Journal: Cell discovery

Article Title: RBBP6 maintains glioblastoma stem cells through CPSF3-dependent alternative polyadenylation.

doi: 10.1038/s41421-024-00654-3

Figure Lengend Snippet: Fig. 7 Working model of RBBP6 in regulating GSCs. RBBP6 regulates the maintenance of GSCs. Depletion of RBBP6 leads to the elimination of K63-linked ubiquitination of CPSF3, resulting in the destabilization of CPSF3 and the subsequent shortening of the 3’ UTRs of MYC ceRNAs. This shortening promotes the release of miR-590-3p from the truncated UTRs, thereby reducing MYC expression and hindering the survival and progression of GSCs.

Article Snippet: The antibodies used in this study wereRBBP6 (Bethyl Laboratories, Cat# A304-975A), CPSF3 (Proteintech, Cat# 11609-1-AP), CPSF2 (Proteintech, Cat# 17739-1-AP), NUDT21 (Proteintech, Cat# 10322-1-AP), CSTF2 (Proteintech, Cat# 26825-1-AP), HA-tag (Cell signaling Technology, Cat# 3724 S), Flag-tag (Sigma, Cat # F1804), MYC-tag (Proteintech, Cat # 16286- 1-AP), MYC (Cell signaling Technology, Cat# D3N8F), GAPDH (Proteintech, Cat# 60004-1-Ig).

Techniques: Ubiquitin Proteomics, Expressing

Journal: Journal of Molecular Cell Biology

Article Title: Distinct roles of two SEC scaffold proteins, AFF1 and AFF4, in regulating RNA polymerase II transcription elongation

doi: 10.1093/jmcb/mjad049

Figure Lengend Snippet: AFF4 disruption leads to increased CSTF2 occupancy with a 5′ shift. ( A ) The protein levels of CSTF2, CPSF3, SYMPK, XRN2, PPP1CB, and PPP1CC in WT, AFF4 KO, and AFF4 MT HCT-116 cells. α-Tubulin was used as a loading control. ( B ) Metaplot analysis of CSTF2 occupancies at the serum-inducible genes in WT, AFF4 KO, and AFF4 MT HCT-116 cells. CSTF2 densities are plotted in the region from −3 kb of the TSS to +3 kb of the TES within the genes. The metaplot was generated by rep1 ChIP–seq data that were verified by ChIP–qPCR. ( C ) Genome browser tracks of CSTF2 ChIP–seq at the serum-inducible genes FOS and EGR1 in WT, AFF4 KO, and AFF4 MT cells. Purple vertical dotted lines denote the TES and black arrows indicate Pol II peak shift toward 5′ ends. ( D and E ) RT–qPCR analysis of CSTF2 ( D ) and FOS ( E ) gene expression levels after CSTF2 knockdown in WT and AFF4 MT HCT-116 cells. ( F ) ChIP–qPCR analysis of Pol II occupancy at the FOS gene after CSTF2 knockdown in WT and AFF4 MT cells. The HEMO gene acts as a negative control for ChIP–qPCR. Data represent mean ± SEM of three biological replicates. ** P < 0.01, *** P < 0.001, **** P < 0.0001, t -test.

Article Snippet: Pol II Ser2P (ab5095), Pol II Ser5P (ab5131), and PAF1 (ab20662) rabbit polyclonal antibodies were purchased from Abcam; SPT5 (A9193), SPT6 (A16434), CSTF2 (A8116), CPSF3 (A12368), SYMPK (A8722), XRN2 (A18350), PPP1CB (A13528), and PPP1CC (A4025) rabbit polyclonal antibodies were purchased from ABclonal.

Techniques: Disruption, Generated, ChIP-sequencing, Quantitative RT-PCR, Expressing, Negative Control

Journal: BMC Cancer

Article Title: Comprehensive analysis of alternative polyadenylation regulators concerning CD276 and immune infiltration in bladder cancer

doi: 10.1186/s12885-022-10103-7

Figure Lengend Snippet: Analysis of the CPSF3 gene expression in BC. A The Venn diagram suggested that the upregulation of CPSF3 was an unfavorable prognostic factor and positively correlated with the CD276 expression in bladder cancer (BC). B The heat map visualized the relative expression of 26 APA regulators in BC/normal (row 1) and the correlations of 26 regulators with the CD276 expression (row 2) and prognosis (row 3). The red color indicates a high expression in BC, positively correlated with CD276 or unfavorable prognosis. * p < 0.05 and ** p < 0.01. CPSF3 was upregulated in BC tissues compared to normal tissues.TCGA-BLCA cohort ( C ), GSE13507 ( D ), and GSE38264 ( E ). F Relative expression of CPSF3 was detected by qRT -PCR in 16 pairs of BC and normal tissues. G Compared with SV-HUC-1 cell line (normal), CPSF3 was highly expressed in EJ, T24, TCCSUP, UMUC3, 5637, RT4 cell lines (BC). H Immunohistochemistry staining indicated that, compared with normal bladder tissue (left), CPSF3 was significantly elevated in BC tissue (right) in the human protein atlas (antibody HPA034657, × 10). * p < 0.05, ** p < 0.01

Article Snippet: The protein expression level of CPSF3 in BC compared to normal tissue was obtained from the Human Protein Atlas database ( https://www.proteinatlas.org/ ).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining

Journal: BMC Cancer

Article Title: Comprehensive analysis of alternative polyadenylation regulators concerning CD276 and immune infiltration in bladder cancer

doi: 10.1186/s12885-022-10103-7

Figure Lengend Snippet: Upregulated CPSF3 expression is associated with poor outcomes of bladder cancer (BC). A , B , C The Kaplan–Meier analysis of BC patients with high and low CPSF3 expression level in the TCGA cohort for ( A ) overall surviva, ( B ) disease-specific surviva, ( C ) progress-free interval. D , E The stacked bar chart and violin plot visualized the proportion of live/death and survival time in high/low CPSF3 groups in BC patients. F Time-dependent receiver operating characteristics analysis of CPSF3. G , H Forest plots based upon the outcomes of univariate ( G ) and multivariate Cox regression ( H ) of CPSF3 expression and other clinicopathological factors. * p < 0.05

Article Snippet: The protein expression level of CPSF3 in BC compared to normal tissue was obtained from the Human Protein Atlas database ( https://www.proteinatlas.org/ ).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Comprehensive analysis of alternative polyadenylation regulators concerning CD276 and immune infiltration in bladder cancer

doi: 10.1186/s12885-022-10103-7

Figure Lengend Snippet: Correlation analysis of CPSF3 expression with CD276 and infiltrating immune cells in bladder cancer (BC). A , B The enriched Gene Ontology ( A ) and Kyoto Encyclopedia of Genes and Genomes signaling pathways ( B ) analysis of highly expressed CPSF3 in BC. C Pearson correlation analysis of CPSF3 expression and CD276 expression in BC. D The infiltrating levels of immune cells in high and low CPSF3 expression groups in BC patients. ( E ) The heat map visualized the percentage abundance of tumor-infiltrating immune cells in each sample. * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: The protein expression level of CPSF3 in BC compared to normal tissue was obtained from the Human Protein Atlas database ( https://www.proteinatlas.org/ ).

Techniques: Expressing, Protein-Protein interactions

Journal: Nature chemical biology

Article Title: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma

doi: 10.1038/s41589-019-0424-1

Figure Lengend Snippet: a, Full genome siRNA plus compound 1 synergy screen in A-673 cells, with cell viability read-out via CellTiter Glo assay. Per siRNA, the differential effect between DMSO-treated versus compound 1-treated cells was calculated. Plotting gene-level redundant siRNA activity (RSA statistical model, one-sided, down) as a measure of significance of effect versus Q1 Z-score as a measure of magnitude of effect identified siRNA-targeted genes whose knock-down sensitized cells to 0.4 µM compound 1 treatment. The siRNA library was tested as one replicate with each gene represented by n=8 siRNAs on average. Top screening hits, including known regulators of mRNA processing, are highlighted and named. b, Chemical proteomics studies using 6 in NOMO-1 cells. Proteins identified as specifically competed are highlighted and named. c, Photo-affinity labeling with PAL probe 7 in A-673 cells. Specifically enriched proteins are highlighted and named. d, Affinity binding measurement of compound 2 to human CPSF3 (Kd = 370 nM) as determined by SEC-TID. Error bars represent standard deviation of the mean (n=2). e, Western blot against CPSF3 following Cellular Thermal Shift Assay (CETSA) with compound 2 (+) or DMSO (-) treatment using NOMO-1 cell lysates (n=1).

Article Snippet: Blots were probed with either anti-FLI1 antibody (Abcam ab124791, dilution 1:1000), anti-NKX2.2 antibody (Abcam ab187375, dilution 1:1000), anti-MYC antibody (Cell Signaling Technologies 13987, dilution 1:1000), anti-MYB antibody (Cell Signaling Technologies 12319, dilution 1:1000), anti-MEK1/2 (Cell Signaling 8727, dilution 1:1000), anti-Vimentin (Cell Signaling 5741, dilution 1:1000), anti-CPSF3 (Abgent AT1610a, dilution 1:1000), anti-CES1 (Abcam ab68190, dilution 1:1000), or anti β-actin antibody (Sigma A5441, dilution 1:10,000) in 3% non-fat dry milk in TBST (TBS, 0.1% Tween-20) at 4°C overnight (see Supplementary Table 3 for list of antibodies used in this study).

Techniques: Glo Assay, Activity Assay, Labeling, Binding Assay, Standard Deviation, Western Blot, Thermal Shift Assay

Journal: Nature chemical biology

Article Title: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma

doi: 10.1038/s41589-019-0424-1

Figure Lengend Snippet: a, Co-crystal structure of CPSF3 and 2. Ribbon representation of the CPSF3 metallo-β-lactamase domain in cyan (residues 7 to 208) and magenta (396 to 459), and the β-CASP domain in green (209 to 395). Two zinc ions at the active site (orange spheres) are coordinated by a phosphate. 2 is shown as yellow ball-andstick and the interfacial cavity outlined by a grey surface. Insert highlights polar interactions between 2 and CPSF3. The carboxylate group of 2 forms bifurcated hydrogen bonds to the backbone NH of Phe241 and Gly330, respectively. Three ordered water molecules contact the carboxylate. The hydroxyl group is in hydrogen bonding distance to the backbone NH groups of Gly330 and Met331, respectively. b, In vitro cleavage reaction monitored over time using yeast recombinant 8-subunit core CPF complex, cleavage factors (CFIA and CFIB) and a Cyc1 model RNA substrate in the presence of 1% DMSO or 100 µM compound 2. The data was fitted to an exponential plateau model (solid line), error bars represent standard deviation of the mean (n=4). c, Missense mutation distribution in CPSF3 that desensitize to compound 1 in cell viability assay as identified from variomics studies in A-673. d, Cell viability assessment of A-673 cells expressing in trans either wild-type (WT) CPSF3 encoding cDNA or dominant mutant versions of human CPSF3 as derived from variomics study in response to treatment with compound 1. Small black circle on fitted curve for WT condition indicates IC50 value. Center values represent mean, error bars represent standard deviation (n=3).

Article Snippet: Blots were probed with either anti-FLI1 antibody (Abcam ab124791, dilution 1:1000), anti-NKX2.2 antibody (Abcam ab187375, dilution 1:1000), anti-MYC antibody (Cell Signaling Technologies 13987, dilution 1:1000), anti-MYB antibody (Cell Signaling Technologies 12319, dilution 1:1000), anti-MEK1/2 (Cell Signaling 8727, dilution 1:1000), anti-Vimentin (Cell Signaling 5741, dilution 1:1000), anti-CPSF3 (Abgent AT1610a, dilution 1:1000), anti-CES1 (Abcam ab68190, dilution 1:1000), or anti β-actin antibody (Sigma A5441, dilution 1:10,000) in 3% non-fat dry milk in TBST (TBS, 0.1% Tween-20) at 4°C overnight (see Supplementary Table 3 for list of antibodies used in this study).

Techniques: In Vitro, Recombinant, Standard Deviation, Mutagenesis, Viability Assay, Expressing, Derivative Assay

Journal: Nature chemical biology

Article Title: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma

doi: 10.1038/s41589-019-0424-1

Figure Lengend Snippet: Cancers such as EWSFLI translocated Ewing’s sarcoma or MLL-translocated AML have selected for a balance between oncogenic fusion protein-driven aberrant transcription and its associated genomic instability that allows for proliferation. Under such conditions, the mRNA processing and termination machinery, specifically one of its core components, the cleavage and polyadenylation specificity factor 3 (CPSF3), represents a previously undescribed novel synthetic lethal node. Compound 2 inhibition of CPSF3-mediated mRNA cleavage results in RNA Pol II read-through beyond the 3’-UTR and transcript accumulation. As a result, gene expression is perturbed, including downregulation of genes involved in DNA double-strand break repair, while R-loop formation is increased to levels that the cell can no longer buffer or balance, ultimately leading to a cell viability defect.

Article Snippet: Blots were probed with either anti-FLI1 antibody (Abcam ab124791, dilution 1:1000), anti-NKX2.2 antibody (Abcam ab187375, dilution 1:1000), anti-MYC antibody (Cell Signaling Technologies 13987, dilution 1:1000), anti-MYB antibody (Cell Signaling Technologies 12319, dilution 1:1000), anti-MEK1/2 (Cell Signaling 8727, dilution 1:1000), anti-Vimentin (Cell Signaling 5741, dilution 1:1000), anti-CPSF3 (Abgent AT1610a, dilution 1:1000), anti-CES1 (Abcam ab68190, dilution 1:1000), or anti β-actin antibody (Sigma A5441, dilution 1:10,000) in 3% non-fat dry milk in TBST (TBS, 0.1% Tween-20) at 4°C overnight (see Supplementary Table 3 for list of antibodies used in this study).

Techniques: Inhibition, Expressing