Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Schematic representation of the yeast two-hybrid screen performed using Gal4-DBD-Sam68 as bait and a Gal4-AD fusion cDNA library from LNCaP cells, b, Table reporting the Sam68-interacting factors identified by the screen, c, Five clones of the AH109 yeast strain transformed with the plasmid expressing Gal4-AD-XRN2 (1,929–2,842 nt) (clone 177) and Gal4-DBD-Sam68 fusion proteins, or both plasmids co-transformed with empty vectors as controls. Clones were plated in non-stringency (SD without Leu and Trp) and high-stringency (SD without Leu, Trp, His and Ade) medium and grown at 28 °C for four days, d, Scheme of the XRN2 structure with the position of the Sam68-interacting region (red box), e. Representative western-blot analysis of the reciprocal co-immunoprecipitation (co-IP) between endogenous Sam68 and XRN2 from LNCaP nuclear extracts using Sam68 (α-Sam68) or XRN2 (α-XRN2) antibodies (n = 3). Input = 0.25%. f, Representative western-blot analysis of the co-IP of endogenous Sam68 with XRN2, performed using LNCaP nuclear extracts (NE) in the presence (+) or absence (−) of RNaseA (n = 3). A representative agarose gel of RNA degradation is also shown (RNA). In e and f, non-immune rabbit immunoglobulins G (α-IgG) were used as a negative control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Transformation Assay, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Agarose Gel Electrophoresis, Negative Control

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Pearson’s correlation analyses of XRN2 and MYC expression in the PC Jenkins dataset (GSE46691). Pearson’s correlation coefficient (r; two-sided) and P value are reported (95% confidence interval), b, Dot plot showing the distribution of XRN2 expression in patients with PC (Jenkins dataset, GSE46691), classified into Sam68low (blue circles) and Sam68high (red squares) expression groups according to Z-score normalization. The median is shown as a solid horizontal line, c, Representative images of immunohistochemistry analyses of patients with PC (n = 20) with low and high expression of XRN2 and Sam68. Spearman’s correlation is reported (ρ = 0.653; P = 0.002). d, Violin plot showing the correlation between Sam68 and XRN2 expression with Gleason score, in patients with PC (Jenkins dataset, GSE46691). In b and d, statistical significance was calculated by the Mann-Whitney test (two-sided), and P values are reported (95% confidence interval).

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Bar graph showing the percentage of 3’UTR- and CDS-APA events annotated in the genes expressed in LNCaP cells (white columns) and the percentage of those that are differentially regulated in Sam68- and XRN2-depleted cells (gray columns). Statistical significance wascalculated by modified Fisher’s exact test (two-sided, 95% confidence interval), and the exact P values are reported. b,c, Representative western-blot (b) and densitometric analyses (c) of subcellular fractionation experiments (n = 3) performed in control (sh-scr), Sam68 (sh-Sam68) and XRN2 (sh-XRN2) stably depleted LNCaP cells. CE, total cell extract; Cyt, cytoplasmic fraction; Nuc, nucleoplasmic fraction; Chr, chromatin fraction. d,e, Western blot (d) and bar graphs showing qPCR analysis (e) of pA usage of the SCARB2 gene evaluated in cells knocked down for XRN2 targeting 3’UTR (sh-XRN2-3’UIR) and transfected with empty vector (EV), wild-type (WT) and catalytically inactive (D235A) XRN2 (n = 3). LNCaP cells stably depleted with a shRNA targeting CDS (sh-XRN2) were used as control. Fold change of distal (d-pA) relative to the proximal pA (p-pA) in the 3’UTR was calculated by the ACq method. The representative western blot (d) shows the expression of endogenous (XRN2) and recombinant (FLAG) proteins; β-actin was used as loading control. f,g, CLIP assays performed in LNCaP cells stably depleted for XRN2 (sh-XRN2) (n = 3) (f) or transfected as in d (n = 3) (g) using the Sam68 antibody or control IgCs. The RNA associated with Sam68 was quantified by qPCR using primers located upstream of regulated and non-regulated pAs and is represented as percentage (%) of input. Inc and e-g, statistical significance was calculated by unpaired Student’s t-test (two-sided). In c, sh-XRN2/Cyt P = 0.324, sh-XRN2/Nuc P = 0.058, sh-XRN2/Chr P = 0.035, sh-Sam68/Cyt P = 0.8119, sh-Sam68/Nuc P = 0.7612, sh-Sam68/Chr p = 0.6481. In e, sh-XRN2/EV p = 3.4 ×10−3, sh-XRN2-UTR/EVP = 2.1 × 10−3, sh-XRN2-UTR/XRN2WT P = 0.4198, sh-XRN2-UTR/XRN2D235A P = 0.2456. In f, Sam68(sh-scr-downreg/sh-scr-upreg) p = 4.34 ×10−5, Sam68downreg(sh-scr/sh-XRN2) P = 1.7 × 10−3, Sam68upreg(sh-scr/sh-XRN2) P = 3 × 10−4. In g, downregulated: Sam68(sh-scr+EV/sh-XRN2-3’UTR + EV) P = 2 × 10−3, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2WT) P = 0.0215, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2D235A) P = 0.1502, Sam68(sh-XRN2-3’UTR + XRN2WT/sh-XRN2-3’UTR + EV) P = 0.0252, Sam68(sh-XRN2-3’UTR + XRN2D235A/sh-XRN2-3’UTR + EV) P = 0.0157; upregulated: Sam68(sh-scr + EV/sh-XRN2-3’UTR + EV) P = 7.3 × 10−5, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2WT) P = 0.036, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2D235A) P = 0.031, Sam68(sh-XRN2-3’UTR + XRN2WT/sh-XRN2-3’UTR + E V) p = 3.3 × 10−3, Sam68(sh-XRN2-3’UTR + XRN2D235A/sh-XRN2-3’UTR + EV) P = 0.0141. In c and e-g, the bars represent mean + s.d. of three biological replicates; statistical value is reported as *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Modification, Western Blot, Fractionation, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Expressing, Recombinant

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Pearson’s correlation analysis of XRN2 and MYC expression in the jenkins dataset (GSE46691). Pearson’s correlation coefficient (r; two-sided) and P values are reported (95% confidence interval), b, Distribution of XRN2 expression in patients with PC classified as MYCflow (blue circles) and MYChigh (red squares) groups according to Z-score normalization of expression data retrieved from the jenkins dataset (GSE46691). Statistical significance was calculated by Mann-Whitney test (two-sided), and the P value is reported, c, Representative semiquantitative (sq) PCR analysis of ChIP experiments (n = 3) performed in LNCaP cells using MYC antibody and IgG, or no antibody (−), as negative controls. MYC binding was evaluated on the XRN2 promoter. Binding to the sam68 promoter and 16q22 intergenic region were used as positive and negative control, respectively. A schematic representation of the indicated promoters and 16q22 intergenic region is also shown. MYC binding sites (boxes), and positions of primers used for PCR analyses (arrows) are reported. d,e, qPCR (d) and western-blot (e) analyses of MYC, XRN2 and Sam68 expression in LNCaP and 22Rv1 cells lines transfected with control (si-scr#l) and MYC (si-MYC#1) siRNAs (n = 3). Expression was reported as fold change (ΔΔCq) with respect to control. Data represent mean + s.d. of three biological replicates, and statistical significance was calculated by unpaired Student’s t-test (two-sided) (MYC/LNCaP P = 3.8 × 10−5, MYC/22Rv1 P = 5.1 × 10−6; XRN2/LNCaP P = 3.7 × 10−3, XRN2/22Rv1 P = 1.4 × 10−3; Sam68/LNCaP P = 8.4 × 10−5, Sam68/22Rv1P = 7.7 × 10−5). In d, statistical value is reported as **P < 0.01, ***P < 0.001. In e, β-actin was used as loading control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, MANN-WHITNEY, Binding Assay, Negative Control, Western Blot, Transfection

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Meta-transcriptome profiles of Sam68 binding across mRNA transcripts retrieved from two replicates of CLIP-seq experiments (GSE85164). TSS, transcription start site; TES, transcription end site; RPM, reads per million, b, Representative western-blot analysesofthe co-IP ofSam68 and XRN2 with componentsoftheC/P complex from LNCaP nuclear extracts using Sam68 (α-Sam68) and XRN2 (α-XRN2) antibodies, or rabbit immunoglobulins G (α-IgG) as negative control (n = 2). c, Bar graphs representing the percentage of genes (left) and polyadenylation sites (pAs; right graph) undergoing APA regulation in Sam68 (si-Sam68)- and XRN2 (si-XRN2)-depleted LNCaP cells, d, Venn diagram showing the overlap between regulated APA events identified in Sam68- or XRN2-depleted cells. Statistical significance was calculated by hypergeometric test and the P value is shown. e, Venn diagram showing the number of unique and common up- (purple) and downregulated (orange) APA events identified in Sam68- and XRN2-depleted cells. f,g, Bar graphs showing qPCR analysis of pA usage evaluated in two representative genes undergoing 3’UTR-APA (f) and CDS-APA (g) regulation in cells knocked down for Sam68 (si-Sam68), XRN2 (si-XRN2) or both proteins. Fold change of distal (d-pA) (f) or intronic (g) pA relative to the proximal pA (p-pA) in the 3’UTR was calculated by the ΔCq method. Data represent mean + s.d. of three biological replicates. Statistical significance was calculated by unpaired Student’s t-test (two-sided). In f, SCARB2: si-Sam68/si-scr P = 1.5 × 10−3, si-XRN2/si-scr P = 2.0 × 10−3, si-Sam68si-XRN2/si-scr P = 0.017; FLNB: si-Sam68/si-scr P = 0.015, si-XRN2/si-scr P = 2.1 × 10−3, si-Sam68si-XRN2/si-scr P = 3 × 10−4. In g, RNF130: si-Sam68/si-scr P = 0.013, si-XRN2/si-scr P = 5.5 × 10−3, si-Sam68si-XRN2/si-scr P = 5.4 × 10−3; CEP70: si-Sam68/si-scr P = 4.3 × 10−3, si-XRN2/si-scr P = 0.0112, si-Sam68si-XRN2/si-scr P = 0.0147. In f and g, statistical values are reported as *P < 0.05; **P < 0.01; ***P < 0.001. UCSC genome browser tracks showing APA regulation of the events analyzed are also shown on the left side of each graph. Purple and orange boxes in the schemes indicate up- and downregulated events, respectively. Schematic representations of these CDS- and 3’UTR-APA events are shown in the upper panels.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: Genome-wide regulation of APA by XRN2 and Sam68 in PC cells (Related to Fig. 4).

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Genome Wide

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Percentage and number of up- (purple) and downregulated (orange) 3’UTR-APA events regulated by Sam68 and XRN2 (pA position is shown as F, proximal-most; M, intermediate; L, distal-most), b, Changes of 3’UTR pA isoform abundance (ΔAbn) at both p-pA and d-pA sites in si-Sam68 and si-XRN2 cells. Mean values and number of pA events (n) are reported, c, Percentage of up- and downregulated canonical and non-canonical PAS sequences in 3’UTR-APA events regulated by Sam68 and XRN2. d, AAUAAA frequency profile in up- (purple), down- (orange) and unregulated (black) 3’UTR pAs evaluated between −100 and +100 nt from the CS (shading represents 95% confidence interval). Statistical significance (unpaired Student’s t-test, two-sided) was calculated between −15 and −25 nt (boxplot). e, A- and G-base frequency distribution in up- (purple), down-grange) and unregulated (black) pAs between −100 and +100 nt from the CS (0). f, Scheme of cis-elements and CS position. Hexamers enriched between −100 and +100 nt from the CS in up- and downregulated pAs with respect to unregulated pAs. Motif (H), number (N) and significance score (P) of hexamers are indicated. Significance score was calculated by –log10(P)xS, where P is based on the Fisher’s exact test and the S value was 1 or −1 for enrichment and depletion, respectively, g, APA isoform abundance (Abn) of si-Sam68/si-XRN2 up- (mean = 28.6) and downregulated (mean = 47.2) isoforms. Values refer to expression in control cells, h, Scheme of the FLNB minigene comprising the genomic region from the second-last exon to 200 nt downstream of the d-pA (source data). i,j, Semiquantitative (micrographs) and quantitative (bar graphs) analyses of pA usage in LNCaP transfected with the FLNB minigene and indicated plasmids (n = 3). Protein expression was evaluated by western blot, k, CLIP assays performed in sh-Sam68 and sh-XRN2 cells using CPSF30 antibody or IgGs (n = 3). Statistical significance was calculated by unpaired Student’s t-test, two-sided (b, g, i-k) and with Fisher’s exact test, two-sided (a, c). (l-k) Bar graphs represent mean + s.d. When not indicated, P values are reported as *P < 0.05, ***P < 0.001, ****P < 0.0001 (exact P values are reported in the source data). In the boxplots (b, d, g), the center line and box indicate the median and the 25th and 75th percentiles, respectively. Whiskers indicate ±1.5x interquartile range.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, Transfection, Western Blot

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Enrichment of Gene Ontology (GO) terms (dot plot) in genes regulated by 3’UTR-APA upon depletion of Sam68 or XRN2. Dot size and color indicate the number of genes and statistical significance (Fisher’s exact test, two-sided), respectively, b, Cytometric analyses showing DNA content versus BrdU incorporation upon stable depletion of Sam68 (sh-Sam68) and XRN2 (sh-XRN2) in LNCaP cells. The bar graph shows the percentage of BrdU-positive (S phase) cells, c. Percentage (mean + s.d.) of BrdU-positive LNCaP cells described in b at the indicated time points after release from G1/S synchronization. d,e, Western blot (d) and qPCR (e) analyses of MCM10 and ORC2 expression level in sh-Sam68 and sh-XRN2 LNCaP cells (n = 3). f, PCR strategy used to evaluate 3’UTR-APA isoforms distribution on a 15–50% sucrose gradient, g, sqPCR analysis of the indicated p-pA and d-pA isoform abundance within the polysomal and non-polysomal fractions obtained from sucrose gradient. The graphs show the densitometric analysis of the band signal in each fraction, expressed as a percentage of that detected in all fractions, h, Relative luciferase activity (Renilla/Firefly ratio) of long and short MCM10 3’UTR in LNCaP cells. i, Representative western-blot analysis (n = 3) of the indicated proteins performed in LNCaP cells depleted for the indicated genes, j, Cytometric analyses showing DNA content versus BrdU incorporation in control (si-scr), si-MCMlO and si-ORC2 LNCaP cells. The bar graph shows the percentage of S-phase BrdU-positive cells, k, Kaplan-Meier curves comparing progression-free survival of494 patients with PC (Prostate Adenocarcinoma, TCGA, PanCancer Atlas; https://www.cbioportal.org) stratified according to MCM10 (right), ORC2 (middle) and MCM10/ORC2 (left) expression level. I, Schematic model showing the impact of the functional interaction between Sam68 and XRN2 on cell cycle regulation. The Sam68/XRN2 complex promotes 3’UTR shortening of cell cycle-related genes, increasing their mRNA translation efficiency and cell proliferation. Conversely, Sam68/XRN2 knockdown induces 3’UTR lengthening, reduces translation efficiency of transcripts and causes cell cycle arrest. In b, e, h and j, the bar graphs represent the mean + s.d. In b, c, e, g, h and j, statistical significance was calculated by unpaired Student’sf-test, two-sided (n = 3; *P < 0.05, **P < 0.01,***P < 0.001; NS, not significant; exactPvalues are reported in the source data). In d and I, β-actin was used as loading control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: BrdU Incorporation Assay, Western Blot, Expressing, Luciferase, Activity Assay, Functional Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenvlation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Genes & Development

Article Title: SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA

doi: 10.1101/gad.309138.117

Figure Lengend Snippet: SAM68 binds NORAD repeats. ( A ) Secondary structure of NRU#7 predicted by RNAfold with the elements marked. ( B ) MS intensities for the indicated proteins and the indicated sequence variants (all from the same experiment). ( C ) RIP of the indicated proteins quantified using qRT–PCR for the indicated genes ( left ; n ≥ 3, normalized to the input and the GAPDH mRNA; average ± SEM is shown) and visualized by Western blot ( right ). Negative control (NC) in Western blot is a lysis buffer to control for bands originating from the antibody. Twenty micrograms was loaded as input for the Western blot. ( D ) The distribution of SAM68-binding sites in the different NRUs and eCLIP (enhanced cross-linking immunoprecipitation) data of SAM68 and PUM2 from K562 cells in NRU#7 and NRU#9. The color shadings highlight the different conserved elements. (Light green) PREs; (yellow) short hairpin; (green) U-rich region; (blue) long hairpin. ( E ) PhyloP conservation scores (based on the 100-way vertebrate alignment of the hg19 human genome in the University of California at Santa Cruz [UCSC] genome browser) for all of the bases in NORAD and just the bases overlapping the TAAA or TGTRTATA motifs. ( F ) Pull-down of four indicated fragments from NRU#8 and NRU#9 followed by Western blot for SAM68. The sequences of the fragments are annotated in Supplemental Figure 1B . ( G ) Pull-down of SAM68 using in vitro transcribed RNA from the indicated variants of NRU#7 (sequences in Supplemental Table 1 ).

Article Snippet: The beads were prewashed and coated with antibodies against GAPDH (diluted 1:1000; Santa Cruz Biotechnology, SC-32233), PUM2 and SAM68 (each diluted 1:1000; Bethyl Laboratories, A300-201A and A300-202A, respectively), and RUVBL1 (diluted 1:1000; Proteintech, 10210-2-AP) overnight at 4°C with gentle rotation.

Techniques: Sequencing, Quantitative RT-PCR, Western Blot, Negative Control, Lysis, Control, Binding Assay, Cross-linking Immunoprecipitation, In Vitro

Journal: Genes & Development

Article Title: SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA

doi: 10.1101/gad.309138.117

Figure Lengend Snippet: The effect of SAM68 on NORAD- and PUM2-regulated genes. ( A ) The effects of PUM1, PUM2, NORAD, and SAM68 perturbation on genes bearing PREs. For each protein, we computed fold changes in gene expression following overexpression and knockdown (two replicates each). The shown values are the differences between log 2 transformed fold changes following overexpression and knockdown conditions (OE-KD). The diagonal plots show changes in RNA levels of the indicated genes in each of the perturbations. The scatter plots above the diagonal compare the indicated perturbations for genes having two or more PREs and report the Spearman R between the values. Colors indicate local point density. The bar charts below the diagonal show the Spearman correlation coefficients between the fold changes when considering genes with the indicated number of PREs. ( B ) The effects of the indicated perturbations on luciferase activity from psiCHECK1-PREx3 (psiCheck-1 containing 3× wild-type PREs) and psiCHECK1-mPREx3 (psiCheck-1 containing 3× mutated PREs) after transfection of an empty plasmid and plasmids for PUM2, SAM68, or SAM68 without the KH domain (SAM68ΔKHD). Average ± SEM is shown, based on at least three independent replicates. (*) P < 0.05; (**) P < 0.005, t -test.

Article Snippet: The beads were prewashed and coated with antibodies against GAPDH (diluted 1:1000; Santa Cruz Biotechnology, SC-32233), PUM2 and SAM68 (each diluted 1:1000; Bethyl Laboratories, A300-201A and A300-202A, respectively), and RUVBL1 (diluted 1:1000; Proteintech, 10210-2-AP) overnight at 4°C with gentle rotation.

Techniques: Gene Expression, Over Expression, Knockdown, Transformation Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Genes & Development

Article Title: SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA

doi: 10.1101/gad.309138.117

Figure Lengend Snippet: SAM68 interacts with PUM2 and facilitates the interaction between PUM2 and NORAD. ( A , B ) Interaction between SAM68 and PUM2 in lysates derived from HCT116 wild type and NORAD −/− was examined by co-IP using anti-SAM68 and PUM2 antibodies followed by Western blot. Five percent of the lysate used for immunoprecipitation was loaded as input. ( C ) RIP of PUM2 in HCT116 wild-type or SAM68 −/− cells quantified using qRT–PCR for the indicated genes. n ≥ 3, normalized to the input and the IgG control. Average ± SEM. “Control” indicates cells transfected with an empty pcDNA3.1 plasmid, and “SAM68 OE” and “Rescue” indicate cells transfected with a SAM68 expression plasmid. Asterisks indicate P < 0.05 compared with the “wild-type control” sample ( t -test).

Article Snippet: The beads were prewashed and coated with antibodies against GAPDH (diluted 1:1000; Santa Cruz Biotechnology, SC-32233), PUM2 and SAM68 (each diluted 1:1000; Bethyl Laboratories, A300-201A and A300-202A, respectively), and RUVBL1 (diluted 1:1000; Proteintech, 10210-2-AP) overnight at 4°C with gentle rotation.

Techniques: Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Control, Transfection, Plasmid Preparation, Expressing

Journal: Genes & Development

Article Title: SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA

doi: 10.1101/gad.309138.117

Figure Lengend Snippet: PUM2 regulation by NORAD is SAM68-dependent. ( A ) Regulation of expression of a luciferase with three consensus (PREx3) or mutated (mPREx3) PREs in its 3′ UTR in HCT116 cells with the indicated genotype following transfection of the indicated vector. Average ± SEM. n = 4 independent experiments. (*) P < 0.05; (**) P < 0.005, t -test. ( B ) Fold changes in expression of the indicated gene group following siRNA-mediated knockdown of NORAD in wild-type and SAM68 −/− HCT116 cells. P -values were computed with Wilcoxon rank sum test.

Article Snippet: The beads were prewashed and coated with antibodies against GAPDH (diluted 1:1000; Santa Cruz Biotechnology, SC-32233), PUM2 and SAM68 (each diluted 1:1000; Bethyl Laboratories, A300-201A and A300-202A, respectively), and RUVBL1 (diluted 1:1000; Proteintech, 10210-2-AP) overnight at 4°C with gentle rotation.

Techniques: Expressing, Luciferase, Transfection, Plasmid Preparation, Knockdown

Journal: Genes & Development

Article Title: SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA

doi: 10.1101/gad.309138.117

Figure Lengend Snippet: Chromosome segregation errors in the cells depleted of SAM68 and NORAD. ( A ) siRNA-mediated knockdown of SAM68 and NORAD in HeLa Kyoto cells. Expression levels were compared with the control nontargeting siRNA and normalized to the indicated controls. P -values were computed using two-sided t -test. ( B ) Quantification results of chromosome segregation errors from time-lapse microscopy of HeLa Kyoto cells progressing through mitosis after treatment with the indicated siRNAs. Each bar represents the mean of five independent experiments ±SEM. The numbers in parentheses indicate the number of anaphase cells analyzed per condition. ( C ) Representative still images from the time-lapse microscopy. White arrows depict chromosome segregation errors. Asterisk depicts magnified images at the indicated times. Bar, 5 µm.

Article Snippet: The beads were prewashed and coated with antibodies against GAPDH (diluted 1:1000; Santa Cruz Biotechnology, SC-32233), PUM2 and SAM68 (each diluted 1:1000; Bethyl Laboratories, A300-201A and A300-202A, respectively), and RUVBL1 (diluted 1:1000; Proteintech, 10210-2-AP) overnight at 4°C with gentle rotation.

Techniques: Knockdown, Expressing, Control, Time-lapse Microscopy

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: Arginine methylation of HNRNPUL2, KHDRBS1(SAM68) and SRSF1 is reduced in HD ISPNs ( A ) Total cell lysates from non-differentiated control (33CAG) and HD (180CAG) ISPNs were prepared as described in the Experimental Section. IPs with a mixture of mono-methyl (MMA) and asymmetric dimethyl (ADMA) antibodies were performed followed by western blot analysis with indicated protein-specific antibodies. Protein bands were visualized and quantified using the Licor System and Image Studio software. ( B ) – ( D ) Graphs (left) show the mean intensity values (±SEM) of the signal detected with antibodies to total HNRNPUL2, KHDRBS1(SAM68) and SRSF1 in the IPs normalized to the signal obtained from input lysates with the same antibodies. Total protein levels normalized to β-tubulin were also quantified (right graphs). n = 3 (biological replicates), Normality Test (Shapiro–Wilk): Passed, Equal Variance Test: Passed. T -test with equal variances was performed: (B) * 33CAG versus 180CAG, P 0.04. (C) * 33CAG versus 180CAG, P 0.0046. * * 33CAG versus 180CAG, P 0.0039.

Article Snippet: For evaluation of arginine methylation of HNRNPUL2, KHDRBS1 (SAM68) and SRSF1 we performed methyl-IP with the lysates of non-differentiated control and HD ISPNs using a mixture of pan-specific mono-methyl (MMA) and asymmetric dimethyl (ADMA) antibodies (both obtained from Cell Signaling Technology) followed by western blot analysis with protein-specific antibodies to HNRNPUL2 (E1B-AP5A300-863A, Bethyl Laboratories), KHDRBS1 (SAM68, A302-110A, Bethyl Laboratories) and SRSF1(SF2/ASF, 96, sc-33652, Santa-Cruz Biotechnology).

Techniques: Methylation, Western Blot, Software

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: RNA association of hypomethylated RBPs is decreased in HD cells. RNA-binding assay in ISPNs. RNA pull-downs were performed as described in the Experimental Section according to Castello et al . (2013) . ( A ) Validation of RNA-binding assay in ISPNs demonstrating detection of indicated RNA-bound proteins in cross-linked cells (but not in the eluates of nonirradiated control cells) with protein-specific antibodies. ( B ) The amounts of mRNA within RNA-protein complexes were estimated by absorbance at A260, and equal amounts were analyzed by western blotting with indicated protein-specific antibodies. Representative experiment is shown. ( C ) – ( G ) Quantitation of blots shown in (B). Graphs show the mean intensity values (±SD) of the signal with antibodies to indicated proteins detected in the RNA-bound fraction normalized to the signal obtained from input lysates with the same antibodies. n = 3 (biological replicates). Normality test (Shapiro–Wilk): Passed, equal variance test: Passed. One-way ANOVA was performed with pairwise multiple comparison procedures (Holm–Sidak method). (C) HNRNPUL2, * 33EE versus 180, P 0.002; * * 33DG versus 180, P 0.002. (D) KHDRBS1, * 33EE versus 180, P 0.004; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001. (E) FUS, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.008. (F) SRSF1, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.011. (G) TAF15, * 33EE versus 180, P < 0.001; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001.

Article Snippet: For evaluation of arginine methylation of HNRNPUL2, KHDRBS1 (SAM68) and SRSF1 we performed methyl-IP with the lysates of non-differentiated control and HD ISPNs using a mixture of pan-specific mono-methyl (MMA) and asymmetric dimethyl (ADMA) antibodies (both obtained from Cell Signaling Technology) followed by western blot analysis with protein-specific antibodies to HNRNPUL2 (E1B-AP5A300-863A, Bethyl Laboratories), KHDRBS1 (SAM68, A302-110A, Bethyl Laboratories) and SRSF1(SF2/ASF, 96, sc-33652, Santa-Cruz Biotechnology).

Techniques: RNA Binding Assay, Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: Sequences and product numbers for target specific siRNA primers.

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: ( A) RT-qPCR validation of siRNA knockdown for U2AF1, SYNCRIP, ILF2, and SRRM2. Error bars represent mean ± S.D. (n = 6). Immunoblot analysis shows reduced protein levels for U2AF1 with protein expression quantified and normalized to tubulin (representative experiment). (B) Histogram depicts expression of the cell activation markers CD25 and CD62L from FACS analysis of resting and activated T cells treated with negative control siRNA (blue) or U2AF1 siRNA (green). (C) Proliferation of resting and activated CD4 T cells after siRNA treatment as measured by MTT assays. Error bars represent mean ± S.D. (n = 4). (D) ELISA results for secretion of 11 cytokines after knockdown of U2AF2 interactome members plotted as log 2 fold changes. Error bars represent mean ± S.E.M. (n = 3). Statistical significance determined by Student’s t-test (*: p <0.05).

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Activation Assay, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: (A) Schematic for knockdown of U2AF2 interactome members and whole exome splicing microarray analysis in activated CD4 T cell cultures. (B) Volcano plots of significantly differentially expressed genes (up in siRNA–red; down in siRNA–green) after U2AF1, SRRM2, ILF2, or SYNCRIP knockdowns. Statistical significance determined by edgeR analysis (FDR adjusted p-value <0.05, n = 3). (C) RT-qPCR validation of select differentially expressed genes in U2AF1 (green) or SYNCRIP (orange) knockdowns. Error bars represent mean ± S.D. (n = 3). (D) Total genes differentially expressed and alternatively spliced in knockdowns. Alternatively spliced genes determined by Alt-Analyze (ASPIRE > 0.2); differentially expressed genes same as (B). (E) Cytoscape map of differentially expressed genes within significantly enriched immune related pathways (hypergeometric test adjusted p-value <0.05) affected by knockdown of U2AF1 (blue). Genes from these pathways differentially expressed in ILF2 (yellow), SYNCRIP (red), or all (green) knockdowns are highlighted.

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Microarray, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: Sequences and product numbers for target specific siRNA primers.

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: ( A) RT-qPCR validation of siRNA knockdown for U2AF1, SYNCRIP, ILF2, and SRRM2. Error bars represent mean ± S.D. (n = 6). Immunoblot analysis shows reduced protein levels for U2AF1 with protein expression quantified and normalized to tubulin (representative experiment). (B) Histogram depicts expression of the cell activation markers CD25 and CD62L from FACS analysis of resting and activated T cells treated with negative control siRNA (blue) or U2AF1 siRNA (green). (C) Proliferation of resting and activated CD4 T cells after siRNA treatment as measured by MTT assays. Error bars represent mean ± S.D. (n = 4). (D) ELISA results for secretion of 11 cytokines after knockdown of U2AF2 interactome members plotted as log 2 fold changes. Error bars represent mean ± S.E.M. (n = 3). Statistical significance determined by Student’s t-test (*: p <0.05).

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Activation Assay, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: (A) Schematic for knockdown of U2AF2 interactome members and whole exome splicing microarray analysis in activated CD4 T cell cultures. (B) Volcano plots of significantly differentially expressed genes (up in siRNA–red; down in siRNA–green) after U2AF1, SRRM2, ILF2, or SYNCRIP knockdowns. Statistical significance determined by edgeR analysis (FDR adjusted p-value <0.05, n = 3). (C) RT-qPCR validation of select differentially expressed genes in U2AF1 (green) or SYNCRIP (orange) knockdowns. Error bars represent mean ± S.D. (n = 3). (D) Total genes differentially expressed and alternatively spliced in knockdowns. Alternatively spliced genes determined by Alt-Analyze (ASPIRE > 0.2); differentially expressed genes same as (B). (E) Cytoscape map of differentially expressed genes within significantly enriched immune related pathways (hypergeometric test adjusted p-value <0.05) affected by knockdown of U2AF1 (blue). Genes from these pathways differentially expressed in ILF2 (yellow), SYNCRIP (red), or all (green) knockdowns are highlighted.

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Microarray, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: (A) Breakdown of differential splicing events affected by knockdown of U2AF1 in whole exon splicing microarray data using Alt-Analyze. (B) Top : RNAseq coverage plot of regions differentially spliced between resting and activated T cells for IL18R1, CASP2, ABI1. Middle : Normalized microarray intensities for junction probes (Exclusion/Inclusion) and exon probes (Cassette and Constitutive) from Control and U2AF1 siRNA samples are used by the Transcript Analysis Console (Affymetrix) to calculate the Splicing Index (SI). Greater probeset level intensity values in the Control siRNA samples correspond to negative SI values and positive SI represents increased intensity in the U2AF1 siRNA samples. Bottom : RT-qPCR Validation of alternative splicing after U2AF1 (green) or SYNCRIP (orange) knockdowns. ASE–Alternatively Spliced Exon. Error bars represent mean ± S.D. (n = 3).

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Microarray, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells

doi: 10.1371/journal.pone.0144409

Figure Lengend Snippet: (A) Immunoblot analysis for binding of select U2AF2 interactome members after immunoprecipitation with U2AF2 antibody in activated cells with negative controls vs. U2AF1 siRNA. Protein expression quantified using the Li-Cor Odyssey and normalized to tubulin. (B) Totals of genes differentially bound to U2AF2 after knockdown of U2AF1 and SYNCRIP as compared to negative control siRNAs. Statistical significance determined by edgeR analysis (FDR adjusted p-value <0.05, n = 3). (C) RT-qPCR validation of selected differentially expressed genes after U2AF1 knockdown as revealed by U2AF2 RIP. Error bars represent mean ± S.D. (n = 3). (D) Cytoscape map of genes differentially bound to U2AF2 within KEGG pathways, Gene Ontology, and ImmuneMap after knockdown of U2AF1 (blue) or SYNCRIP (red). Statistical significance determined by hypergeometric test (multiple testing corrected p-value <0.05).

Article Snippet: Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Techniques: Western Blot, Binding Assay, Immunoprecipitation, Expressing, Negative Control, Quantitative RT-PCR