Journal: Nucleic Acids Research

Article Title: PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing

doi: 10.1093/nar/gkac499

Figure Lengend Snippet: The DICER 1 3′UTR contains a function PUM Regulatory Element. ( A ) Schematic of the human DICER1 3′UTR region showing the position of the two putative PREs (TGTAAATA). ( B ) Sequence conservation of the distal PRE sequence in DICER1 -3′UTR across mammalian species and its respective distance from the polyA site. The putative PRE site is underlined in the sequence alignment and highly conserved nucleotides are marked by a * symbol. ( C ) PUM1 in vitro RNA binding assays of the DICER1 WT sequences. Ex: Extract Input alone, Sn:Supernatant flow through alone, DICER1 RNA plus: -: TAP-Tagged PUM1 expressing extract, R1: – + PRE containing non-biotinylated oligo, R2: – + non-PRE containing non-biotinylated oligo, MRAS RNA (non-PUM substrate) and VEGFA (PUM substrate). ( D) Relative E2F4 (negative control), DICER1, DROSHA and DCGR8 RNA bound to IgG, PUM1 and PUM2 from RIP-qPCR analysis from MDA-MB-231 cells. n = 3 biological replicates. Error bars represent standard deviations from these replicates. ** P < 0.01. ( E ) Representative confocal microscopy images of Cy3-labelled WT RNA (red), PUM1 (green) and DAPI (blue) from RPE1 cells. Inset image scale bar = 2 μm. ( F ) Relative Luciferase levels from MDA-MB-231 cells transfected with a Luciferase reporter gene followed by a WT DICER1 3′UTR region (WT), PRE1 (A mut), PRE2 (B mut) or PRE1 and 2 (AB mut) disrupting mutations. ( G ) Relative Luciferase levels from MDA-MB-231 cells infected with shRNAs targeting a Scrambled control sequence (sh-SCR), PUM1 (sh-PUM1) or PUM2 (sh-PUM2) and transfected with a Luciferase gene followed by a WT DICER1 3′UTR region (WT), or PRE1 and 2 (AB mut) disrupting mutations. ( H ) Relative Luciferase levels from MDA-MB-231 cells transfected with a control pcDNA plasmid, HA-PUM1 or HA-PUM2 and transfected a Luciferase gene followed by a WT DICER1 3′UTR region (WT), or PRE1 and 2 (AB mut) disrupting mutations. n = 3 biological replicates. Error bars represent standard deviations from these replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: PUM1, PUM2 and IgG immunoprecipitations were performed O/N using PUM1 (Bethyl) and PUM2 (Abcam) antibodies with IgG antibody (CST) as negative control with cells lysed in Polysome Lysis Buffer (10 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl 2 , 25 mM EDTA (pH 8), and 0.5% NP40.

Techniques: Sequencing, In Vitro, RNA Binding Assay, Expressing, Negative Control, Confocal Microscopy, Luciferase, Transfection, Infection, Control, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing

doi: 10.1093/nar/gkac499

Figure Lengend Snippet: DICER1 and PUM levels correlate. ( A ) Western blots of DICER1, HA and ACT from MDA-MB-231 cells transfected with a control pcDNA plasmid or increasing concentrations of HA-PUM1. ( B ) Western blots of DICER1, PUM1, PUM2 and ACT from CRISPR-Cas9 edited WT , PUM1 –/– , PUM2 –/– and PUM1/2 –/– HCT116 cells. ( C ) Relative pre-miRNA levels of pre-miR-301b, pre-miR-128 and pre-miR-21 (non-DICER1 substrate) from MDA-MB-231 cells infected with shRNAs targeting a scrambled control sequence (sh-SCR), PUM1 (sh-PUM1) or PUM2 (sh-PUM2). ( D ) Relative mature miRNA levels of miR-10a, miR-454 and miR-129 from MDA-MB231 cells transfected with pCDNA (control) and HA-PUM1 plasmids. ( E ) Relative DICER1 RNA levels from MDA-MB-231 cells infected with shRNAs targeting a scrambled control sequence (sh-SCR), PUM1 (sh-PUM1) or PUM2 (sh-PUM2). ( F ) Relative DICER1 RNA levels from MDA-MB231 cells transfected with pCDNA (control), HA-PUM1 and HA-PUM2 plasmids. ( G ) Heat map of R values from linear regression analysis between DICER1 and PUM1, PUM2 and TUB (control) RNA levels in 30 TCGA tumor datasets. ( H ) Linear regression between DICER1 and PUM1 (blue), PUM2 (green) and TUB (black) (control) RNA levels across 30 TCGA tumor datasets. List of abbreviation along with R values and p values are found in . ( I ) Scatter plots, linear regression and correlation analysis of DICER1 and PUM1 levels from each tumor in the pan-cancer TCGA dataset. ( J ) Scatter plots, linear regression and correlation analysis of DICER1 and PUM2 levels from each tumor in the pan-cancer TCGA dataset. ( K ) Scatter plots, linear regression and correlation analysis of DICER1 and TUB levels from each tumor in the pan-cancer TCGA dataset. n = 3 biological replicates. Error bars represent standard deviations from these replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: PUM1, PUM2 and IgG immunoprecipitations were performed O/N using PUM1 (Bethyl) and PUM2 (Abcam) antibodies with IgG antibody (CST) as negative control with cells lysed in Polysome Lysis Buffer (10 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl 2 , 25 mM EDTA (pH 8), and 0.5% NP40.

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, CRISPR, Infection, Sequencing

Journal: Nucleic Acids Research

Article Title: PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing

doi: 10.1093/nar/gkac499

Figure Lengend Snippet: Disruption of the DICER1 distal PRE, alters DICER1 protein levels and miRNA production. ( A ) The PRE sequence and rs1252940486 (A > G) patient mutation. ( B ) RNA pull-down assays of the DICER1 3′UTR PRE2 region comparing WT and the A > G sequence. ( C ) Representative confocal microscopy images of Cy3-labelled DICER1 WT (DICER1-WT) or A > G (DICER1-A > G) RNA (red), PUM1 and PUM2 (green) and DAPI (blue) from RPE1 cells. ( D ) Relative Luciferase levels from WT, PUM1 –/– and PUM2 –/– HCT116 cells transfected with a Luciferase reporter gene followed by a WT DICER1 3′UTR region (WT) or A > G sequence. n = 3 biological replicates. Error bars represent standard deviations from these replicates. ** P < 0.01. ( E ) Schematic of CRISPR-Cas9 mediated alteration of PRE2 within the DICER1 3′UTR. ( F ) Western blots of DICER1, PUM1, PUM2 and ACT from WT, and C1-C3 MDA-MB-231 cells. ( G ) Relative mRNA levels of DICER1 from WT, and C1-C3 MDA-MB-231 cells. ( H ) Volcano plots of log 2 fold mature miRNA changes in C1 CRISPR-Cas9 edited MDA-MB-231 cells relative to WT. Blue dots highlight significantly altered miRNAs. ( I ) Volcano plots of log 2 fold mature miRNA changes in C2 CRISPR-Cas9 edited MDA-MB-231 cells relative to WT. Green dots highlight significantly altered miRNAs. ( J ) Volcano plots of log 2 fold mature miRNA changes in C3 CRISPR-Cas9 edited MDA-MB-231 cells relative to WT. Orange dots highlight significantly altered miRNAs.

Article Snippet: PUM1, PUM2 and IgG immunoprecipitations were performed O/N using PUM1 (Bethyl) and PUM2 (Abcam) antibodies with IgG antibody (CST) as negative control with cells lysed in Polysome Lysis Buffer (10 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl 2 , 25 mM EDTA (pH 8), and 0.5% NP40.

Techniques: Disruption, Sequencing, Mutagenesis, Confocal Microscopy, Luciferase, Transfection, CRISPR, Western Blot

Journal: Nucleic Acids Research

Article Title: PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing

doi: 10.1093/nar/gkac499

Figure Lengend Snippet: The DICER1 mRNA is destabilized by AUF1 in the absence of PUM-mediated regulation. ( A ) Volcano plot of log 2 (Fold Change) of endogenous RBPs from in vitro RNA binding-LC/MS analysis of proteins bound to the DICER1-3′UTR A > G RNA sequence relative DICER1-3′UTR WT RNA sequence. Significantly upregulated genes (log 2 FC > 1, P < 0.05) are shown in green and significantly downregulated genes (log 2 FC < 1, P < 0.05) are shown in red. ( B ) DICER1 mRNA levels in WT, C1 and C2 MDA-MB-231 cells depleted of AUF1 or SCR (control) by shRNA. ( C ) PUM1 and AUF1 in vitro RNA binding assays of the DICER1 WT and pMUT sequences. ( D ) Relative binding of the DICER1 RNA from RIP-qPCR analysis of IgG (non-specific antibody) and AUF1 in MDA-MB-231 cells. ( E ) Relative DICER1 RNA half-life in MDA-MB-231 cells depleted of SCR, PUM1, PUM2 or over-expressing AUF1. ( F ) RNA half-life of DICER1 mRNA in WT, C1 and C2 MDA-MB-231 cells. ( G ) Relative binding of the DICER1 RNA from RIP-qPCR analysis of IgG (non-specific antibody) and AUF1 in WT, C1 and C2 MDA-MB-231 cells. ( H ) RNA half-life of DICER1 mRNA in WT, C1 and C2 MDA-MB-231 cells depleted with shRNAs targeting control, SCR, or AUF1. ( I ) EMSA from WT DICER1 pre-coated with AUF1 followed by increasing amounts of PUM1. ( J ) EMSA from A > G DICER1 pre-coated with AUF1 followed by increasing amounts of PUM1. ( K ) EMSA from WT DICER1 pre-coated with PUM1 followed by increasing amounts of AUF1. ( L) EMSA from A > G DICER1 pre-coated with PUM1 followed by increasing amounts of AUF1. n = 3, error bar represents standard deviation. * P < 0.05, ** P < 0.01.

Article Snippet: PUM1, PUM2 and IgG immunoprecipitations were performed O/N using PUM1 (Bethyl) and PUM2 (Abcam) antibodies with IgG antibody (CST) as negative control with cells lysed in Polysome Lysis Buffer (10 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl 2 , 25 mM EDTA (pH 8), and 0.5% NP40.

Techniques: In Vitro, RNA Binding Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Control, shRNA, Binding Assay, Expressing, Standard Deviation

Journal: Nucleic Acids Research

Article Title: PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing

doi: 10.1093/nar/gkac499

Figure Lengend Snippet:

Article Snippet: PUM1, PUM2 and IgG immunoprecipitations were performed O/N using PUM1 (Bethyl) and PUM2 (Abcam) antibodies with IgG antibody (CST) as negative control with cells lysed in Polysome Lysis Buffer (10 mM HEPES (pH 7.0), 100 mM KCl, 5 mM MgCl 2 , 25 mM EDTA (pH 8), and 0.5% NP40.

Techniques:

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 1: SPIN paralogues differentially influence TCam-2 cell apoptosis. SPIN1 and SPIN3 were overexpressed or silenced in TCam-2 cells and apoptosis was assessed using flow cytometry. Representative western blot showing SPIN overexpression compared to VINCULIN (A). Apoptosis was analyzed in TCam-2 cells overexpressing SPINs (B) and in cells in which SPINs were silenced (C) CYCD1 expression was measured via real-time qPCR in cells overexpressing SPIN1 and SPIN3 (D). Cells transfected with an empty vector (overexpression) or control siRNA (knockdown) were the baselines in (B) and (C). *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

Article Snippet: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

Techniques: Flow Cytometry, Western Blot, Over Expression, Expressing, Transfection, Plasmid Preparation, Control, Knockdown

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 2: SPIN1 and SPIN3 promote TCam-2 cell cycle progression. TCam-2 cell cycle analysis was performed following siRNA-mediated SPIN1 or SPIN3 knockdown (A) Cells transfected with control siRNA represented the baseline. *P ≤ 0.05. TCam-2 cell cycle analysis was performed following SPIN1 or SPIN3 overexpression (B) Values higher than the baseline indicate an increase in a given cell population, while values below the baseline indicate a decrease. The p16 and p21 CDK inhibitors were used as positive controls (C).

Article Snippet: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

Techniques: Cell Cycle Assay, Knockdown, Transfection, Control, Over Expression

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 3: PUM1 and PUM2 proteins bind and regulate SPIN1 and SPIN3 mRNAs. Schematic of full-length human SPIN1 and SPIN3 3ʹUTRs (A). PBE-like motifs responsible for PUF-domain binding are in red and UGUA core motifs are in black (SPIN3). Short 3ʹUTR fragments containing PBE motifs, which were used for luciferase reporter assays, and full-length 3ʹUTRs are indicated in brackets, with position within the 3ʹUTR starting from the end of the stop codon. Enrichment of SPIN1 and SPIN3 in RIP-PUM1 and RIP-PUM2 (indicated by RIP P1 and RIP P2, respectively) was measured via RT-qPCR and was compared to the negative control (nonimmune IgG, indicated by RIP IgG) (B). Influence of PUM1 and PUM2 proteins on endogenous SPIN mRNA level (C). PUM1 and PUM2 siRNA knockdown efficiencies are shown on the left. Total RNA was isolated from TCam-2 cells in the presence of actinomycin D. SPIN expression was measured via RT-qPCR and was compared to that in untransfected cells. PUM1 or PUM2 overexpression downregulated endogenous SPIN1 as measured by western blotting (D). Graphs represent average values with standard errors. P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

Article Snippet: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Negative Control, Knockdown, Isolation, Expressing, Over Expression, Western Blot

Journal: Oncotarget

Article Title: SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

doi: 10.18632/oncotarget.25977

Figure Lengend Snippet: Figure 4: Influence of PUM1 and PUM2 proteins on luciferase reporter constructs carrying SPIN1 or SPIN3 3ʹUTRs. The effects of PUM proteins on SPIN expression were assessed using a dual luciferase assay. Luciferase reporter constructs carrying full-length 3ʹUTRs for SPIN1 (upper panel) or SPIN3 (lower panel) were tested with PUM1 (P1) or PUM2 (P2) overexpression or empty pCMV6-entry vector (pC) (A). Effects of PUM overexpression on luciferase reporter construct carrying full-length GAPDH mRNA 3ʹUTR, which lacks PBE motifs (negative control) (B). Effects of siRNA-mediated PUM1 (P1 KD) or PUM2 (P2 KD) knockdown (KD) on luciferase reporter constructs carrying full-length SPIN 3ʹUTRs (C). Effects of PUM1 or PUM2 overexpression (D). or knockdown (E). on luciferase constructs containing short SPIN1 or SPIN3 3ʹUTR fragments. **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.00005.

Article Snippet: Cells were washed with 1× PBS (Lonza), and then visualized using a Leica DMi8 IVD microscope under UV (for Hoechst 33258 detection) and visible light. siRNA knockdown For transient knockdowns, TCam-2 cells were transfected with one of the following specific siRNAs at 10 nM final concentration: SPIN1 (sc-92696), SPIN3 (sc91032), PUM1 (sc-62912), PUM2 (sc-44773) or control (sc-37007) (Santa Cruz Biotechnology, USA).

Techniques: Luciferase, Construct, Expressing, Over Expression, Plasmid Preparation, Negative Control, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model

doi: 10.3390/ijms23126592

Figure Lengend Snippet: FOXM1 and TAF1 act as transcription factors for NANOS1 and NANOS3 downregulated genes. ( A ) Cumulative distribution analysis of differentially expressed genes upon NANOS3 RNA-Seq. Common differentially expressed mRNA distribution shown in blue and labeled as targets. The rest of the differentially expressed genes are shown in red and labeled as non-targets. ( B ) Common differentially expressed genes that are repressed by both NANOS3 and PUM1 identified as downregulated (log2FC < 0) upon NANOS3 overexpression and upregulated (log2FC > 0) upon PUM1 knockdown. ( C ) iRegulon transcription factor prediction analysis for NANOS3/PUM1 common repressed genes. ( D ) Cell cycle related biological processes identified by gene ontology enrichment analysis for NANOS3-PUM1 common repressed genes.

Article Snippet: Transient overexpression was performed by transfecting cells with pCMV6-entry vector (OriGene) containing NANOS1 (#RC219123), NANOS3 (#RC221867), and PUM1 (#RC201219) ORF tagged with FLAG (DYKDDDDK) peptide in the C-terminus.

Techniques: RNA Sequencing Assay, Labeling, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model

doi: 10.3390/ijms23126592

Figure Lengend Snippet: FOXM1 mRNA is regulated by the NANOS3/PUM1 post-transcriptional complex through its’ 3′UTR. ( A ) Western-blot visualization of GST pull-down of NANOS3 with and without RNase A. ( B ) Schematic representation of FOXM1 3′UTR containing the Pumilio binding element (PBE-UGUAHAUA) and 2 PBE-like motifs (containing core UGUA motif). ( C ) Dual luciferase assay to measure Renilla/Firefly ratio upon PUM1 knockdown and NANOS3 overexpression using 3′UTR of FOXM1 in the downstream of Renilla ORF. Standard error is visualized as error bars. * = p -value ≤ 0.05, *** = p -value ≤ 0.001.

Article Snippet: Transient overexpression was performed by transfecting cells with pCMV6-entry vector (OriGene) containing NANOS1 (#RC219123), NANOS3 (#RC221867), and PUM1 (#RC201219) ORF tagged with FLAG (DYKDDDDK) peptide in the C-terminus.

Techniques: Western Blot, Binding Assay, Luciferase, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model

doi: 10.3390/ijms23126592

Figure Lengend Snippet: The NANOS3–PUM1–FOXM1 axis coordinates the expression of the cell cycle genes. ( A ) Dual luciferase assay to measure Renilla/Firefly ratio upon PUM1 knockdown and NANOS3 overexpression using 3′UTR of CCNA2 , KIF20B , and RAD21 in the downstream of Renilla ORF. ( B ) Relative fold change of FOXM1 binding events per 1000 cells for the promoter region of the target genes measured by ChIP-qPCR. ( C ) Differential correlation analysis of NANOS3 , PUM1 , and FOXM1 in publicly available healthy testis (GTEx) and testicular cancer datasets (TCGA). Each dot represents a single GTEx or TCGA dataset. Expression values are transformed as log(expression +1). Standard error is visualized as error bars. ** = p -value ≤ 0.01, *** = p -value ≤ 0.001.

Article Snippet: Transient overexpression was performed by transfecting cells with pCMV6-entry vector (OriGene) containing NANOS1 (#RC219123), NANOS3 (#RC221867), and PUM1 (#RC201219) ORF tagged with FLAG (DYKDDDDK) peptide in the C-terminus.

Techniques: Expressing, Luciferase, Over Expression, Binding Assay, Transformation Assay

Journal: International Journal of Molecular Sciences

Article Title: Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model

doi: 10.3390/ijms23126592

Figure Lengend Snippet: Model of NANOS3-PUM1-FOXM1 axis regulating G2/M phase of the cell cycle in TCam-2 cell line. Created with BioRender.com.

Article Snippet: Transient overexpression was performed by transfecting cells with pCMV6-entry vector (OriGene) containing NANOS1 (#RC219123), NANOS3 (#RC221867), and PUM1 (#RC201219) ORF tagged with FLAG (DYKDDDDK) peptide in the C-terminus.

Techniques: