Journal: iScience

Article Title: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal

doi: 10.1016/j.isci.2022.104804

Figure Lengend Snippet: UBE3D positively regulates CPSF73 but not other C/P proteins at the post-translational level (A) Western blot verification at the protein level of UBE3D KO in HEK293 cells and UBE3D addback to KO cells. (B) CPSF73 was depleted in UBE3D KO cells and its level was restored by adding back UBE3D. Other C/P proteins were not depleted. A representative Western blot performed using antibodies specific to the subunits of the C/P complex is shown on the left panel. Quantitation of the log2-fold changes in protein levels compared to wild-type (WT) using two replicates is shown on the right. (C) CPSF73 mRNA level remained unchanged in UBE3D -negative and positive cells. Three biological replicates were analyzed by RT-qPCR, and values normalized to RPL13A expression. (D) Cycloheximide (CHX) chase assay revealed that CPSF73 is less stable in UBE3D KO cells. Left panel, representative Western Blot and right panel, quantification of protein levels from three replicates, with values normalized to GAPDH. (E) The level of CPFS73 in UBE3D KO cells was increased by the proteasome inhibitor MG132 (10 μM) and the E1 ubiquitin-activating enzyme inhibitor TAK243 (1 μM), demonstrating the degradation of CPFS73 was mediated by the ubiquitin-proteasome pathway. CPSF73 protein level with each treatment was normalized to the untreated control from two replicates. For all panels, error bars show the mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (Student’s t test, unpaired, two-tailed).

Article Snippet: CPSF73 Human Tagged ORF Clone , ORIGENE , Cat#RC205834.

Techniques: Western Blot, Quantitation Assay, Quantitative RT-PCR, Expressing, Ubiquitin Proteomics, Control, Two Tailed Test

Journal: iScience

Article Title: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal

doi: 10.1016/j.isci.2022.104804

Figure Lengend Snippet: UBE3D depletion inhibits breast cancer cell motility in a CPFS73-dependent manner (A) Analysis of mRNA 3′ end processing efficiency of ACTB and MYC transcripts in MDA-MB-231 after UBE3D KD from two biological replicates, as described in Figure 2 A. (B) GO term analysis of the genes downregulated in MDA-MB-231 UBE3D KD samples was performed using Metascape. (C) Migration and invasion assessments in MDA-MB-231 cells upon UBE3D KD. Representative images of the migrated and invaded cells. Scale bar = 200 μm. (D) Quantification of migratory and invasive cells upon UBE3D KD. (E) CPSF73 overexpression in UBE3D KD cells was determined by Western blot. (F and G) CPSF73 overexpression reversed the UBE3D KD-mediated MDA-MB-231 migration and invasion defects. Scale bar = 200 μm. For all panels, error bars show the mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (Student’s t test, unpaired, two-tailed).

Article Snippet: CPSF73 Human Tagged ORF Clone , ORIGENE , Cat#RC205834.

Techniques: Migration, Over Expression, Western Blot, Two Tailed Test

Journal: iScience

Article Title: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal

doi: 10.1016/j.isci.2022.104804

Figure Lengend Snippet: Catalytic inhibition of CPSF73 leads to breast cancer cell migration and invasion defects (A) ACTB and MYC mRNA 3′ end processing in MDA-MB-231 cells treated with JTE-607, determined from three replicates. (B and C) Transwell assays to evaluate the effects of JTE-607 treatment on cell migration and invasion. Representative images of the migrated and invaded cells (B) and quantitation data (C). Scale bar = 200 μm. (D) JTE-607 reduced MDA-MB-231 cell branches in a 3D invasion assay. Right panel: quantification of branches from 30 to 50 colonies in each group. (E) JTE-607 effectively inhibited MDA-MB-231 sphere formation in Matrigel. Spheres with a diameter over 50 μm were counted and quantified from three replicates. Representative images are shown. For all panels, error bars show the mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (Student’s t test, unpaired, two-tailed).

Article Snippet: CPSF73 Human Tagged ORF Clone , ORIGENE , Cat#RC205834.

Techniques: Inhibition, Migration, Quantitation Assay, Invasion Assay, Two Tailed Test

Journal: iScience

Article Title: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal

doi: 10.1016/j.isci.2022.104804

Figure Lengend Snippet: CPSF73 expression level correlates with breast cancer cell self-renewal properties (A) CPSF73 knockdown by three independent inducible shRNAs in MDA-MB-231 cells. (B and C) CPSF73 knockdown effectively inhibited MDA-MB-231 sphere formation in Matrigel. Spheres with a diameter over 50 μm were counted and quantified from three replicates. Representative images are shown. Scale bar = 200 μm. (D) Doxycycline-inducible overexpression of CPSF73 in MDA-MB-231 cells. (E and F) CPSF73 overexpression (OE) promoted MDA-MB-231 sphere formation in Matrigel. Spheres with a diameter over 50 μm were counted and quantified from three replicates. Representative images are shown. Scale bar = 200 μm. For all panels, error bars show the mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (Student’s t test, unpaired, two-tailed).

Article Snippet: CPSF73 Human Tagged ORF Clone , ORIGENE , Cat#RC205834.

Techniques: Expressing, Knockdown, Over Expression, Two Tailed Test

Journal: iScience

Article Title: Targeting the mRNA endonuclease CPSF73 inhibits breast cancer cell migration, invasion, and self-renewal

doi: 10.1016/j.isci.2022.104804

Figure Lengend Snippet:

Article Snippet: CPSF73 Human Tagged ORF Clone , ORIGENE , Cat#RC205834.

Techniques: Recombinant, Cell Viability Assay, shRNA, Control, Plasmid Preparation, Software

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: 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