel) form, or PABPC1 together with CSDE1 residues 2–186 in either wild-type (WT, top panel) or mutant (mut, bottom panel) form. (B) Repression of deadenylation by full-length CSDE1. Shown are time courses of in vitro deadenylation of a UCUAAAU(A)□□ substrate by the full CCR4–NOT complex. Reaction conditions are similar to (A) except full-length CSDE1 was used in these experiments. (C) Reduction of viral mRNA poly(A)-tail length in cells depleted of CSDE1. Shown is an RNase H northern blot measuring poly(A)-tail lengths of viral mRNAs in virions for each passage of a serial infection in control and CSDE1 KD NIH-3T3 cells. (D) CDF plot of the poly(A)-tail lengths of viral mRNAs in virions quantified from (C). Plotted are the average values of two independent nontargeting sgRNAs or two sgRNAs targeting Csde1 .

Article Snippet: The primary antibodies were used at the following dilutions: PABPC1 (Cell Signaling Technology, #4992S, 1:1000), Histone H3 (Cell Signaling Technology, #9715S, 1:5000), nsp9 (MHV; Thermo Fisher, #200-301-A56, 1:1000), LC3 (Cell Signaling Technology, #2775S, 1:1000), CALR (Cell Signaling Technology, #12238S, 1:1000), CTSB (Cell Signaling Technology, #31718S, 1:1000), Golgin-97 (Cell Signaling Technology, #13192S, 1:1000), CSDE1 (Abcam, #ab201688, 1:1000), Vinculin (Cell Signaling Technology, #13901S, 1:5000), GFP (Cell Signaling Technology, #2956S, 1:2000), and Puromycin (MilliporeSigma, #MABE343, 1:1000).

Techniques: In Vitro, Mutagenesis, Northern Blot, Infection, Control

Journal: bioRxiv

Article Title: mRNA poly(A)-tail length is a battleground for coronavirus–host competition

doi: 10.1101/2025.10.09.680815

Figure Lengend Snippet: (A) A very weak relationship between changes in half-lives of host mRNAs observed upon infection with SARS-CoV-2 in Calu-3 cells and changes in half-lives of mRNAs observed upon expression of SARS-CoV-2 nsp1 in HEK-293T cells . (B) Impact of MHV nsp1 on global translation levels. Shown are translating proteins labeled by puromycylation in L2 cells expressing a doxycycline-inducible version of MHV nsp1 for the indicated times. The lack of signal in the presence of cycloheximide is shown as a control. (C) Degradation of mRNAs by MHV nsp1. Plotted are abundances of cytoplasmic mRNAs in L2 cells expressing a doxycycline-inducible version of MHV nsp1 for the indicated time. (D) The correlation between changes in mRNA abundances in L2 cells infected with MHV for either 9 or 11 h (top and bottom, respectively) and changes in mRNA abundances of L2 cells expressing a dox-inducible version of nsp1 from MHV for 6 h. (E) The correlation between changes in mRNA abundances in L2 cells infected with MHV for either 9 or 11 h (top and bottom, respectively) and changes in mRNA abundances of L2 cells expressing a dox-inducible version of nsp1 from MHV for 12 h. (F) The correlation between changes in half-lives of host mRNAs upon infection with SARS-CoV-2 in Calu-3 cells and changes in half-lives of mRNAs upon knockdown of PABPC1 and PABPC4 in HeLa cells . (G) The correlation between changes in mRNA abundances upon infection of HCT116 cells with MHV (18 hpi) and changes in mRNA abundances upon PABPC1 depletion in HCT116 cells. (H) Ectopic increase of PABPC1 levels during MHV-A59 infection. Shown are Immunoblots of L2 cells expressing a PABPC1 variant in which the C term of PABPC1 is fused to the destabilizing moiety ecDHFR in mock and infected cells treated with either DMSO or stabilizing ligand TMP. (I) Sensitivity of infection-induced cytoplasmic mRNA degradation to increased PABPC1 levels. Plotted is the correlation between changes in cytoplasmic host mRNA abundances of L2 cells upon infection with MHV and changes in cytoplasmic host mRNA abundances of MHV-infected L2 cells upon ectopic PABPC1 expression upon treatment with TMP.

Article Snippet: The primary antibodies were used at the following dilutions: PABPC1 (Cell Signaling Technology, #4992S, 1:1000), Histone H3 (Cell Signaling Technology, #9715S, 1:5000), nsp9 (MHV; Thermo Fisher, #200-301-A56, 1:1000), LC3 (Cell Signaling Technology, #2775S, 1:1000), CALR (Cell Signaling Technology, #12238S, 1:1000), CTSB (Cell Signaling Technology, #31718S, 1:1000), Golgin-97 (Cell Signaling Technology, #13192S, 1:1000), CSDE1 (Abcam, #ab201688, 1:1000), Vinculin (Cell Signaling Technology, #13901S, 1:5000), GFP (Cell Signaling Technology, #2956S, 1:2000), and Puromycin (MilliporeSigma, #MABE343, 1:1000).

Techniques: Infection, Expressing, Labeling, Control, Knockdown, Western Blot, Variant Assay

Journal: bioRxiv

Article Title: mRNA poly(A)-tail length is a battleground for coronavirus–host competition

doi: 10.1101/2025.10.09.680815

Figure Lengend Snippet: (A) Tail-length distributions of zebrafish mRNA poly(A) tags, used as spike-in controls for the experiment shown in . (B) Domination of host cytoplasm by viral mRNAs. Plotted are the proportion of host and viral mRNAs in the total cytoplasmic mRNA population over the course of infection. (C) Preferential degradation of short-tailed mRNAs upon PABPC1 knockdown in NIH-3T3 cells . Otherwise, this panel is as in . (D) Preferential degradation of short-tailed mRNAs upon PABPC1 knockdown in NIH-3T3 cells , analyzed after grouping mRNAs by gene; otherwise, as in . (E) Tail-length distributions of zebrafish mRNA poly(A) tags, used as spike-in controls for the experiment shown in . (F) AF3 predicted model of nsp1 from MHV. The putative ribosome-binding domain at the C terminus is indicated in a dashed box. The regions deleted in the MHV-Δ99 strain are shown in red. (G) Impact of nsp1-Δ99 on global translation levels compared to nsp1; otherwise, as in . (H) Tail-length distributions of zebrafish mRNA poly(A) tags, used as spike-in controls, for the experiment shown in .

Article Snippet: The primary antibodies were used at the following dilutions: PABPC1 (Cell Signaling Technology, #4992S, 1:1000), Histone H3 (Cell Signaling Technology, #9715S, 1:5000), nsp9 (MHV; Thermo Fisher, #200-301-A56, 1:1000), LC3 (Cell Signaling Technology, #2775S, 1:1000), CALR (Cell Signaling Technology, #12238S, 1:1000), CTSB (Cell Signaling Technology, #31718S, 1:1000), Golgin-97 (Cell Signaling Technology, #13192S, 1:1000), CSDE1 (Abcam, #ab201688, 1:1000), Vinculin (Cell Signaling Technology, #13901S, 1:5000), GFP (Cell Signaling Technology, #2956S, 1:2000), and Puromycin (MilliporeSigma, #MABE343, 1:1000).

Techniques: Infection, Knockdown, Binding Assay

Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Inhibition of tRNA charging activates the ISR without inducing RNP granules. ( A ) U-2 OS cells were treated with 0.2% DMSO carrier, 20 µM halofuginone (HF), 250 µM sodium arsenite (As), or 1.25 µM thapsigargin (Tg) for 1 h, and Western blotting for phosphorylated and total eIF2α was done. Total protein ( n = 3 independent replicates) and molecular weights (in kilodaltons) are shown. ( B ) Translation activity was measured using bioorthogonal noncanonical amino acid tagging in cells treated as in A . Methionine or azidohomoalanine (AHA) was added 10 min prior to collection. The ratio of nascent to total protein is shown from n = 3 independent replicates. ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A and fixed, and nuclei were stained with Hoechst (blue). The percentage of cells with SGs was quantified from n = 3 independent experiments; ≥305 cells were counted in each condition. Scale bars, 10 µm. ( D ) Cells were treated as in C , and immunofluorescence and fluorescence in situ hybridization were done to detect UBAP2L (magenta), PABPC1 (cyan), and poly(A) RNA with oligo(dT) probes (yellow) ( n = 2 independent experiments). ( E ) Cells were treated with 0.2% DMSO, 20 µM HF plus 0.2% DMSO, 250 µM arsenite, or 1.25 µM thapsigargin for 1 h, and the percentage of cells with SGs was quantified ( n = 3 independent experiments); ≥287 cells were counted per treatment. ( F ) Cells were treated, and immunofluorescence microscopy was done as in D to detect the P-body marker DCP1A. Quantification of the percentage of cells with P-bodies (PBs) from n = 3 independent experiments is shown; ≥364 cells were counted per treatment. ( G ) Cells were treated as in C to detect XRN1 (magenta) and EDC4 (yellow) by immunofluorescence microscopy ( n = 2 independent experiments). ( H ) U-2 OS cells were treated as in E , and immunofluorescence microscopy was done to detect DCP1A. The percentage of cells with PBs from n = 3 independent experiments is shown; ≥351 cells were counted per treatment across all replicates. Representative images are shown with the average ± SEM for each experiment, and green, gray, and pink points represent the average of each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, (****) P < 0.001.

Article Snippet: PABPC1 , Proteintech , 66809 , 1:100 (immunofluorescence).

Techniques: Inhibition, Western Blot, Activity Assay, Stable Transfection, Expressing, Staining, Immunofluorescence, Fluorescence, In Situ Hybridization, Microscopy, Marker

Journal: Journal of experimental & clinical cancer research : CR

Article Title: EIF4A3-mediated biogenesis of circSTX6 promotes bladder cancer metastasis and cisplatin resistance.

doi: 10.1186/s13046-023-02932-6

Figure Lengend Snippet: Fig. 9 Schematic diagram describes the mechanism by which circSTX6 promotes metastasis and chemoresistance of BCa through rehulation of SUZ12 by sponging miR-515-3p and interacting with PABPC1, respectively

Article Snippet: Short hairpin RNAs targeting circSTX6, SUZ12 and PABPC1 were synthesized by BIOMED, and were cloned into the pLKO.1 vector (addgene, USA).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Deer thymosin beta 10 functions as a novel factor for angiogenesis and chondrogenesis during antler growth and regeneration

doi: 10.1186/s13287-018-0917-y

Figure Lengend Snippet: Antibodies

Article Snippet: Vascular endothelial growth factor-D (VEGFD) , Rabbit , Polyclonal antibody , Elabscience, China , E-AB-33213 , WB: 1 μg/ml (1:1000).

Techniques: Control, Labeling