Journal: RNA Biology

Article Title: WDR33 alternative polyadenylation is dependent on stochastic poly(a) site usage and splicing efficiencies

doi: 10.1080/15476286.2024.2408708

Figure Lengend Snippet: CFIm25 variably regulates V2 and V3 PAS usage.

Article Snippet: Primary antibodies were: rabbit-anti-NUDT21 (CFIm25) (ABclonal, A4482); rabbit-anti-FIP1L1 (FIP1) (ABclonal, A7138), rabbit-anti-CstF64 [ ]; rabbit anti-Actin (Sigma-Aldrich, A2066) and rabbit anti-GAPDH (Sigma-Aldrich, G9545).

Techniques:

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) SYBR Green staining of PCR product and DNA fragments resulted from T7 Endonuclease I assay. PCR product amplified from CFIm25 gene locus is indicated by the arrow and digested DNA fragments is indicated by the bracket. Two representative replicates experiments are shown. (B) Sanger sequencing of CFIm25 gene locus to confirm the genomic mutations/deletions in three CRISPR-Cas9 system-mediated H9 cell clones. Start codon ‘ATG’ is colored green, nucleotides colored in red represent mutations, and symbol “-” stands for nucleotide deletion at the corresponding position. Genomic positions targeted by gRNAs are underlined. (C) Cell proliferation rate measurement by CCK-8 kit in mock and three CFIm25-mutant H9 cell lines. The starting cell density in this experiment is 7500 cell per well of 96 well plates. Three independent experiments have been carried out and representative results are shown. (D) Quantifications of the percentages of cells at different stages during cell cycles. The results are from three independent experiments. A representative result is shown in (m: mutant; OE: overexpression). Student’s t-test was used to estimate the significance of the change. *P<0.05; n.s.: non-significant. (E) RT-qPCR analysis of the expression level of four pluripotency-associated markers in mock and CFIm25-m hESCs. (F) Immunostaining analysis of pluripotency marker OCT4 in mock and CFIm25-m hESCs. (G) Phase-contrast images of mock and CFIm25-m hESC clones.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: SYBR Green Assay, Staining, T7EI Assay, Amplification, Sequencing, CRISPR, Clone Assay, CCK-8 Assay, Mutagenesis, Over Expression, Quantitative RT-PCR, Expressing, Immunostaining, Marker

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 knockdown/mutation impacts the cell proliferation rate and differentiation potential of H9 cell line. (A) Western blot analysis of CFIm25 and CFIm59/68 proteins in cell lysates prepared from two controls and three CFIm25 gene-edited H9 cell lines. GAPDH serves as sample loading control (Control: H9 cells; Mock control: eCRISPR empty vector-transfected H9 cells; CFIm25 m1-3: eCRISPR-CFIm25 gRNAs transfected H9 cells). The primary antibody 1 against CFIm25 is from Santa Cruz Company, and antibody 2 is from Proteintech. (B) Western blot analysis of CFIm25 and indicated protein (peptide) in cell lysates prepared from Mock, CFIm25-m, and CFIm25-m plus 3XFIag-CFIm25 overexpression cells. At least three independent experiments have been carried out and representative images are shown. The primary antibody for CFIm25 is from santa cruz (sc-81109). (C) Cell proliferation rate measurement by CCK-8 kit for indicated cell lines. The growth rate of hESCs is largely dependent on starting cell density. The starting cell density in this experiment is 5000 per well of 96 well plates. Three independent experiments have been carried out and representative results are shown. (D) Flow Cytometry analysis of cell cycle using a Propidium Iodide Flow Cytometry Kit in the indicated cell lines (m: mutant; OE:overexpression). The right panel shows the representative result of the percentages of cells during different stages of cell cycle. The quantification of three independent experiments is shown in . (E) RT-qPCR analysis of the expression level of corresponding lineage differentiation markers in indicated cell lines during trilineage differentiation. Three independent experiments have been carried out and quantified. Student’s t-test was used to estimate the significance of the change. *p<0.05. ns: non-significant. (F) Quantification of the yield of cardiomyocytes by performing fluorescence-activated cell sorting (FACS) analysis in the indicated cell lines during cardiomyocytes differentiation from three independent experiments. cTnT antibodies were used in FACS experiment. Bottom panel is the quantification from three representative experiments (m: mutant; OE: overexpression). Student’s t-test was used to estimate the significance: * p < 0.05.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Knockdown, Mutagenesis, Western Blot, Control, Plasmid Preparation, Transfection, Over Expression, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR, Expressing, Fluorescence, FACS

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: Effect of CFIm25 knockdown/mutation on the global mRNA alternative polyadenylation (APA) profile and expression level of poly(A+) transcripts in H9 cell line. (A) mRNA alternative polyadenylation (APA) change in mock and CFIm25-m(1-3) H9 cell lines. 3’-seq analysis of APA in mock and CFIm25-m H9 cells, Log2(proximal/distal ratio) are plotted for mock (y-axis) and CFIm25-m H9 cells (x-axis). Statistically significant changes are highlighted in blue (distal to proximal shift) and red (proximal to distal shift). The numbers of APA changes are shown in the column graph. (B) Volcano plot showing the expression level change of poly(A+) mRNA in mock and CFIm25-m (1-3) H9 cells. Significant changes (p<0.05, fold change>l) were colored red (up-regulated in CFIm25-m cells in comparison to mock cells) or green (down-regulated in CFIm25-m cells in comparison to mock cells), blue dots shows the changes either not statistically significant (p>0.05) or less reliable (fold change<1). Genes for subsequent studies are circled. (C) IGV track screen shots showing mRNA-seq and 3’-seq results for rex1 gene in mock and CFIm25-m H9 cells. (D) Gene ontology analysis of the group of down-regulated genes (277 genes) upon CFIm25-m using the Gene Ontology Consortium platform ( http://geneontology.org/ ). Gene ontology terms (y axis) and corresponding p-values (x axis) are shown. (E) RT-qPCR analysis of the expression level of indicated genes in mock and CFIm25-m (1-3) H9 cells. The results of three independent experiments have been quantified. Student’s t-test was used to estimate the significance of the change. *P<0.05.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Knockdown, Mutagenesis, Expressing, Comparison, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) IGV track screen shots showing the 3’-seq results for dicer1 and ccnd1 genes in mock and CFIm25-m H9 cells. Two predominant PASs within 3’UTR regions are indicated with arrows. Proximal or distal PAS are named according to their positions relative to gene 5’ end. (B) Measurement of canonical SVL PAS processing efficiency using pPASPORT system in mock and CFIm25-m cells, SVL-m (AAUAAA core hexamer was replaced as AACAAA) serves as negative control. SVL or SVL(m) PAS were inserted into multiple cloning sites between Renilla luciferase (Rlu) gene and IRES (internal ribosome entry site). Downstream of the IRES is the Firefly luciferase (Flu) gene. Relative PAS processing efficiency was quantified by calculating the Rlu/Flu ratio. Results from three independent experiments are quantified and represented. Student’s t-test was used to estimate the significance: ns: non-significant. (C) Schematic representation of the SVL RNA substrates used in the biotin–streptavidin pull-down assay (top). The AAUAAA hexamer in wild-type RNA substrate and AACAAA in mutant substrate (boxes) are shown. The asterisk is used to highlight the single nucleotide change. Bottom panel shows the western blot results of known core 3’ processing factors in the RNA-biotin based pull-down experiment using nuclear extracts (NEs) prepared from mock and CFIm25-m H9 cells. Two independent experiments have been carried out and representative results are shown. 5% of the lysate was kept as input. The primary antibody for CFIm25 is from santa cruz (sc-81109). (D) Comparison of gene expression profiling by 3’-seq and mRNA-seq. X axis: total read count for each gene in CFIm25-m sample based on mRNA-seq data; Y axis: total read count for each gene in CFIm25-m based on mRNA-seq results of control and 3’-seq analysis. The mRNA-seq read count for a gene in CFIm25-m sample=(mRNA-seq read count for this gene in control H9) x (expression fold change for this gene based on 3’-seq analyses: CFIm25-m/control). Both X axis and Y axis are in log scale. Pearson’s r=0.65. (E) IGV track screen shots showing mRNA-seq and 3’-seq results for chchd2 and znf717 genes in mock and CFIm25-m H9 cells. (F) Venn diagram showing the number of overlapping and non-overlapping genes that display expression level change upon CFIm25 (m+OE) (blue) and CFIm25-m (yellow) (m: mutant; OE: overexpression). (G) Comparison of the expression level of indicated genes in mock (m+OE) and CFIm25 (m+OE) H9 cells. Data comes from RNA-seq analysis listed in Supplemental Table 4. Student’s t-test was used to estimate the significance: *p<0.05; ns: non-significant.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Negative Control, Cloning, Luciferase, Pull Down Assay, Mutagenesis, Western Blot, Comparison, Gene Expression, Control, Expressing, Over Expression, RNA Sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 knockdown/mutation impacts rex1 gene transcription in H9 cells. (A-B) A pair of primers (F1/R1) was designed to detect the gene transcription readthrough beyond rex1 PAS. The relative expression of extended transcript in mock and CFIm25-m H9 cells was estimated by RT-qPCR analysis shown in . Gapdh gene expression serves as internal control. Student’s t-test was used to estimate the significance of the change. *p<0.05. (C) RT-qPCR analysis of the expression level of rex1 pre-mRNA and mRNA using indicated primers in mock and CFIm25-m cells. Student’s t-test was used to estimate the significance of the change. *P<0.05. (D) Outline of the 3C procedure used to detect chromatin interactions between promoter and terminator region for rex1 gene. (E) PCR product resulting from 3C library amplification using the primers located in the promoter and terminator regions. PCR product targeting gapdh gene serves as input. Cells used for 3C library preparation are indicated above the gel image. (F) Sanger sequencing shows that the PCR products correspond to the ligated DNA fragments of the two regions located at the promoter and terminator of rex1.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Knockdown, Mutagenesis, Expressing, Quantitative RT-PCR, Gene Expression, Control, Library Amplification, Sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) Mock and CFIm25-m (1-3) H9 cells were subjected to Actinomycin D treatment and total RNAs were extracted at the indicated time. RT-qPCR was used to calculate the percentage of the rex1 mRNA left, 28s rRNA serves as normalization control. (B-C) RT-qPCR analysis of rex1 pre-mRNA abundance using chromatin-associated RNAs (B) and BrU-incorporated pre-mRNAs (C) in mock and CFIm25-m H9 cells. gapdh gene product serves as internal normalization control. (D) Comparison of rex1 gene promoter activity in control and CFIm25-m H9 cells using pGL3-basic reporter system. Student’s t-test was used to estimate the significance of the change. ns: non-significant. (E) ChIP-qPCR analysis using primary antibody against CFIm25 and indicated primers targeting different position of rex1 gene locus. For the bar graph, y axis represents the fold change of ChIP signal in mock H9 cells in comparison to that of CFIm25-m cells, x axis stands for the indicated positions across rex1 gene locus. (F) RT-qPCR analysis of rex1 gene expression in undifferentiated H9 cells, and trilineage differentiated cells. Gapdh mRNA was assayed as normalization control. Student’s t-test was used to estimate the significance of the change. *P<0.05.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Quantitative RT-PCR, Control, Comparison, Activity Assay, ChIP-qPCR, Gene Expression

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 knockdown/mutation globally impacts transcription dynamics in H9 cells. (A) (left panel) Metagene plots of RNAPII ChIP-seq reads in mock and CFIm25-m H9 cells for actively transcribed genes (FPKM>1 based on mRNA-seq, 14274 genes in total), and its corresponding cumulative frequency plot (right panel). K-S test was used to examine the significance of the difference between the two plots. (B) (left panel) Pie plot showing the genomic annotations of 4024 sites that displayed differential RNAPII binding. Peak calling was performed using MACS2 software and DiffBind package was used to identify the differential binding events. (right panel) Venn diagram showing the numbers of overlapping and non-overlapping genes that displayed differential RNAPII binding and mRNA expression level change. (C) Comparison of RNAPII ChIP-seq and ChIP-qPCR results in mock and CFIm25-m H9 cells for the tested genomic sites. Y axis represents the average fold changes from replicates (ChIP-seq: two replicates; ChIP-qPCR: three replicates). (D) Metagene plots of RNAPII Ser5 ChIP-seq and RNAPII Ser2 ChIP-seq reads for actively transcribed genes in mock and CFIm25-m H9 cells. K-S test was used to examine the significance of the difference between the two plots. (E) Metagene plots of CFIm25 CUT-Tag (top), CFIm59 ChIP-seq (middle), and CFIm68 ChIP-seq (bottom) reads for actively expressed genes in mock and CFIm25-m H9 cells. CFIm25 CUT-Taq profile in CFIm25-m cells was used as normalization control. For CFIm59 and CFIm68 ChIP-seq, K-S test was used to examine the significance of the difference between the two plots. (F) Nuclear run-on assay on the nascent ccdc152 transcript. The gene structure and the primer positions are indicated on the top. The diagram for the nuclear run-on assay is shown in the middle. A representative set of RT-PCR data are shown in the bottom panel. Left gel image: nuclear run on assays followed RT-PCR using primers targeting P1-P5 region. ‘CFIm25-’ represents CFIm25-m cell nuclei, whereas ‘CFIm25 +’ represents mock cell nuclei. Right Bar graph represents RT-qPCR data from three independent experiments. U1 snRNA was assayed as normalization control. Student’s t-test was used to estimate the significance of the change. *P<0.05.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Knockdown, Mutagenesis, ChIP-sequencing, Binding Assay, Software, Expressing, Comparison, ChIP-qPCR, Control, Nuclear Run-on Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) Metagene plots of RNAPII ChIP-seq reads for highly expressed genes (top 2000 genes based on mRNA-seq FPKM value), lowly expressed gene (the rest of the genes), 277 down-regulated genes and 310 up-regulated genes upon CFIm25 gene editing. K-S test was used to examine the significance of the difference between the two plots. (B) IGV track screen shot showing RNAPII ChIP-seq result for gapdh gene in mock and CFIm25-m H9 cells. (C) Metagene plots of RNAPII ChIP-seq reads for actively expressed genes in mock and rex1 RNAi H9 cells. K-S test was used to examine the significance of the difference between the two plots. The rex1 gene knockdown efficiency was estimated by RT-qPCR and western blot analysis. Student’s t-test was used to estimate the significance of the change. *P<0.05. (D) Venn diagram showing the number of genes that displayed differential RNAPII/Ser5/Ser2 binding upon CFIm25 depletion. (E) IGV track screen shots showing RNAPII, RNAPII Ser5 and Ser2 ChIP-seq results for dctn5/ccdc152 gene in mock and CFIm25-m H9 cells. (F) Plots showing the normalized CFIm25 CUT&Tag signals in the specific group of genes (up-regulated or down-regulated genes) or individual gene (ccdcl52, dctn5). (G) Nuclear run-on assay on the nascent dctn5 transcript. The gene structure and the probe positions are indicated on the top. The diagram for the nuclear run-on assay is shown in the . A representative set of RT-PCR data are shown in the middle panel. RT-PCR products from mock and CFIm25-m H9 cells are indicated below each set. ‘CFIm25-’ represents CFIm25-m cell nuclei, whereas ‘CFIm25 +’ represents mock cell nuclei. Bar graph represents RT-qPCR data from three independent experiments. U1 snRNA was assayed as normalization control. Student’s t-test was used to estimate the significance of the change. *P<0.05. (H) Comparison of global mRNA APA (left) and gene expression profiles (middle) in control and CFIm25 RNAi cells using the previously reported dataset. The plots are similar to that in . Venn diagram shows that the two groups of genes showing changes do not overlap extensively.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: ChIP-sequencing, Knockdown, Quantitative RT-PCR, Western Blot, Binding Assay, Nuclear Run-on Assay, Reverse Transcription Polymerase Chain Reaction, Control, Comparison, Gene Expression

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 interacts with LEO1 and impacts the DNA genomic binding profile of LEO1. (A) Western blot analysis of the abundance of indicated proteins in Flag-IPed sample. Flag-IP was performed using extracts from CFIm25-m3 and CFIm25 (m3+3XFIag-CFIm25 overexpression) H9 cells. Input: 1% of the lysates for IP. The primary antibody for CFIm25 is from santa cruz (sc-81109). (B) Schematic representation of full length human LEO1 protein and truncation fragements (F1-F6) (upper). Truncation fragments were fused to pET-28a vector (BamHI and XhoI) for recombinant His-tag protein expression. At least three independent experiments have been performed, and a representative western blotting result of GST-pull down assay using anti-His antibody is shown in the middle picture. As the bait protein, recombinant GST-CFIm25 was stained with Colloidal Coomassie G-250 (Bottom). The percentage of input is indicated in the bracket. (C) Schematic representation of human CFIm25 protein and its N-terminus deletion/mutation derivatives m1-12d/m2-17d/m3-13d represent the 12/17/13 amino acids deletion/mutation proteins produced in CFIm25-mutant (m1-m3) ceils respectively) (upper). Full length and mutant CFIm25 proteins were fused to pGEX-4T3 vector (BamHI and XhoI) for recombinant GST-tag protein expression. In the GST pull-down assay, recombinant His-tag LEO1-F6 protein was used as the prey protein. At least three independent experiments have been performed, and a representative western blotting result of GST-pull down assay using anti-His antibody is shown in the middle picture. GST-fused bait proteins are stained with Colloidal Coomassie G-250 (Bottom). The percentage of input is indicated in the bracket. (D) Metagene plots of LEO1 ChIP-seq and HNRNPL ChIP-seq reads for actively expressed genes in mock and CFIm25-m cells. K-S test was used to examine the significance of the difference between the two plots.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Binding Assay, Western Blot, Over Expression, Plasmid Preparation, Recombinant, Expressing, Pull Down Assay, Staining, Mutagenesis, Produced, ChIP-sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) Western blot analysis of the abundance of LEO1 protein in Flag-IPed sample. Flag-IP was performed using extracts from CFIm25-m3 and CFIm25 (m3+3XFIag-CFIm25 overexpression) H9 cells in the presence of 5 ug/ml RNAse A. Input: 1% of the lysates for IP. (B) Commassie blue staining of purified GST-CFIm25, His-LEO1 (truncation fragments 1-6) fusion proteins (left), and three GST-CFIm25 mutants (the N terminus mutations are based on sequences listed in ). (C) Metagene plots of LEO1 ChIP-seq reads for highly expressed genes (top 2000 genes based on mRNA-seq FPKM value), lowly expressed gene (the rest of the genes), and 277 down-regulated genes upon CFIm25 gene editing. K-S test was used to examine the significance of the difference between the two plots. (D) IGV track screen shot showing LEO1 ChIP-seq result for gapdh gene in mock and CFIm25-m H9 cells.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Western Blot, Over Expression, Staining, Purification, ChIP-sequencing

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: CFIm25 targets play roles in hESCs cell proliferation and pluripotency. (A-B; D) RT-qPCR analysis of the expression level of 6 endoderm lineage differentiation markers in indicated cell lines during endoderm lineage differentiation (OE: overexpression; ASO: antisense oligo). Three independent experiments have been performed and quantified results are shown. Student’s t-test was used to estimate the significance of the change. *P<0.05; ns: non-significant. (C) Cell proliferation rate measurement by CCK-8 kit for mock and tusc1 gene overexpression H9 cell lines. Three independent experiments have been performed and quantified results are shown. (E) A schematic model summarizing the key finding in this study. Mutations in human CFIm25 protein N-terminus did not significantly affect cellular mRNA alternative polyadenylation profile, but rather affected transcription process, thereby decreasing the expression level of a group of transcripts associated with pluripotency (such as rex1 gene) and cell proliferation (such as tusc1 gene). CFIm25 depletion/mutation predominantly caused defects in the endoderm/mesoderm differentiation and accelerated the rate of cell growth in H9 cells.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Quantitative RT-PCR, Expressing, Over Expression, CCK-8 Assay, Mutagenesis

Journal: bioRxiv

Article Title: CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

doi: 10.1101/2021.12.08.471721

Figure Lengend Snippet: (A) RT-qPCR analysis of the expression level of four pluripotency-associated markers in indicated cell lines. Student’s t-test was used to estimate the significance of the change. *P<0.05; ns: non-significant. (B) Representative phase-contrast images of indicated cell lines. (C) Bar graph showing the percentages of poly(A+) RNAs among total RNAs in mock and CFIm25-m H9 cells. Poly (A+) RNAs were purified by OligodT magnetic beads from total RNAs. Quantification was performed with three independent experiments. Student’s t-test was used to estimate the significance of the change. ns: non-significant.

Article Snippet: After antibiotics selection, we picked more than 300 clones for western blot analysis (Figure 1-source data file 2), and results showed that at least three of them showed gene KO using a CFIm25 primary antibody (sc-81109, santa cruz) ( ; Figure 1-source data file 3), which recognizes the N-terminus of CFIm25 protein.

Techniques: Quantitative RT-PCR, Expressing, Purification, Magnetic Beads