flag sam68  (OriGene)

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, Control, 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, 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: 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, Control, 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, Control, Functional Assay, Knockdown

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: Genome biology

Article Title: Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing.

doi: 10.1186/s13059-020-02199-6

Figure Lengend Snippet: Fig. 6 NimtRNA protein interaction analysis. a Biotinylated transcripts containing or lacking nimtRNAs were incubated with nuclear protein extracts. Subsequently, binding proteins were isolated and analyzed by PAGE; differential band patterns were excised and analyzed by MS. b GO analysis of nimtRNA transcript interacting proteins. c The top 10 nimtRNA transcript interacting proteins ranked by absolute abundance. d–f An electrophoretic mobility shift assay was performed with increasing concentrations of KHDRBS1 being incubated with a transcript of the nimtRNATyr (d), nimtRNATyr delT (e), or snoRNA SNORD115 (f). The unbound transcript is indicated by an asterisk; the transcript-protein complexes are indicated by triangles

Article Snippet: Human recombinant KHDRBS1 (Sam68; CAT#: TP300263) was ordered from Origene (Rockville, Maryland, USA).

Techniques: Incubation, Binding Assay, Isolation, Electrophoretic Mobility Shift Assay