The poly A polymerase Star-PAP controls 3'-end cleavage by promoting CPSF interaction and specificity toward the pre-mRNA

Star-PAP is a poly (A) polymerase (PAP) that is putatively required for 3'-end cleavage and polyadenylation of a select set of pre-messenger RNAs (mRNAs), including heme oxygenase (HO-1) mRNA. To investigate the underlying mechanism, the cleavage and polyadenylation of pre-mRNA was reconstituted with nuclear lysates. siRNA knockdown of Star-PAP abolished cleavage of HO-1, and this phenotype could be rescued by recombinant Star-PAP but not PAPα. Star-PAP directly associated with cleavage and polyadenylation specificity factor (CPSF) 160 and 73 subunits and also the targeted pre-mRNA. In vitro and in vivo Star-PAP was required for the stable association of CPSF complex to pre-mRNA and then CPSF 73 specifically cleaved the mRNA at the 3'-cleavage site. This mechanism is distinct from canonical PAPα, which is recruited to the cleavage complex by interacting with CPSF 160. The data support a model where Star-PAP binds to the RNA, recruits the CPSF complex to the 3'-end of pre-mRNA and then defines cleavage by CPSF 73 and subsequent polyadenylation of its target mRNAs.     

The Cleavage and Polyadenylation Specificity Factor in Xenopus laevis Oocytes Is a Cytoplasmic Factor Involved in Regulated Polyadenylation

During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.     

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.     

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF

Processing of the 3′ end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3′ termini is not sensitive to small changes in cpsf and su(f) dosage. Received: 6 June 1997 / Accepted: 5 November 1997     

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF

Processing of the 3′ end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3′ termini is not sensitive to small changes in cpsf and su(f) dosage.     

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.     

Inhibition of glycogen synthase kinase-3β counteracts ligand-independent activity of the androgen receptor in castration resistant prostate cancer

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.     

Alternative polyadenylation writer CSTF2 forms a positive loop with FGF2 to promote tubular epithelial-mesenchymal transition and renal fibrosis

Effective therapies for renal fibrosis, the common endpoint for most kidney diseases, are lacking. We previously reported that alternative polyadenylation (APA) drives transition from acute kidney injury to chronic kidney disease, suggesting a potential role for APA in renal fibrogenesis. Here, we found that among canonical APA writers, CSTF2 expression was upregulated in tubular epithelial cells (TEC) of fibrotic kidneys. CSTF2 was also identified as a TGF-β-inducible pro-fibrotic gene. Further analysis revealed that CSTF2 promoted epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) overproduction in TEC by inducing 3’UTR shortening and upregulation of the expression of basic fibroblast growth factor 2 (FGF2). Additionally, 3’UTR shortening stabilised FGF2 mRNA through miRNA evasion. Interestingly, FGF2 enhanced CSTF2 expression, leading to the forming of a CSTF2-FGF2 positive loop in TEC. Furthermore, CSTF2 knockdown alleviated unilateral ureteral obstruction-induced renal fibrosis in vivo. Finally, we developed a CSTF2-targeted antisense oligonucleotide (ASO) and validated its effectiveness in vitro. These results indicate that the expression of the APA writer, CSTF2, is upregulated by TGF-β and CSTF2 facilitates TGF-β-induced FGF2 overexpression, forming a TGF-β-CSTF2-FGF2 pro-fibrotic axis in TEC. CSTF2 is a potentially promising target for renal fibrosis that does not directly disrupt TGF-β.     

A Complex Containing the CPSF73 Endonuclease and Other Polyadenylation Factors Associates with U7 snRNP and Is Recruited to Histone Pre-mRNA for 3′-End Processing

Animal replication-dependent histone pre-mRNAs are processed at the 3′ end by endonucleolytic cleavage that is not followed by polyadenylation. The cleavage reaction is catalyzed by CPSF73 and depends on the U7 snRNP and its integral component, Lsm11. A critical role is also played by the 220-kDa protein FLASH, which interacts with Lsm11. Here we demonstrate that the N-terminal regions of these two proteins form a platform that tightly interacts with a unique combination of polyadenylation factors: symplekin, CstF64, and all CPSF subunits, including the endonuclease CPSF73. The interaction is inhibited by alterations in each component of the FLASH/Lsm11 complex, including point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with the endogenous U7 snRNP and are recruited in a U7-dependent manner to histone pre-mRNA. Collectively, our studies identify the molecular mechanism that recruits the CPSF73 endonuclease to histone pre-mRNAs, reveal an unexpected complexity of the U7 snRNP, and suggest that in animal cells polyadenylation factors assemble into two alternative complexes—one specifically crafted to generate polyadenylated mRNAs and the other to generate nonpolyadenylated histone mRNAs that end with the stem-loop.     

Selective degradation of AR-V7 to overcome castration resistance of prostate cancer

Androgen receptor splice variant 7 (AR-V7), a form of ligand-independent and constitutively activating variant of androgen receptor (AR), is considered as the key driver to initiate castration-resistant prostate cancer (CRPC). Because AR-V7 lacks ligand-binding domain, the AR-targeted therapies that aim to inactivate AR signaling through disrupting the interaction between AR and androgen are limited in CRPC. Thus, the emergence of AR-V7 has become the greatest challenge for treating CRPC. Targeting protein degradation is a recently proposed novel avenue for cancer treatment. Our previous studies have been shown that the oncoprotein AR-V7 is a substrate of the proteasome. Identifying novel drugs that can trigger the degradation of AR-V7 is therefore critical to cure CRPC. Here we show that nobiletin, a polymethoxylated flavonoid derived from the peel of Citrus fruits, exerts a potent anticancer activity via inducing G0/G1 phase arrest and enhancing the sensitivity of cells to enzalutamide in AR-V7 positive PC cells. Mechanically, we unravel that nobiletin selectively induces proteasomal degradation of AR-V7 (but not AR). This effect relies on its selective inhibition of the interactions between AR-V7 and two deubiquitinases USP14 and USP22. These findings not only enrich our understanding on the mechanism of AR-V7 degradation, but also provide an efficient and druggable target for overcoming CRPC through interfering the stability of AR-V7 mediated by the interaction between AR-V7 and deubiquitinase.Subject terms: Drug development, Translational research     

Assembly of a processive messenger RNA polyadenylation complex.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.     

A CPSF-73 homologue is required for cell cycle progression but not cell growth and interacts with a protein having features of CPSF-100

Formation of the mature 3' ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent beta-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the beta-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G(1) phase, but surprisingly the arrested cells continue growing and reach the size typical of G(2) cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3' end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G(1) progression and entry into S phase.     

Similarities and Distinctions in Actions of Surface-Directed and Classic Androgen Receptor Antagonists

The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists (MDV3100, ARN-509) at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC.     

Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation

Cleavage and polyadenylation specific factor 4 (CPSF4), a member of CPSF complex, plays a key role in mRNA polyadenylation and mRNA 3′ ends maturation. However, its possible role in lung cancer pathogenesis is unknown. In this study, we investigated the biological role and clinical significance of CPSF4 in lung cancer growth and survival and elucidated its underlying molecular mechanisms. We found that CPSF4 was highly expressed in lung adenocarcinoma cell lines and tumor tissue but was undetectable in 8 normal human tissues. We also found that CPSF4 overexpression was correlated with poor overall survival in patients with lung adenocarcinomas (P<0.001). Multivariate survival analyses revealed that higher CPSF4 expression was an independent prognostic factor for overall survival of the patients with lung adenocarcinomas. Suppression of CPSF4 by siRNA inhibited lung cancer cells proliferation, colony formation, and induced apoptosis. Mechanism studies revealed that these effects were achieved through simultaneous modulation of multiple signaling pathways. Knockdown of CPSF4 expression by siRNA markedly inhibited the phosphorylation of PI3K, AKT and ERK1/2 and JNK proteins. In contrast, the ectopic expression of CPSF4 had the opposite effects. Moreover, CPSF4 knockdown also induced the cleavage of caspase-3 and caspse-9 proteins. Collectively, these results demonstrate that CPSF4 plays a critical role in regulating lung cancer cell proliferation and survival and may be a potential prognostic biomarker and therapeutic target for lung adenocarcinoma.