CPEB1 induces autophagy and promotes apoptosis in ovarian granulosa cells of polycystic ovary syndrome.

Inflammatory damage in ovarian granulosa cells (GCs) is a key mechanism in polycystic ovary syndrome (PCOS), cytoplasmic polyadenylation element binding protein-1 (CPEB1) is important in inflammatory regulation, however, its role in PCOS is unclear. We aim to research the mechanism of CPEB1 in ovarian GCs in PCOS using dehydroepiandrosterone (DHEA)-induced PCOS rat models and testosterone-incubated GC models. The pathophysiology in PCOS rats was analyzed. Quantitative-realtime-PCR, TUNEL, immunohistochemistry, and Western blot were applied for quantification. Additionally, cell counting kit-8, flow cytometry, immunofluorescence, Western blot, and Monodansylcadaverine staining were performed. We found that PCOS rat models exhibited a disrupted estrus cycle, elevated serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), increased LH/FSH ratio, and heightened ovarian index. Furthermore, reduced corpus luteum and increased follicular cysts were observed in ovarian tissue. In ovarian tissue, autophagy and apoptosis were activated and CPEB1 was overexpressed. In vitro, CPEB1 overexpression inhibited cell viability and sirtuin-1 (SIRT1), activated tumor necrosis factor-α, and interleukin-6 levels, as well as apoptosis and autophagy; however, CPEB1 knockdown had the opposite effect. In conclusion, overexpression of CPEB1 activated autophagy and apoptosis of ovarian GCs in PCOS.

Downregulation of CPSF6 leads to global mRNA 3' UTR shortening and enhanced antiviral immune responses.

Alternative polyadenylation (APA) is a widespread mechanism of gene regulation that generates mRNA isoforms with alternative 3' untranslated regions (3' UTRs). Our previous study has revealed the global 3' UTR shortening of host mRNAs through APA upon viral infection. However, how the dynamic changes in the APA landscape occur upon viral infection remains largely unknown. Here we further found that, the reduced protein abundance of CPSF6, one of the core 3' processing factors, promotes the usage of proximal poly(A) sites (pPASs) of many immune related genes in macrophages and fibroblasts upon viral infection. Shortening of the 3' UTR of these transcripts may improve their mRNA stability and translation efficiency, leading to the promotion of type I IFN (IFN-I) signalling-based antiviral immune responses. In addition, dysregulated expression of CPSF6 is also observed in many immune related physiological and pathological conditions, especially in various infections and cancers. Thus, the global APA dynamics of immune genes regulated by CPSF6, can fine-tune the antiviral response as well as the responses to other cellular stresses to maintain the tissue homeostasis, which may represent a novel regulatory mechanism for antiviral immunity.

Disruption of Zar1 leads to arrested oogenesis by regulating polyadenylation via Cpeb1 in tilapia (Oreochromis niloticus).

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.

CPSF6 regulates alternative polyadenylation and proliferation of cancer cells through phase separation.

Cancer cells usually exhibit shortened 3' untranslated regions (UTRs) due to alternative polyadenylation (APA) to promote cell proliferation and migration. Upregulated CPSF6 leads to a systematic prolongation of 3' UTRs, but CPSF6 expression in tumors is typically higher than that in healthy tissues. This contradictory observation suggests that it is necessary to investigate the underlying mechanism by which CPSF6 regulates APA switching in cancer. Here, we find that CPSF6 can undergo liquid-liquid phase separation (LLPS), and elevated LLPS is associated with the preferential usage of the distal poly(A) sites. CLK2, a kinase upregulated in cancer cells, destructs CPSF6 LLPS by phosphorylating its arginine/serine-like domain. The reduction of CPSF6 LLPS can lead to a shortened 3' UTR of cell-cycle-related genes and accelerate cell proliferation. These results suggest that CPSF6 LLPS, rather than its expression level, may be responsible for APA regulation in cancer cells.

YY1-regulated lncRNA SOCS2-AS1 suppresses HCC cell stemness and progression via miR-454-3p/CPEB1.

BACKGROUND: Cancer stem cells are one fundamental reason for the high recurrence rate of hepatocellular carcinoma (HCC) and its resistance to treatment. This study explored the mechanism by which SOCS2-AS1 affects HCC cell stemness. METHODS: Stem cells of HCC cell lines Huh7 and SNU-398 were sorted as NANOG-positive by flow cytometry. Stem cell sphere formation ability was detected. Stem cell viability, migration, invasion, and apoptosis were assessed by colony formation assays, Transwell assays, wound-healing assays, and TUNEL assays, respectively. The binding sites for SOCS2-AS1, miR-454-3p, miR-454-3p, and CPEB1 mRNA were assessed by dual-luciferase reporter assays. Quantitative real-time PCR (qPCR) and Western blot studies were performed to evaluate gene expression levels. ChIP and EMSA assays were conducted to confirm that YY1 binds with the SOCS2-AS1 promoter. A subcutaneous xenograft model was used to verify results in vivo. Tumor tissues were analyzed by H&E and TUNEL staining. RESULTS: SOCS2-AS1 was expressed at low levels in NANOG+ HCC stem cells, and HCC patients with a high level of SOCS2-AS1 expression had a higher survival rate. SOCS2-AS1 inhibited HCC cell stemness, migration, and invasion, and increased the cisplatin sensitivity of HCC cells by regulating miR-454-3p/CPEB1. YY1 was confirmed as a transcription factor of SOCS2-AS1, and served to inhibit SOCS2-AS1 transcription. YY1 knockdown suppressed HCC stemness via SOCS2-AS1. The role of SOCS2-AS1 was confirmed in a subcutaneous xenograft model, and SOCS2-AS1 overexpression enhanced the inhibitory effect of cisplatin on HCC in vivo. CONCLUSIONS: YY1-regulated lncRNA SOCS2-AS1 suppresses HCC cell stemness and progression via miR-454-3p/CPEB1.

Elevated pre-mRNA 3' end processing activity in cancer cells renders vulnerability to inhibition of cleavage and polyadenylation.

Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets CPSF-73, is the first known CPA inhibitor (CPAi) in mammalian cells. Here we show that JTE-607 perturbs gene expression through both transcriptional readthrough and alternative polyadenylation (APA). Sensitive genes are associated with features similar to those previously identified for PCF11 knockdown, underscoring a unified transcriptomic signature of CPAi. The degree of inhibition of an APA site by JTE-607 correlates with its usage level and, consistently, cells with elevated CPA activities, such as those with induced overexpression of FIP1, display greater transcriptomic disturbances when treated with JTE-607. Moreover, JTE-607 causes S phase crisis and is hence synergistic with inhibitors of DNA damage repair pathways. Together, our data reveal CPA activity and proliferation rate as determinants of CPAi-mediated cell death, raising the possibility of using CPAi as an adjunct therapy to suppress certain cancers.

An increase in polyadenylation of histone isoforms, Hist1h2ah and Hist2h3c2, is governed by 3'-UTR in de-differentiated and undifferentiated hepatocyte.

Replication-dependent histones have a stem-loop structure at the 3' end of messenger RNA (mRNA) and are stabilized by stem-loop binding protein (SLBP). Moreover, loss of SLBP and imbalance in the level of ARE (adenylate-uridylate-rich elements)-binding proteins, HuR, and BRF1 are associated with the polyadenylation of canonical histone mRNAs under different physiological conditions. Previous studies from the lab have shown increased protein levels of H2A1H and H3.2 in N-nitrosodiethylamine (NDEA)-induced hepatocellular carcinoma (HCC). In this study, we report that increase in the polyadenylation of histone mRNA contributes to increased levels of H2A1H and H3.2 in NDEA-induced HCC. The persistent exposure to carcinogen with polyadenylation of histone mRNA increases the total histone pool resulting in aneuploidy. The embryonic liver has also shown increased polyadenylated histone isoforms, Hist1h2ah and Hist2h3c2, primarily contributing to their increased protein levels. The increase in polyadenylation of histone mRNA in HCC and e15 are in coherence with the decrease in SLBP and BRF1 with an increase in HuR. Our studies in neoplastic CL38 cell line showed that direct stress on the cells induces downregulation of SLBP with enhanced histone isoform polyadenylation. Moreover, the polyadenylation is related to increase in activated MAP kinases, p38, ERK, and JNK in HCC liver tumor tissues and CL38 cells treated with arsenic. Our data suggest that SLBP degrades under stress, destabilizing the stem-loop, elongating histone isoforms mRNA with 3' polyadenylated tail with increase of HuR and decrease of BRF1. Overall, our results indicate that SLBP may play an essential part in cell proliferation, at least in persistent exposure to stress, by mediating the stabilization of histone isoforms throughout the cell cycle.

3'-end mRNA processing within apicomplexan parasites, a patchwork of classic, and unexpected players.

The 3'-end processing of mRNA is a co-transcriptional process that leads to the formation of a poly-adenosine tail on the mRNA and directly controls termination of the RNA polymerase II juggernaut. This process involves a megadalton complex composed of cleavage and polyadenylation specificity factors (CPSFs) that are able to recognize cis-sequence elements on nascent mRNA to then carry out cleavage and polyadenylation reactions. Recent structural and biochemical studies have defined the roles played by different subunits of the complex and provided a comprehensive mechanistic understanding of this machinery in yeast or metazoans. More recently, the discovery of small molecule inhibitors of CPSF function in Apicomplexa has stimulated interest in studying the specificities of this ancient eukaryotic machinery in these organisms. Although its function is conserved in Apicomplexa, the CPSF complex integrates a novel reader of the N6-methyladenosine (m6A). This feature, inherited from the plant kingdom, bridges m6A metabolism directly to 3'-end processing and by extension, to transcription termination. In this review, we will examine convergence and divergence of CPSF within the apicomplexan parasites and explore the potential of small molecule inhibition of this machinery within these organisms. This article is categorized under: RNA Processing > 3' End Processing RNA Processing > RNA Editing and Modification.

Detection of FIP1L1-PDGFRA fusion gene-positive cells in the skin lesion of a patient with hypereosinophilic syndrome.

Hypereosinophilic syndrome (HES) is a heterogeneous group of diseases, characterized by persistent hypereosinophilia and end-organ damage. The FIP1L1-PDGFRA (F/P) fusion gene is found in 3-25% of patients with HES and is an oncogenic driver of myeloid neoplasms with clonal eosinophilia. Although cutaneous symptoms are the most common type of symptom in patients who have F/P fusion gene-positive HES (F/P HES), histological reports are limited. We herein present the case of a 78-year-old man with erythematous macules and severe pruritus on his trunk and extremities. Laboratory investigations revealed marked eosinophilia and elevated serum vitamin B12. A histological examination showed massive infiltration of eosinophils and mast cells around the vessels in the upper dermis. Fluorescence in situ hybridization revealed F/P fusion genes in nuclei in the peripheral blood and the skin lesion. The patient was diagnosed with F/P HES, and showed an excellent clinical and haematological response to imatinib.

FIP1L1-PDGFRA fusion gene in T-lymphoblastic lymphoma: A case report.

BACKGROUND: T-lymphoblastic lymphoma (T-LBL) is an aggressive malignancy of T-lymphoid precursors, rarely co-occurring with myeloid/lymphoid neoplasms with eosinophilia (M/LNs-Eo), with consequent rearrangement of tyrosine kinase (TK)-related genes. The FIP1L1-PDGFRA fusion gene is the most frequent molecular abnormality seen in eosinophilia-associated myeloproliferative disorders, but is also present in acute myeloid leukemia (AML), T-lymphoblastic leukemia/lymphoma (TLL), or both simultaneously. T-LBL mainly affects children and young adults, involving lymph node, bone marrow, and thymus. It represents about 85% of all immature lymphoblastic lymphomas, whereas immature B-cell lymphomas comprise approximately 15% of all cases of LBL. CASE: In this case report, we present an example of T cell lymphoblastic lymphoma with coexistent eosinophelia, treated successfully with a tyrosine-kinase inhibitor (TKI). CONCLUSION: FIP1L1-PDGFRA-positive T-LBL and myeloproliferative disorders have excellent response to low-dose treatment with (TKI) imatinib. Most patients achieve rapid and complete hematologic and molecular remission within weeks.

The roles of CPSF6 in proliferation, apoptosis and tumorigenicity of lung adenocarcinoma.

Cleavage and polyadenylation specific factor 6 (CPSF6), a member of serine/arginine-rich protein family, is implicated in HIV-1-infection and replication. Overexpression of CPSF6 predicts poor prognostic outcomes of breast cancer. However, the expression and possible function of CPSF6 in lung adenocarcinoma (LUAD) still needs to be explored. Here, we found that CPSF6 is significantly higher expressed in tumor tissues than normal tissues in multiple cancer types. Besides, CPSF6 plays a significant risky role in LUAD that is associated with overall survival (HR=1.337, P=0.051) and disease-specific survival (HR=1.4739, P=0.042). CPSF6 mRNA was up-regulated in LUAD tissues by analyzing publicly available datasets from Gene Expression Omnibus (GEO). Further survival analysis on The Cancer Genome Atlas (TCGA) dataset suggested a close correlation between CPSF6 expression and overall survival, and disease-free survival of LUAD patients. Inhibition of CPSF6 expression by lentivirus-mediated RNA interference (RNAi) in two LUAD cell lines (A549 and NCH-H1299) caused a significant reduction in cell proliferation, colony formation and a notable induction in apoptotic rate. CPSF6 knockdown in xenograft tumors inhibited LUAD cell growth in vivo. Moreover, we identified differentially expressed genes with CPSF6 inhibition by Microarray analysis, and pathway analyses revealed that CPSF6 knockdown resulted in the dysregulation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. Collectively, our results are the first to demonstrate that CPSF6 functions as an oncoprotein by regulating cancer-related pathways in LUAD.

Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade.

Precise maintenance of PTEN dosage is crucial for tumor suppression across a wide variety of cancers. Post-transcriptional regulation of Pten heavily relies on regulatory elements encoded by its 3'UTR. We previously reported the important diversity of 3'UTR isoforms of Pten mRNAs produced through alternative polyadenylation (APA). Here, we reveal the direct regulation of Pten APA by the mammalian cleavage factor I (CFIm) complex, which in turn contributes to PTEN protein dosage. CFIm consists of the UGUA-binding CFIm25 and APA regulatory subunits CFIm59 or CFIm68. Deep sequencing analyses of perturbed (KO and KD) cell lines uncovered the differential regulation of Pten APA by CFIm59 and CFIm68 and further revealed that their divergent functions have widespread impact for APA in transcriptomes. Differentially regulated genes include numerous factors within the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway that PTEN counter-regulates. We further reveal a stratification of APA dysregulation among a subset of PTEN-driven cancers, with recurrent alterations among PI3K/Akt pathway genes regulated by CFIm. Our results refine the transcriptome selectivity of the CFIm complex in APA regulation, and the breadth of its impact in PTEN-driven cancers.

MiR-122-5p regulates the pathogenesis of childhood obesity by targeting CPEB1.

BACKGROUND: Childhood obesity is strongly associated with inflammation which contributes to the development of several obesity-related disorders. Accumulating evidence has suggested that microRNAs (miRNAs) are involved in the pathogenesis of multiple human diseases, including childhood obesity. MiR-122-5p was reported to be related to obesity in childhood, however, the detailed function and mechanism of miR-122-5p are still obscure. METHODS: Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were cocultured with macrophage cell line THP-1 or macrophage-conditioned medium (MacCM) to promote cytokine expression. Oil Red O staining was used to detect the accumulation of lipid droplets in SGBS cells. The expression of interleukin 6 (IL-6), IL-8, and monocyte chemoattractant protein 1 (MCP-1) at mRNA and protein levels was assessed by RT-qPCR and ELISA, respectively. Western blotting was used for measuring protein levels of target genes of miR-122-5p. The luciferase reporter assay was applied for detecting the binding relation between miR-122-5p and cytoplasmic polyadenylation element binding protein 1 (CPEB1). RESULTS: Coculture of SGBS adipocytes and THP-1 macrophages/MacCM promoted IL-6, IL-8, and MCP-1 expression at mRNA and protein levels. Overexpression of miR-122-5p inhibited IL-6, IL-8, and MCP-1 expression in SGBS adipocytes, and this inhibitory effect was rescued by CPEB1 upregulation. CPEB1 3'-untranslated region was directly targeted by miR-122-5p. CONCLUSION: MiR-122-5p suppresses cytokine expression in SGBS adipocytes by targeting CPEB1.

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis.

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3' UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.

Reconstitution of 3' end processing of mammalian pre-mRNA reveals a central role of RBBP6.

The 3' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

Cleavage-Polyadenylation Factor Cft1 and SPX Domain Proteins Are Agents of Inositol Pyrophosphate Toxicosis in Fission Yeast.

Inositol pyrophosphate (IPP) dynamics govern expression of the fission yeast phosphate homeostasis regulon via their effects on lncRNA-mediated transcription interference. The growth defects (ranging from sickness to lethality) elicited by fission yeast mutations that inactivate IPP pyrophosphatase enzymes are exerted via the agonistic effects of too much 1,5-IP8 on RNA 3'-processing and transcription termination. To illuminate determinants of IPP toxicosis, we conducted a genetic screen for spontaneous mutations that suppressed the sickness of Asp1 pyrophosphatase mutants. We identified a missense mutation, C823R, in the essential Cft1 subunit of the cleavage and polyadenylation factor complex that suppresses even lethal Asp1 IPP pyrophosphatase mutations, thereby fortifying the case for 3'-processing/termination as the target of IPP toxicity. The suppressor screen also identified Gde1 and Spx1 (SPAC6B12.07c), both of which have an IPP-binding SPX domain and both of which are required for lethality elicited by Asp1 mutations. A survey of other SPX proteins in the proteome identified the Vtc4 and Vtc2 subunits of the vacuolar polyphosphate polymerase as additional agents of IPP toxicosis. Gde1, Spx1, and Vtc4 contain enzymatic modules (glycerophosphodiesterase, RING finger ubiquitin ligase, and polyphosphate polymerase, respectively) fused to their IPP-sensing SPX domains. Structure-guided mutagenesis of the IPP-binding sites and the catalytic domains of Gde1 and Spx1 indicated that both modules are necessary to elicit IPP toxicity. Whereas Vtc4 polymerase catalytic activity is required for IPP toxicity, its IPP-binding site is not. Epistasis analysis, transcriptome profiling, and assays of Pho1 expression implicate Spx1 as a transducer of IP8 signaling to the 3'-processing/transcription termination machinery. IMPORTANCE Impeding the catabolism of the inositol pyrophosphate (IPP) signaling molecule IP8 is cytotoxic to fission yeast. Here, by performing a genetic suppressor screen, we identified several cellular proteins required for IPP toxicosis. Alleviation of IPP lethality by a missense mutation in the essential Cft1 subunit of the cleavage and polyadenylation factor consolidates previous evidence that toxicity results from IP8 action as an agonist of RNA 3'-processing and transcription termination. Novel findings are that IP8 toxicity depends on IPP-sensing SPX domain proteins with associated enzymatic functions: Gde1 (glycerophosphodiesterase), Spx1 (ubiquitin ligase), and Vtc2/4 (polyphosphate polymerase). The effects of Spx1 deletion on phosphate homeostasis imply a role for Spx1 in communicating an IP8-driven signal to the transcription and RNA processing apparatus.

DRAIC promotes growth of breast cancer by sponging miR-432-5p to upregulate SLBP.

Mounting evidence suggests that lncRNAs can exert functions in cancer progression in multiple manners. In recent years, competing endogenous RNA (ceRNA) has been widely reported in human cancers as a lncRNA-dominant molecular pathway. The current study aimed at proving the role of lncRNA downregulated RNA in cancer (DRAIC) in breast cancer (BRCA) progression. To be specific, qRT-PCR assay was conducted to measure the expression of DRAIC and other downstream target genes. It was uncovered that DRAIC was expressed at a high level in BRCA cells. Functional analyses, including CCK-8, colony formation, and EdU assays demonstrated that DRAIC depletion suppressed BRCA cell proliferation. In addition, cell apoptosis was promoted due to DRAIC knockdown. The inhibitory effect of DRAIC reduction on BRCA cell migration and invasion was proven by transwell assays. Mechanistically, DRAIC was confirmed to predominantly distribute in the cytoplasm and could interact with miR-432-5p. In addition, stem-loop binding protein (SLBP) was verified to be a downstream target of miR-432-5p and was positively regulated by DRAIC. Taken together, DRAIC sponged miR-432-5p to enhance SLBP expression, by which malignant behaviors of BRCA cells were promoted. Our findings may help to provide a promising therapeutic target for BRCA patients.