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

Nature Structural & Molecular Biology volume 29, pages 1101–1112 (2022)Cite this article

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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.

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Fig. 1: XRN2 physically interacts with Sam68.
Fig. 2: XRN2 and Sam68 expression are positively correlated in PC.
Fig. 3: MYC positively controls XRN2 expression in PC.
Fig. 4: Genome-wide regulation of APA by XRN2 and Sam68 in PC cells.
Fig. 5: Sam68 and XRN2 globally modulate pA selection in the 3′UTR of target transcripts.
Fig. 6: Sam68 and XRN2 repress strong, canonical target pAs.
Fig. 7: XRN2 and Sam68 promotes cell cycle progression through APA modulation.

Data availability

The sequencing data generated in this study are deposited in the Gene Expression Omnibus at GSE198872. Public sequencing data used in this study are deposited under GSE37401 (https://doi.org/10.1016/j.celrep.2012.05.003)54, GSE85164 (https://doi.org/10.1038/nature21715)45, GSE46691(https://doi.org/10.1371/journal.pone.0066855) 41, GSE29079 (https://doi.org/10.1186/1471-2407-11-507)43 and GSE21034 (https://doi.org/10.1016/j.ccr.2010.05.026)42. Prostate adenocarcinoma (TGCA, PanCancer Atlas, 494 samples) gene expression data (mRNA) and clinical data (Progression-Free Survival) were downloaded from cBioPortal (https://www.cbioportal.org/study/summary?id=prad_tcga_pan_can_atlas_2018). Source data are provided with this paper.

Code availability

Code used to analyze the 3′READS-seq is available at https://github.com/DinghaiZ/3-prime-READS-plus.

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Acknowledgements

We thank all members of Sette’s laboratory for fruitful discussions throughout this study. The research reported in this paper was supported by Ministero della Salute ‘Ricerca Finalizzata 2011’ (GR-2011-02348423 to P.B.) and ‘Ricerca Finalizzata 2016’ (RF-2016-02363460 to C.S.), by the Associazione Italiana Ricerca sul Cancro (AIRC IG23416 to C.S.), Ministero della Salute – Ricerca Corrente 2021 and 2022 to IRCCS Fondazione Policlinico Gemelli, and by the National Institutes of Health (GM084089 and GM129069 to B.T.). We acknowledge financial support from the Università Cattolica del Sacro Cuore (UCSC) for the execution and publication of this study.

Author information

Author notes

  1. These authors contributed equally: Claudio Sette, Pamela Bielli.

Authors and Affiliations

  1. Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Hearth, Rome, Italy

    Marco Pieraccioli, Cinzia Caggiano & Claudio Sette

  2. GSTEP-Organoids Core Facility, Fondazione Policlinico Agostino Gemelli IRCCS, Rome, Italy

    Marco Pieraccioli, Cinzia Caggiano, Gabriele Babini & Claudio Sette

  3. Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy

    Luca Mignini & Pamela Bielli

  4. Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA, USA

    Chuwei Zhong & Bin Tian

  5. Department of Innovative Technologies in Medicine & Dentistry, G. d’Annunzio University, Chieti, Italy

    Rossano Lattanzio

  6. Center for Advanced Studies and Technology (CAST), G. d’Annunzio University, Chieti, Italy

    Rossano Lattanzio

  7. Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy

    Savino Di Stasi

  8. Laboratory of Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy

    Pamela Bielli

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  1. Marco Pieraccioli
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  2. Cinzia Caggiano
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Contributions

C.S. and P.B. conceived the study. M.P., P.B. and C.S. wrote the manuscript. M.P., C.C., P.B. and C.S. designed the experiments. M.P., C.C., L.M. and P.B. performed the experiments. B.T., M.P., G.B. and C.Z. performed the 3′READS data and bioinformatic analyses. R.L. and S.D.S. performed PC immunohistochemistry analysis.

Corresponding authors

Correspondence to Claudio Sette or Pamela Bielli.

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Nature Structural & Molecular Biology thanks David Elliott and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Beth Moorefield, Tiago Faial and Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

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

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).

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).

Source data

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

a-d, Pearson’s correlation between XRN2 and Sam68 expression (a,c) and XRN2 expression in Sam68low (blue circles) and Sam68high (red squares) patient 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) 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 expression of XRN2 and Sam68 proteins in PC specimens.

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

a-d, Pearson’s correlation between XRN2 and MYC expression (a,c) and distribution of XRN2 expression in MYClow (blue circles) and MYChigh (red squares) 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 XRN2 gene. RNAPII (POLR2A), MYC and MAX binding regions are indicated (dark box). f, Schematic representation of the putative XRN2 promoter cloned upstream of the luciferase-based report pGL3-basic plasmid. The putative MYC binding site (E-box) is indicated in bold. g, Bar graph (left panel) represents luciferase activity of XRN2 promoter compared to an intergenic region (intergenic), used as negative control. The luciferase 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-MYC#2) siRNAs. The expression was reported as fold enrichment (ΔΔCq) of Histone 3. g-i, Data represent mean ± 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.

Source data

Extended Data Fig. 5 Genome-wide regulation of APA by XRN2 and Sam68 in PC cells (Related to Fig. 4).

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 pAs are indicated in orange and purple, respectively.

Source data

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

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.

Source data

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

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 qPCR 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-labeled 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.

Source data

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

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.

Source data

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

a,b, Cell cycle (a) and sub-G1 (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: G1: 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, G1: si-MCM10/si-scr P = 2.6 × 10−6, si-ORC2/si-scr P = 3.6 × 10−3; S: si-MCM10/si-scr P = 3.0 × 10−4, si-ORC2/si-scr P = 0.1679; G2-M: si-MCM10/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.

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Supplementary methods, Tables 1–3 and Fig. 1.

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Source Data Fig. 1

Unprocessed western blots and agarose gel related to Fig. 1.

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Pieraccioli, M., Caggiano, C., Mignini, L. et al. The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer. Nat Struct Mol Biol 29, 1101–1112 (2022). https://doi.org/10.1038/s41594-022-00853-0

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