RESUMEN

Systemic sclerosis (SSc) is a life-threatening autoimmune disease characterized by widespread fibrosis in the skin and several internal organs. Nudix Hydrolase 21 (NUDT2 or CFIm25) downregulation in fibroblasts is known to play detrimental roles in both skin and lung fibrosis. This study aims to investigate the upstream mechanisms that lead to NUDT21 repression in skin fibrosis. We identified transforming growth factor ß (TGFß1) as the primary cytokine that downregulated NUDT21 in normal skin fibroblasts. In the bleomycin-induced dermal fibrosis model, consistent with the peak activation of TGFß1 at the late fibrotic stage, NUDT21 was downregulated at this stage, and delayed NUDT21 knockdown during this fibrotic phase led to enhanced fibrotic response to bleomycin. Further investigation suggested TGFß downregulated NUDT21 through microRNA (miRNA) 181a and 181b induction. Both miR-181a and miR-181b were elevated in bleomycin-induced skin fibrosis in mice and primary fibroblasts isolated from SSc patients, and they directly targeted NUDT21 and led to its downregulation in skin fibroblasts. Functional studies demonstrated that miR-181a and miR-181b inhibitors attenuated bleomycin-induced skin fibrosis in mice in association with decreased NUDT21 expression, while miR-181a and miR-181b mimics promoted bleomycin-induced fibrosis. Overall, these findings suggest a novel role for miR-181a/b in SSc pathogenesis by repressing NUDT21 expression.

Asunto(s)

Bleomicina , Fibroblastos , Fibrosis , MicroARNs , Esclerodermia Sistémica , Piel , MicroARNs/genética , MicroARNs/metabolismo , Animales , Humanos , Ratones , Fibrosis/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/inducido químicamente , Bleomicina/toxicidad , Bleomicina/efectos adversos , Piel/patología , Piel/metabolismo , Femenino , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Ratones Endogámicos C57BL , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Células Cultivadas , Regulación hacia Abajo

RESUMEN

OBJECTIVE: Alternative polyadenylation (APA) is a co-transcriptional process that leads to isoform diversity in the 3' ends of mRNAs. APA is known to occur during differentiation, and its dysregulation is observed in diseases like cancer and autoimmune disorders. It has been previously reported that differentiation of 3T3-L1 cells to adipocytes leads to an overall lengthening of mRNAs, but the proteins involved in this regulation have not been identified. The expression levels of subunits of the cleavage and polyadenylation (C/P) complex can regulate the choice of poly(A) site, which in turn can affect different cellular activities. In this paper, we studied the change in levels of C/P proteins during 3T3-L1 differentiation. RESULTS: We observed that while the RNA expression of these proteins is unchanged during differentiation, the protein levels of some subunits do change, including a decrease in levels of CPSF73, the nuclease that cuts at the poly(A) site. However, overexpression of CPSF73 alone does not affect the efficiency and rate of differentiation.

Asunto(s)

Células 3T3-L1 , Adipogénesis , Diferenciación Celular , Animales , Ratones , Adipogénesis/genética , Poliadenilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adipocitos/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética

RESUMEN

Under warm temperatures, plants adjust their morphologies for environmental adaption via precise gene expression regulation. However, the function and regulation of alternative polyadenylation (APA), an important fine-tuning of gene expression, remains unknown in plant thermomorphogenesis. In this study, we found that SUMOylation, a critical post-translational modification, is induced by a long-term treatment at warm temperatures via a SUMO ligase SIZ1 in Arabidopsis. Disruption of SIZ1 altered the global usage of polyadenylation signals and affected the APA dynamic of thermomorphogenesis-related genes. CPSF100, a key subunit of the CPSF complex for polyadenylation regulation, is SUMOylated by SIZ1. Importantly, we demonstrated that SUMOylation is essential for the function of CPSF100 in genome-wide polyadenylation site choice during thermomorphogenesis. Further analyses revealed that the SUMO conjugation on CPSF100 attenuates its interaction with two isoforms of its partner CPSF30, increasing the nuclear accumulation of CPSF100 for polyadenylation regulation. In summary, our study uncovers a regulatory mechanism of APA via SIZ1-mediated SUMOylation in plant thermomorphogenesis.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Factor de Especificidad de Desdoblamiento y Poliadenilación , Regulación de la Expresión Génica de las Plantas , Poliadenilación , Sumoilación , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Ligasas/metabolismo , Ligasas/genética , Vernalización

RESUMEN

INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, we find that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. Our studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes.

Asunto(s)

Núcleo Celular , Citoplasma , Proteínas de Drosophila , Unión Proteica , Humanos , Animales , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Citoplasma/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Endonucleasas/metabolismo , Endonucleasas/genética , Células HEK293 , Neurogénesis/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Dominio Catalítico

RESUMEN

The cleavage and polyadenylation specificity factor (CPSF) complex plays a central role in the formation of mRNA 3' ends, being responsible for the recognition of the poly(A) signal sequence, the endonucleolytic cleavage step, and recruitment of poly(A) polymerase. CPSF has been extensively studied for over three decades, and its functions and those of its individual subunits are becoming increasingly well-defined, with much current research focusing on the impact of these proteins on the normal functioning or disease/stress states of cells. In this review, we provide an overview of the general functions of CPSF and its subunits, followed by a discussion of how they exert their functions in a surprisingly diverse variety of biological processes and cellular conditions. These include transcription termination, small RNA processing, and R-loop prevention/resolution, as well as more generally cancer, differentiation/development, and infection/immunity.

Asunto(s)

Factor de Especificidad de Desdoblamiento y Poliadenilación , ARN Mensajero , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Poliadenilación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Terminación de la Transcripción Genética , Procesamiento de Término de ARN 3'

RESUMEN

Platinum-based chemotherapy is the standard postoperative adjuvant treatment for ovarian cancer (OC). Despite the initial response to chemotherapy, 85% of advanced OC patients will have recurrent disease. Relapsed disease and platinum resistance are the major causes of death in OC patients. In this study, we compared the global regulation of alternative polyadenylation (APA) in platinum-resistant and platinum-sensitive tissues of OC patients by analyzing a set of single-cell RNA sequencing (scRNA-seq) data from public databases and found that platinum-resistant patients exhibited global 3' untranslated region (UTR) shortening due to the different usage of polyadenylation sites (PASs). The APA regulator CSTF3 was the most significantly upregulated gene in epithelial cells of platinum-resistant OC. CSTF3 knockdown increased the sensitivity of OC cells to platinum. The lncRNA NEAT1 has two isoforms, short (NEAT1_1) and long (NEAT1_2) transcript, because of the APA processing in 3'UTR. We found that CSTF3 knockdown reduced the usage of NEAT1 proximal PAS to lengthen the transcript and facilitate the expression of NEAT1_2. Downregulation of the expression of NEAT1 (NEAT1_1/_2), but not only NEAT1_2, also increased the sensitivity of OC cells to platinum. Overexpressed NEAT1_1 reversed the platinum resistance of OC cells after knocking down CSTF3 expression. Furthermore, downregulated expression of CSTF3 and NEAT1_1, rather than NEAT1_2, was positively correlated with inactivation of the PI3K/AKT/mTOR pathway in OC cells. Together, our findings revealed a novel mechanism of APA regulation in platinum-resistant OC. CSTF3 directly bound downstream of the NEAT1 proximal PAS to generate the short isoform NEAT1_1 and was conducive to platinum resistance, which provides a potential biomarker and therapeutic strategy for platinum-resistant OC patients.

Asunto(s)

Resistencia a Antineoplásicos , Neoplasias Ováricas , Poliadenilación , ARN Largo no Codificante , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Platino (Metal)/farmacología , Platino (Metal)/uso terapéutico , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transducción de Señal

RESUMEN

Alternative polyadenylation (APA), an important post-transcriptional regulatory mechanism, is aberrantly activated in cancer，but how APA functions in tumorigenesis remains elusive. We analyzed APA events in RNA-seq data in TCGA and reported 3'UTR alterations associated with esophageal squamous cell carcinoma (ESCC) patient prognosis and gene expression changes involving loss of tumor-suppressive miRNA binding sites. Moreover, we investigated the expression and function of cleavage and polyadenylation specific factor 3 (CPSF3), a key APA regulator in ESCC. By immunohistochemistry and qRT-PCR, we found that CPSF3 was highly expressed in ESCC tissues and associated with poor patient prognosis. Overexpression of CPSF3 enhanced, while knockdown of CPSF3 inhibited ESCC cell proliferation and migration in vitro and in vivo, as determined by colony formation, transwell assays and animal experiments. Iso-Seq and RNA-seq data analysis indicated that knockdown of CPSF3 favored use of the distal poly (A) site in the 3'UTR of Cornichon family AMPA receptor auxiliary protein 2 (CNIH2), resulting in a long-3'UTR CNIH2 isoform that produced less CNIH2 protein due to miR-125a-5p targeting and downregulating CNIH2 mRNA through a miR-125a-5p binding site in the long CNIH2 mRNA 3'UTR. Moreover, CPSF3-induced ESCC tumorigenicity was mediated by CNIH2. Taken together, CPSF3 promotes ESCC progression by upregulating CNIH2 expression through loss of miR-125a-5p-mediated CNIH2 repression through alternative splicing and polyadenylation of the CNIH2 mRNA 3'UTR.

Asunto(s)

Proliferación Celular , Factor de Especificidad de Desdoblamiento y Poliadenilación , Progresión de la Enfermedad , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Regulación Neoplásica de la Expresión Génica , Poliadenilación , Animales , Femenino , Humanos , Masculino , Ratones , Regiones no Traducidas 3' , Línea Celular Tumoral , Movimiento Celular , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Pronóstico , Receptores AMPA/genética , Receptores AMPA/metabolismo

RESUMEN

The role of alternative polyadenylation of mRNA in sustaining aggressive features of tumors is quite well established, as it is responsible for the 3'UTR shortening of oncogenes and subsequent relief from miRNA-mediated repression observed in cancer cells. However, the information regarding the vulnerability of cancer cells to the inhibition of cleavage and polyadenylation (CPA) machinery is very scattered. Only few recent reports show the antitumor activity of pharmacological inhibitors of CPSF3, one among CPA factors. More in general, the fact that deregulated CPA can be seen as a new hallmark of cancer and as a potential reservoir of novel therapeutic targets has never been formalized. Here, to extend our view on the potential of CPA inhibition (CPAi) approaches as anticancer therapies, we systematically tested the fitness of about one thousand cell lines of different cancer types upon depletion of all known CPA factors by interrogating genome-scale CRISPR and RNAi dependency maps of the DepMap project. Our analysis confirmed core and accessory CPA factors as novel vulnerabilities for human cancer, thus highlighting the potential of CPAi as anticancer therapy. Among all, CPSF1 appeared as a promising actionable candidate for drug development, as it showed low dependency scores pancancer and particularly in highly proliferating cells. In a personalized medicine perspective, the observed differential vulnerability of cancer cell lines to selected CPA factors may be used to build up signatures to predict response of individual human tumors to CPAi approaches.

Asunto(s)

Neoplasias , Poliadenilación , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Línea Celular Tumoral , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Regulación Neoplásica de la Expresión Génica

RESUMEN

3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.

Asunto(s)

Microscopía por Crioelectrón , Polinucleotido Adenililtransferasa , Factores de Escisión y Poliadenilación de ARNm , Humanos , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/genética , Polinucleotido Adenililtransferasa/metabolismo , Polinucleotido Adenililtransferasa/genética , Polinucleotido Adenililtransferasa/química , Unión Proteica , Poliadenilación , Modelos Moleculares , Precursores del ARN/metabolismo , Precursores del ARN/genética , Precursores del ARN/química , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/química

RESUMEN

Background: Cleavage factor Im25 (CFIm25) regulates cell function by affecting mRNA editing processes and plays diverse roles in various diseases. Studies have found that peripheral blood monocytes are valuable in diagnosing and prognosing coronary atherosclerosis. However, no studies have examined the predictive value of CFIm25 expression in peripheral blood monocytes for coronary atherosclerosis. Methods and Results: We collected the coronary angiography results of 267 patients and calculated the Gensini score to evaluate their degree of coronary atherosclerosis. We isolated peripheral blood monocytes and detected CFIm25 RNA expression. Based on their Gensini score, we divided the patients into negative (0, n = 46), mild lesion (≤ 8, n = 71), moderate lesion (8-23, n = 76), and severe lesion (≥ 23, n = 74) groups. Results showed that CFIm25 expression correlated negatively with the Gensini score and the number of involved coronary vessels. Univariate and multivariate binary logistic regression analyses showed that CFIm25 expression in peripheral blood monocytes was a protective factor for severe lesions, ≥ 50% stenosis, and three-vessel lesions. The areas under the receiver operating characteristic curve of CFIm25 expression for predicting lesions, severe lesions, ≥50% stenosis, and three-vessel lesions were 0.743, 0.735, 0.791, and 0.736, respectively. Conclusions: CFIm25 expression in peripheral blood monocytes correlates negatively with the degree of coronary atherosclerosis and helps predict the severity and number of coronary artery lesions.

Asunto(s)

Factor de Especificidad de Desdoblamiento y Poliadenilación , Enfermedad de la Arteria Coronaria , Humanos , Constricción Patológica , Angiografía Coronaria , Monocitos , Índice de Severidad de la Enfermedad , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética

RESUMEN

BACKGROUND: NUDT21 (Nudix Hydrolase 21) has been shown to play an essential role in multiple biological processes. Pancreatic adenocarcinoma (PAAD) is one of the most fatal cancers in the world. However, the biological function of NUDT21 in PAAD remains rarely understood. The aim of this research was to identify the prediction value of NUDT21 in diagnosis, prognosis, immune infiltration, and signal pathway in PAAD. METHODS: Combined with the data in online databases, we analyzed the expression, immune infiltration, function enrichment, signal pathway, diagnosis, and prognosis of NUDT21 in PAAD. Then, the biological function of NUDT21 and its interacted protein in PAAD was identified through plasmid transduction system and protein mass spectrometry. Expression of NUDT21 was further verified in clinical specimens by immunofluorescence. RESULTS: We found that NUDT21 was upregulated in PAAD tissues and was significantly associated with the diagnosis and prognosis of pancreatic cancer through bioinformatic data analysis. We also found that overexpression of NUDT21 enhanced PAAD cells proliferation and migration, whereas knockdown NUDT21 restored the effects through in vitro experiment. Moreover, NDUFS2 was recognized as a potential target of NUDT21.We further verified that the expression of NDUFS2 was positively correlated with NUDT21 in PAAD clinical specimens. Mechanically, we found that NUDT21 stabilizes NDUFS2 and activates the PI3K-AKT signaling pathway. CONCLUSION: Our investigation reveals that NUDT21 is a previously unrecognized oncogenic factor in the diagnosis, prognosis, and treatment target of PAAD, and we suggest that NUDT21 might be a novel therapeutic target in PAAD.

Asunto(s)

Adenocarcinoma , Factor de Especificidad de Desdoblamiento y Poliadenilación , NADH Deshidrogenasa , Neoplasias Pancreáticas , Humanos , Adenocarcinoma/genética , Proliferación Celular , NADH Deshidrogenasa/genética , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética

RESUMEN

Alternative polyadenylation (APA) plays a major role in controlling transcriptome diversity and therapeutic resistance of cancers. However, long non-coding RNAs (lncRNAs) involved in pathological APA remain poorly defined. Here, we functionally characterize LINC00921, a MED13L/P300-induced oncogenic lncRNA, and show that it is required for global regulation of APA in non-small cell lung cancer (NSCLC). LINC00921 shows significant potential for reducing NSCLC radiosensitivity, and high LINC00921 levels are associated with a poor prognosis for patients with NSCLC treated with radiotherapy. LINC00921 controls NUDT21 stability by facilitating binding of NUDT21 with the E3 ligase TRIP12. LINC00921-induced destabilization of NUDT21 promotes 3' UTR shortening of MED23 mRNA via APA, which, in turn, leads to elevated MED23 protein levels in cancer cells and nuclear translocation of ß-catenin and thereby activates expression of multiple ß-catenin/T cell factor (TCF)/lymphoid enhancer-binding factor (LEF)-regulated core oncogenes (c-Myc, CCND1, and BMP4). These findings highlight the importance of functionally annotating lncRNAs controlling APA and suggest the clinical potential of therapeutics for advanced NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , ARN Largo no Codificante , Humanos , Regiones no Traducidas 3' , beta Catenina/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Proteínas Portadoras/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/metabolismo , Poliadenilación , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

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.

Asunto(s)

Factor de Especificidad de Desdoblamiento y Poliadenilación , Encephalitozoon cuniculi , Factores de Escisión y Poliadenilación de ARNm , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética

RESUMEN

Transcriptional dysregulation is a recurring pathogenic hallmark and an emerging therapeutic vulnerability in ovarian cancer. Here, we demonstrated that ovarian cancer exhibited a unique dependency on the regulatory machinery of transcriptional termination, particularly, cleavage and polyadenylation specificity factor (CPSF) complex. Genetic abrogation of multiple CPSF subunits substantially hampered neoplastic cell viability, and we presented evidence that their indispensable roles converged on the endonuclease CPSF3. Mechanistically, CPSF perturbation resulted in lengthened 3'-untranslated regions, diminished intronic polyadenylation and widespread transcriptional readthrough, and consequently suppressed oncogenic pathways. Furthermore, we reported the development of specific CPSF3 inhibitors building upon the benzoxaborole scaffold, which exerted potent antitumor activity. Notably, CPSF3 blockade effectively exacerbated genomic instability by down-regulating DNA damage repair genes and thus acted in synergy with poly(adenosine 5'-diphosphate-ribose) polymerase inhibition. These findings establish CPSF3-dependent transcriptional termination as an exploitable driving mechanism of ovarian cancer and provide a promising class of boron-containing compounds for targeting transcription-addicted human malignancies.

Asunto(s)

Recurrencia Local de Neoplasia , Neoplasias Ováricas , Femenino , Humanos , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética

RESUMEN

Alternative polyadenylation (APA), a posttranscriptional mechanism of gene expression via determination of 3'UTR length, has an emerging role in carcinogenesis. Although abundant APA reprogramming is found in kidney renal clear cell carcinoma (KIRC), which is one of the major malignancies, whether APA functions in KIRC remains unknown. Herein, we found that chromatin modifier MORC2 gained oncogenic potential in KIRC among the genes with APA reprogramming, and moreover, its oncogenic potential was enhanced by 3'UTR shortening through stabilization of MORC2 mRNA. MORC2 was found to function in KIRC by downregulating tumor suppressor DAPK1 via DNA methylation. Mechanistically, MORC2 recruited DNMT3A to facilitate hypermethylation of the DAPK1 promoter, which was strengthened by 3'UTR shortening of MORC2. Furthermore, loss of APA regulator NUDT21, which was induced by DNMT3B-mediated promoter methylation, was identified as responsible for 3'UTR shortening of MORC2 in KIRC. Additionally, NUDT21 was confirmed to act as a tumor suppressor mainly depending on downregulation of MORC2. Finally, we designed an antisense oligonucleotide (ASO) to enhance NUDT21 expression and validated its antitumor effect in vivo and in vitro. This study uncovers the DNMT3B/NUDT21/APA/MORC2/DAPK1 regulatory axis in KIRC, disclosing the role of APA in KIRC and the crosstalk between DNA methylation and APA.

Asunto(s)

Carcinoma de Células Renales , Factor de Especificidad de Desdoblamiento y Poliadenilación , Neoplasias Renales , Factores de Transcripción , Humanos , Regiones no Traducidas 3' , Carcinogénesis/genética , Carcinoma de Células Renales/genética , Metilación de ADN , Neoplasias Renales/genética , Poliadenilación , Factores de Transcripción/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética

RESUMEN

Although early diagnosis and therapeutic advances have transformed the living quality and outcome of cancer patients, the poor prognosis for metastatic patients has not been significantly improved. Mechanisms underlying the complexity of metastasis cannot be simply determined by the straightforward 'cause-and-effect relationships'. We have developed a 'dry-lab-driven knowledge discovery and wet-lab validation' approach to embrace the complexity of cancer and metastasis. We have revealed for the first time that polymetastatic (POL) melanoma cells can utilize both the secretory protein pathway (S100A11-Sec23a) and the exosomal crosstalk (miR-487a-5p) to transfer their 'polymetastatic competency' to the oligometastatic (OL) melanoma cells, via synergistic co-targeting of the tumor-suppressor Nudt21. The downstream deregulated glycolysis was verified to regulate metastatic colonization efficiency. Further, two gene sets conferring independent prognosis in melanoma were identified, which have the potential for clinical translation and merit future clinical validation.

Asunto(s)

Exosomas , Melanoma , MicroARNs , Humanos , Melanoma/patología , MicroARNs/genética , MicroARNs/metabolismo , Transporte Biológico , Exosomas/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas S100/genética , Proteínas S100/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo

RESUMEN

Cleavage and polyadenylation specificity factor (CPSF) is a protein complex that plays an essential biochemical role in mRNA 3'-end formation, including poly(A) signal recognition and cleavage at the poly(A) site. However, its biological functions at the organismal level are mostly unknown in multicellular eukaryotes. The study of plant CPSF73 has been hampered by the lethality of Arabidopsis (Arabidopsis thaliana) homozygous mutants of AtCPSF73-I and AtCPSF73-II. Here, we used poly(A) tag sequencing to investigate the roles of AtCPSF73-I and AtCPSF73-II in Arabidopsis treated with AN3661, an antimalarial drug with specificity for parasite CPSF73 that is homologous to plant CPSF73. Direct seed germination on an AN3661-containing medium was lethal; however, 7-d-old seedlings treated with AN3661 survived. AN3661 targeted AtCPSF73-I and AtCPSF73-II, inhibiting growth through coordinating gene expression and poly(A) site choice. Functional enrichment analysis revealed that the accumulation of ethylene and auxin jointly inhibited primary root growth. AN3661 affected poly(A) signal recognition, resulted in lower U-rich signal usage, caused transcriptional readthrough, and increased the distal poly(A) site usage. Many microRNA targets were found in the 3' untranslated region lengthened transcripts; these miRNAs may indirectly regulate the expression of these targets. Overall, this work demonstrates that AtCPSF73 plays important part in co-transcriptional regulation, affecting growth, and development in Arabidopsis.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transcripción Genética , Regulación de la Expresión Génica , Plantas/metabolismo , Poliadenilación/genética

RESUMEN

The initial pre-mRNA transcript in eukaryotes is processed by a large multi-protein complex in order to correctly cleave the 3' end, and to subsequently add the polyadenosine tail. This cleavage and polyadenylation specificity factor (CPSF) is composed of separate subunits, with structural information available for both isolated subunits and also larger assembled complexes. Nevertheless, certain key components of CPSF still lack high-resolution atomic data. One such region is the heterodimer formed between the first and second C-terminal domains of the endonuclease CPSF73, with those from the catalytically inactive CPSF100. Here we report the backbone and sidechain resonance assignments of a minimal C-terminal heterodimer of CPSF73-CPSF100 derived from the parasite Encephalitozoon cuniculi. The assignment process used several amino-acid specific labeling strategies, and the chemical shift values allow for secondary structure prediction.

Asunto(s)

Factor de Especificidad de Desdoblamiento y Poliadenilación , Procesamiento de Término de ARN 3' , Resonancia Magnética Nuclear Biomolecular , Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Precursores del ARN/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética

RESUMEN

Alternative polyadenylation (APA) gives rise to transcripts with distinct 3' untranslated regions (3' UTRs), thereby affecting the fate of mRNAs. APA is strongly associated with cell proliferation and differentiation status, and thus likely plays a critical role in the embryo development. However, the pattern of APA in mammalian early embryos is still unknown. Here, we analyzed the 3' UTR lengths in human and mouse pre-implantation embryos using available single cell RNA-seq datasets and explored the underlying mechanism driving the changes. Although human and mouse early embryos displayed distinct patterns of 3' UTR changing, RNA metabolism pathways were involved in both species. The 3' UTR lengths are likely determined by the abundance of the cleavage factor I complex (CFIm) components NUDT21 and CPSF6 in the nucleus. Importantly, depletion of either component resulted in early embryo development arrest and 3' UTR shortening. Collectively, these data highlight an essential role for APA in the development of mammalian early embryos.

Asunto(s)

Mamíferos , Poliadenilación , Humanos , Ratones , Animales , Regiones no Traducidas 3' , ARN Mensajero/genética , ARN Mensajero/metabolismo , Diferenciación Celular , Proliferación Celular , Mamíferos/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo

RESUMEN

The mammalian cleavage factor I subunit CFIm25 (NUDT21) binds to the UGUA sequences of precursor RNAs. Traditionally, CFIm25 is known to facilitate 3' end formation of pre-mRNAs resulting in the formation of polyadenylated transcripts. Recent studies suggest that CFIm25 may be involved in the cyclization and hence generation of circular RNAs (circRNAs) that contain UGUA motifs. These circRNAs act as competing endogenous RNAs (ceRNAs) that disrupt the ceRNA-miRNA-mRNA axis. Other emerging roles of CFIm25 include regulating both alternative splicing and alternative polyadenylation (APA). APA generates different sized transcripts that may code for different proteins, or more commonly transcripts that code for the same protein but differ in the length and sequence content of their 3' UTRs (3' UTR-APA). CFIm25 mediated global changes in 3' UTR-APA affect human physiology including spermatogenesis and the determination of cell fate. Deregulation of CFIm25 and changes in 3' UTR-APA have been implicated in several human diseases including cancer. In many cancers, CFIm25 acts as a tumor suppressor. However, there are some cancers where CFIm25 has the opposite effect. Alterations in CFIm25-driven 3' UTR-APA may also play a role in neural dysfunction and fibrosis. CFIm25 mediated 3' UTR-APA changes can be used to generate specific signatures that can be used as potential biomarkers in development and disease. Due to the emerging role of CFIm25 as a regulator of the aforementioned RNA processing events, modulation of CFIm25 levels may be a novel viable therapeutic approach. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Disease.

Asunto(s)

Factor de Especificidad de Desdoblamiento y Poliadenilación , Neoplasias , Animales , Humanos , Masculino , Regiones no Traducidas 3' , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Neoplasias/genética , Poliadenilación , ARN Circular/genética , ARN Circular/metabolismo