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.

Non-canonical isoforms of the mRNA polyadenylation factor WDR33 regulate STING-mediated immune responses.

The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two, WDR33v2 (V2) and WDR33v3 (V3), have not been studied. Here, we report that V2 and V3 are generated by alternative polyadenylation, and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly, V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING), the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-ß induction by preventing STING disulfide oligomerization but promotes autophagy, likely by recruiting WIPI2 isoforms. V3, on the other hand, functions to increase STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated, indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated.

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.

Mutations in yeast Pcf11, a conserved protein essential for mRNA 3' end processing and transcription termination, elicit the Environmental Stress Response.

The Pcf11 protein is an essential subunit of the large complex that cleaves and polyadenylates eukaryotic mRNA precursor. It has also been functionally linked to gene-looping, termination of RNA Polymerase II (Pol II) transcripts, and mRNA export. We have examined a poorly characterized but conserved domain (amino acids 142-225) of the Saccharomyces cerevisiae  Pcf11 and found that while it is not needed for mRNA 3' end processing or termination downstream of the poly(A) sites of protein-coding genes, its presence improves the interaction with Pol II and the use of transcription terminators near gene promoters. Analysis of genome-wide Pol II occupancy in cells with Pcf11 missing this region, as well as Pcf11 mutated in the Pol II CTD Interacting Domain, indicates that systematic changes in mRNA expression are mediated primarily at the level of transcription. Global expression analysis also shows that a general stress response, involving both activation and suppression of specific gene sets known to be regulated in response to a wide variety of stresses, is induced in the two pcf11 mutants, even though cells are grown in optimal conditions. The mutants also cause an unbalanced expression of cell wall-related genes that does not activate the Cell Wall Integrity pathway but is associated with strong caffeine sensitivity. Based on these findings, we propose that Pcf11 can modulate the expression level of specific functional groups of genes in ways that do not involve its well-characterized role in mRNA 3' end processing.

Molecular details of the CPSF73-CPSF100 C-terminal heterodimer and interaction with Symplekin.

Eukaryotic pre-mRNA is processed by a large multiprotein complex to accurately cleave the 3' end, and to catalyse the addition of the poly(A) tail. Within this cleavage and polyadenylation specificity factor (CPSF) machinery, the CPSF73/CPSF3 endonuclease subunit directly contacts both CPSF100/CPSF2 and the scaffold protein Symplekin to form a subcomplex known as the core cleavage complex or mammalian cleavage factor. Here we have taken advantage of a stable CPSF73-CPSF100 minimal heterodimer from Encephalitozoon cuniculi to determine the solution structure formed by the first and second C-terminal domain (CTD1 and CTD2) of both proteins. We find a large number of contacts between both proteins in the complex, and notably in the region between CTD1 and CTD2. A similarity is also observed between CTD2 and the TATA-box binding protein (TBP) domains. Separately, we have determined the structure of the terminal CTD3 domain of CPSF73, which also belongs to the TBP domain family and is connected by a flexible linker to the rest of CPSF73. Biochemical assays demonstrate a key role for the CTD3 of CPSF73 in binding Symplekin, and structural models of the trimeric complex from other species allow for comparative analysis and support an overall conserved architecture.

Premature transcription termination complex proteins PCF11 and WDR82 silence HIV-1 expression in latently infected cells.

Postintegration transcriptional silencing of HIV-1 leads to the establishment of a pool of latently infected cells. In these cells, mechanisms controlling RNA Polymerase II (RNAPII) pausing and premature transcription termination (PTT) remain to be explored. Here, we found that the cleavage and polyadenylation (CPA) factor PCF11 represses HIV-1 expression independently of the other subunits of the CPA complex or the polyadenylation signal located at the 5' LTR. We show that PCF11 interacts with the RNAPII-binding protein WDR82. Knock-down of PCF11 or WDR82 reactivated HIV-1 expression in latently infected cells. To silence HIV-1 transcription, PCF11 and WDR82 are specifically recruited at the promoter-proximal region of the provirus in an interdependent manner. Codepletion of PCF11 and WDR82 indicated that they act on the same pathway to repress HIV expression. These findings reveal PCF11/WDR82 as a PTT complex silencing HIV-1 expression in latently infected cells.

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.

A Combinatorial Code for CPEB-Mediated c-myc Repression.

During early embryonic development, the RNA-binding protein CPEB mediates cytoplasmic polyadenylation and translational activation through a combinatorial code defined by the cy-toplasmic polyadenylation element (CPE) present in maternal mRNAs. However, in non-neuronal somatic cells, CPEB accelerates deadenylation to repress translation of the target, including c-myc mRNA, through an ill-defined cis-regulatory mechanism. Using RNA mutagenesis and electrophoretic mobility shift assays, we demonstrated that a combination of tandemly arranged consensus (cCPE) and non-consensus (ncCPE) cytoplasmic polyadenylation elements (CPEs) constituted a combinatorial code for CPEB-mediated c-myc mRNA decay. CPEB binds to cCPEs with high affinity (Kd = ~250 nM), whereas it binds to ncCPEs with low affinity (Kd > ~900 nM). CPEB binding to a cCPE enhances CPEB binding to the proximal ncCPE. In contrast, while a cCPE did not activate mRNA degradation, an ncCPE was essential for the induction of degradation, and a combination of a cCPE and ncCPEs further promoted degradation. Based on these findings, we propose a model in which the high-affinity binding of CPEB to the cCPE accelerates the binding of the second CPEB to the ncCPEs, resulting in the recruitment of deadenylases, acceleration of deadenylation, and repression of c-myc mRNAs.

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.

Fission yeast poly(A) polymerase active site mutation Y86D alleviates the rad24Δ asp1-H397A synthetic growth defect and up-regulates mRNAs targeted by MTREC and Mmi1.

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA-mediated interference is alleviated by genetic perturbations that elicit precocious lncRNA 3'-processing and transcription termination, such as (i) the inositol pyrophosphate pyrophosphatase-defective asp1-H397A allele, which results in elevated levels of IP8, and (ii) absence of the 14-3-3 protein Rad24. Combining rad24Δ with asp1-H397A causes a severe synthetic growth defect. A forward genetic screen for SRA (Suppressor of Rad24 Asp1-H397A) mutations identified a novel missense mutation (Tyr86Asp) of Pla1, the essential poly(A) polymerase subunit of the fission yeast cleavage and polyadenylation factor (CPF) complex. The pla1-Y86D allele was viable but slow-growing in an otherwise wild-type background. Tyr86 is a conserved active site constituent that contacts the RNA primer 3' nt and the incoming ATP. The Y86D mutation elicits a severe catalytic defect in RNA-primed poly(A) synthesis in vitro and in binding to an RNA primer. Yet, analyses of specific mRNAs indicate that poly(A) tails in pla1-Y86D cells are not different in size than those in wild-type cells, suggesting that other RNA interactors within CPF compensate for the defects of isolated Pla1-Y86D. Transcriptome profiling of pla1-Y86D cells revealed the accumulation of multiple RNAs that are normally rapidly degraded by the nuclear exosome under the direction of the MTREC complex, with which Pla1 associates. We suggest that Pla1-Y86D is deficient in the hyperadenylation of MTREC targets that precedes their decay by the exosome.

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.

Formation of nuclear CPSF6/CPSF5 biomolecular condensates upon HIV-1 entry into the nucleus is important for productive infection.

The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection1,2. We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses.

3'UTR of mRNA Encoding CPEB Protein Orb2 Plays an Essential Role in Intracellular Transport in Neurons.

Intracellular trafficking plays a critical role in the functioning of highly polarized cells, such as neurons. Transport of mRNAs, proteins, and other molecules to synaptic terminals maintains contact between neurons and ensures the transmission of nerve impulses. Cytoplasmic polyadenylation element binding (CPEB) proteins play an essential role in long-term memory (LTM) formation by regulating local translation in synapses. Here, we show that the 3'UTR of the Drosophila CPEB gene orb2 is required for targeting the orb2 mRNA and protein to synapses and that this localization is important for LTM formation. When the orb2 3'UTR is deleted, the orb2 mRNAs and proteins fail to localize in synaptic fractions, and pronounced LTM deficits arise. We found that the phenotypic effects of the orb2 3'UTR deletion were rescued by introducing the 3'UTR from the orb, another Drosophila CPEB gene. In contrast, the phenotypic effects of the 3'UTR deletion were not rescued by the 3'UTR from one of the Drosophila α-tubulin genes. Our results show that the orb2 mRNAs must be targeted to the correct locations in neurons and that proper targeting depends upon sequences in the 3'UTR.

Alternative polyadenylation factor CPSF6 regulates temperature compensation of the mammalian circadian clock.

A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3'-end-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1).

Surveillance of 3' mRNA cleavage during transcription termination requires CF IB/Hrp1.

CF IB/Hrp1 is part of the cleavage and polyadenylation factor (CPF) and cleavage factor (CF) complex (CPF-CF), which is responsible for 3' cleavage and maturation of pre-mRNAs. Although Hrp1 supports this process, its presence is not essential for the cleavage event. Here, we show that the main function of Hrp1 in the CPF-CF complex is the nuclear mRNA quality control of proper 3' cleavage. As such, Hrp1 acts as a nuclear mRNA retention factor that hinders transcripts from leaving the nucleus until processing is completed. Only after proper 3' cleavage, which is sensed through contacting Rna14, Hrp1 recruits the export receptor Mex67, allowing nuclear export. Consequently, its absence results in the leakage of elongated mRNAs into the cytoplasm. If cleavage is defective, the presence of Hrp1 on the mRNA retains these elongated transcripts until they are eliminated by the nuclear exosome. Together, we identify Hrp1 as the key quality control factor for 3' cleavage.

CPEB and translational control by cytoplasmic polyadenylation: impact on synaptic plasticity, learning, and memory.

The late 1990s were banner years in molecular neuroscience; seminal studies demonstrated that local protein synthesis, at or near synapses, was necessary for synaptic plasticity, the underlying cellular basis of learning and memory [1, 2]. The newly made proteins were proposed to "tag" the stimulated synapse, distinguishing it from naive synapses, thereby forming a cellular memory [3]. Subsequent studies demonstrated that the transport of mRNAs from soma to dendrite was linked with translational unmasking at synapses upon synaptic stimulation. It soon became apparent that one prevalent mechanism governing these events is cytoplasmic polyadenylation, and that among the proteins that control this process, CPEB, plays a central role in synaptic plasticity, and learning and memory. In vertebrates, CPEB is a family of four proteins, all of which regulate translation in the brain, that have partially overlapping functions, but also have unique characteristics and RNA binding properties that make them control different aspects of higher cognitive function. Biochemical analysis of the vertebrate CPEBs demonstrate them to respond to different signaling pathways whose output leads to specific cellular responses. In addition, the different CPEBs, when their functions go awry, result in pathophysiological phenotypes resembling specific human neurological disorders. In this essay, we review key aspects of the vertebrate CPEB proteins and cytoplasmic polyadenylation within the context of brain function.

PATL2 regulates mRNA homeostasis in oocytes by interacting with EIF4E and CPEB1.

The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes.