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

Abstract

Alternative polyadenylation (APA) yields transcripts differing in their 3'-end, and its regulation is altered in cancer, including prostate cancer. Here we have uncovered a mechanism of APA regulation impinging on the interaction between the exonuclease XRN2 and the RNA-binding protein Sam68, whose increased expression in prostate cancer is promoted by the transcription factor MYC. Genome-wide transcriptome profiling revealed a widespread impact of the Sam68/XRN2 complex on APA. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor for binding to strong polyadenylation signals at distal ends of genes, thus promoting usage of suboptimal proximal polyadenylation signals. This mechanism leads to 3' untranslated region shortening and translation of transcripts encoding proteins involved in G1/S progression and proliferation. Thus, our findings indicate that the APA program driven by Sam68/XRN2 promotes cell cycle progression and may represent an actionable target for therapeutic intervention.

Extended Data Fig. 1:. XRN2 physically interacts with Sam68 (Related to Fig. 1).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer a, Nucleotide sequence alignment between XRN2 (CCDS 13144.1, GRCh38.p13) and Clone 177 (Cln177) retrieved from the two-hybrid screen. b, Nucleotide and aminoacid sequence of the region of interaction of XRN2 with Sam68 identified by the two-hybrid screen.

Extended Data Fig. 2:. XRN2 physically interacts with Sam68 (Related to Fig. 1).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer a,b, Western blot (WB) analysis and Coomassie blue staining of the GST pull-down assay (n = 2) performed using LNCaP nuclear extracts (N.E.) in presence of GST-Sam68 full-length (a) and deletion mutants (b). GST was used as negative control (a,b). A scheme of GST-Sam68 fusion proteins is also shown (b).

Extended Data Fig. 3:. XRN2 and Sam68 expression are positively correlated in PC (Related to Fig. 2).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer a-d, Pearson’s correlation between XRN2 and sam68 expression (a,c) and XRN2 expression in sam68^low (blue circles) and Sam68^high (red squares) patient groups (b,d) retrieved from Sawyers (GSE2104) (a,b) and Sueltman (GSE29079) (c,d) datasets. Pearson’s correlation coefficient (r) (two-sided) and the p-values(P) are reported (95% confidence interval) (a,c). In b and d statistical significance was calculated by Mann-Whitney test (two-sided) and the p-values are reported (95% confidence interval). e, Scatter-plot analysis showing the positive correlation (R² = 0.887) between the expressicn of XRN2 and Sam68 proteins in PC specimens.

Extended Data Fig. 4:. XRN2 and MYC expression are correlated in PC (Related to Fig. 3).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer a-d, Pearson’s correlation between XRN2 and MYC expression (a,c) and distribution of XRN2 expression in MYC^low (blue circles) and, MYC^high (redsquares) groups (b,d) retrieved from Sawyers (GSE21034) (a,b) and Sueltman (GSE29079) (c,d) datasets. Pearson’s correlation coefficient (r) (two-sided) and the p-values (P) of the correlation (95% confidence interval) were reported in a and c panels. In b and d statistical significance was calculated by Mann-Whitney test (two-sided) and the p-values are reported (95% confidence interval). e, UCSC Genome Browser snapshot of RNAPII. H3K27AC and H3K4Me3 ChIP-seq profiles surrounding the TSS of the XKN2 gene. RNAPII (POLR2A). MYC and MAX binding regions are indicated (dark box), f, Schematic representation of the putative XRN2 promoter doned upstream of the luciferase-bosed report pGL3-basic plasmid. The putative MYC binding site (E-box) is indicated in bold. g, Bar graph (left panel) represents ludferase activity of AT?, V2 promoter compared to an intergen ic region (intergen ic). used as negative control. The hiaferase assay was performed in 293 T cells transfected, or not (empty vector. EV), with MYC-pCDNA3 vector (MYC). h,i, qPCR (h) and Western blot (I) analyses of MYC, XRN2 and Sam68 expression in LNCaP and 22Rv1 PC cells lines transfected with Control (si-scr#2) and MYC (si-MYC42) siRNAs. The expression was reported as fold enrichment (AACq) of Histone 3. g-i, Data represent mean i SD of three biological replicates. Statistical significance was calculated by unpaired Student’s t-test (two-sided). In g, the p-values are: intergenic P = 0.686, XRN2 P = 9.6 × 10⁻⁶. In h: MYC/LNCaP P = 2 × 10⁻⁴, MYC/22Rv1 P = 5.1 × 10⁻³, XRN2/LNCaP P = 1.5 × 10⁻³, XRN2/22Rv1 P = 2.7 × 10⁻³. Sam68/LNCaP P = 8.4 × 10⁻⁸, Sam68/22Rv1 P =3.1 × 10⁻³). In the representation of panels, statistical value is reported as ** P < 0.01; *** P < 0.001; n.s. not significant.

Extended Data Fig. 5:

Genome-wide regulation of APA by XRN2 and Sam68 in PC cells (Related to Fig. 4). From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer a, Representative Western-blot analysis of LNCaP cells transfected twice with control (si-Scr), Sam68 (si-Sam68) and XRN2 (si-XRN2) siRNAs. β-actin was used as loading control (n = 3). b, Principal Component Analysis showing variance of 3’READS data from two biological replicates. The red circles, green triangles and blue squares represent pA selection data in control, Sam68 and XRN2 silenced cells, respectively. The proportion of variance (%) for both the first and second principal components is reported, c, 3’READS sample distance analysis. The heatmap show the Euclidean distances between samples. Dendrogram of clustering results are also shown, d, Venn diagram showing the overlap between common regulated genes undergoing to expression (GE) or APA changes in absence of Sam68 (si-Sam68) and XRN2 (si-XRN2) (ns: not significant, modified Fisher’s test), e, Representative 3’RACE PCR analysis (n = 2) of four genes (RCC2, SCAMP2, LAMC1, CD164) undergoing UTR lengthening in absence of Sam68 and XRN2. Downregulated and up-regulated pAsare indicated in orange and purple, respectively.

Extended Data Fig. 6:. Genome-wide regulation of APA by XRN2 and Sam68 in PC cells (Related to Fig. 4).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer a,b, Representative Western blot analysis of LNCaP cells transiently (a) or stably (b) depleted for Sam68 and XRN2 (n = 3). β-actin was used as loading control, c-e, Bar graphs showing qRT-PCR analyses of pA usage evaluated in 24 representative genes undergoing APA regulation in LNCaP cells treated as in a and b. Fold change of d-pA relative to p-pA was calculated by the ΔCq method. In e, unvalidated genes are shown. Data represent mean ± SD of three biological replicates (c-e). In c-e, statistical significance was calculated by unpaired Student’s t-test, two-sided (exact p-values reported in source data). In the representation of panels, statistical value is reported as * P < 0.05; ** P < 0.01; *** P < 0.001. UCSC genome browser tracks showing APA regulation for each event analyzed is also shown on the right side of each graph. Purple and orange boxes indicate up- and down-regulated events, respectively.

Extended Data Fig. 7:. Sam68 and XRN2 globally modulate pA selection in the 3’UTR of target transcripts (Related to Fig. 5).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer a. Representative Western blot and densitometric (bar graphs) analyses of nuclear matrix subcellular fraction isolated in control (sh-scr), Sam68 (sh-Sam68) and XRN2 (sh-XRN2) stably depleted LNCaP cells. Lamin β−1 was used as loading control, b, Bar graphs showing qPCR analysis of pA usage evaluated in three genes undergoing 3’UTR-APA regulation in cells knocked down for XRN2 targeting 3’UTR (sh-XRN2-3’UTR) and transfected with empty vector (EV), XRN2 wild-type (WT) and catalytically-death mutant (D235A). LNCaP cells stably depleted with sh 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 ΔCq method. c, CLIP assays performed in LNCaP cells stably depleted for XRN2 (sh-XRN2) using Sam68 antibody or IgGs, as negative control. RNA associated with Sam68 was quantified by qPC R using primers located upstream of regulated and non-regulated pAs and represented as percentage (%) of input, d, Bar graph showing the qPCR analysis of 4sU-IabeIed RNA isolated from LNCaP cells stably transduced with control (sh-scr) and XRN2 (sh-XRN2) shRNAs. Labeled RNA is represented as percentage (%) of total RNA used for the assay (input). e, CLIP assays performed in LNCaP cells transfected as in b using Sam68 antibody or IgGs, as negative control. RNA associated with Sam68 was reported as in c. a-e. Data represent mean ± SD of three biological replicates. Statistical significance was calculated by unpaired Student’s t-test (two-sided). In panels a,c and b,e the exact p-value is reported in figure and source data, respectively. When not indicated (b,e), p-values are reported as *P < 0.05; ** P < 0.01; ***P < 0.001; n.s.: not significant.

Extended Data Fig. 8:. Sam68 and XRN2 represses strong, distal PAS (Related to Fig. 6).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenylation to cell cycle progression in prostate cancer a, Changes of APA isoform abundance (ΔAbn) of genes presenting at least one regulated pA in LNCaP cells depleted for Sam68 (si-Sam68) or XRN2 (si-XRN2). Mean values (Mean) and number of events (n) are reported. Statistical significance was calculated by unpaired Student’s t-test (two-sided). The p-value is reported. In boxplot, band and box indicate the median and the 25–75th percentile, respectively. Whiskers indicate ±1.5x interquartile range, b, Frequency distribution of the U (upper panel) and C (lower panel) nucleotide in up- (purple line), down- (orange line) and un-regulated (black line) region between −100/+100nt from CS (0). c-e, Metagene analyses of CSTF64 (c), CPSF30 (d), and Sam68 (e) CLIP-binding profile with respect to CS (0) in upregulated (purple), downregulated (orange) and non-regulated (black) PASs. f, Scheme of wild-type (FLNB WT) and mutant (FLNB mut) nucleotide sequence surrounding FLNB distal PAS (highlighted in bold). The putative Sam68 binding sites (underline) and mutated bases (red) are indicated, g, RT-PCR (agarose gel) and qPCR (bar graph) analyses of pA usage of wild-type (WT) and mutant (Mut) FLNB minigene evaluated in LNCaP cells transfected, or not, with Sam68-GFP plasmid. Representative Western blot of protein expression is also shown, h, Western blot analysis of RNA-pulldown assay performed using biotin-labeled FLNB WT or Mut RNA. Streptavidin beads were used as control (−) (n = 1). i,j, CLIP assays performed in sh-Sam68 and sh-XRN2 LNCaP cells using CPSF30 antibody. IgG was used as negative control. FLNB and SCARB2 RNA associated with CPSF30 (i) or CSTF64 (j) factors was quantified by qPCR and represented as percentage (%) of input. In g,i j, statistical significance was calculated by unpaired Student’s t-test, two-sided (n = 3). In g, WT(Sam68-GFP/EV) P= 1.4 × 10⁻³, Mut(Sam68-GFP/EV) P = 0.777, WT Sam68-GFP/Mut Sam68-GFP P = 9.0 × 10⁻³; in i, FLNB: CPSF30(sh-Sam68/sh-scr) P = 5.0 × 10⁻⁴, CPSF30(sh-XRN2/sh-scr) P = 9.0 × 10⁻⁴, SCARB2: CPSF30(sh-Sam68/sh-scr) P = 1.0 × 10⁻⁴, CPSF30(sh-XRN2/sh-scr) P = 9.0 × 10⁻⁴; in j, FLNB downreg: CSTF64(sh-Sam68/sh-scr) P = 0.1999, CSTF64(sh-XRN2/sh-scr) P = 0.2830; FLNB upreg: CSTF64(sh-Sam68/sh-scr) P = 3.1 ×10⁻³, CSTF64(sh-XRN2/sh-scr) P = 0.043; SCARB2 downreg: CSTF64(sh-Sam68/sh-scr) P = 0.4242, CSTF64(sh-XRN2/sh-scr) P = 0.4723; SCARB2 upreg: CSTF64(sh-Sam68/sh-scr) P = 1.0 × 10⁻⁴, CSTF64(sh-XRN2/sh-scr) P = 0.0468). In the representation of panels, statistical value is reported as *P < 0.05; **P < 0.01; ***p < 0.001; n.s. not significant.

Extended Data Fig. 9:. XRN2 and SAM68 promotes cell cycle progression through APA modulation (Related to Fig. 7).

From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenvlation to cell cycle progression in prostate cancer a,b, Cell cycle (a) and sub-Gl (b) distribution assessed by PI staining in asynchronous LNCaP cells stably depleted for Sam68 and XRN2. c, Cell cycle distribution assessed by PI staining in asynchronous LNCaP cells stably depleted for MCM10 and ORC2. a-c, Data represent mean ± SD of three biological replicates. Statistical significance was calculated by unpaired Student’s t-test, two-sided. In a, the p-values are: Gl: sh-Sam68/sh-scr P = 2.2 × 10⁻³, sh-XRN2/sh-scr P = 8.6 × 10⁻³; S: sh-Sam68/sh-scr P = 2.0 × 10⁻⁴, sh-XRN2/sh-scr P = 1.2 × 10⁻³; G2-M: sh-Sam68/sh-scr P = 0.037, sh-XRN2/sh-scr P = 0.036; in b, sh-Sam68,/sh-scr P = 0.2264, sh-XRN2/sh-scr P = 0.6388; in C, Gl: si-MCM10/si-scr P = 2.6 × 10⁻⁶, si-ORC2/si-scr P = 3.6 × 10⁻³; S: si-MCMIO/si-scr P = 3.0 × 10⁻⁴, si-ORC2/si-scr P = 0.1679; G2-M: si-MCMlO/si-scr P = 2 × 10⁻⁴, si-ORC2/si-scr P = 0.0917). In the representation of panels, statistical value is reported as *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 1|. XRN2 physically interacts with Sam68.

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.

Fig. 2|. XRN2 and Sam68 expression are positively correlated in PC.

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 Sam68^low (blue circles) and Sam68^high (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).

Fig. 3|. MYC positively controlsXRN2expression in PC.

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 MYCf^low (blue circles) and MYC^high (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.

Fig. 4|. Genome-wide regulation of APA by XRN2 and Sam68 in PC cells.

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.

Fig. 5|. Sam68 and XRN2 globally modulate pA selection in the 3’UTR of target transcripts.

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.

Fig. 6|. Sam68 and XRN2 repress strong, canonical target pAs.

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.

Fig. 7|. XRN2 and Sam68 promotes cell cycle progression through APA modulation.

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.