RESUMEN
Human diseases are often caused by loss of somatic cells that are incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. In vivo lineage tracing revealed that 15%-20% of adult cardiomyocytes expressing the four factors underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Chemical inhibition of Tgf-ß and Wee1 made CDK1 and cyclin B dispensable. These findings reveal a discrete combination of genes that can efficiently unlock the proliferative potential in cells that have terminally exited the cell cycle.
Asunto(s)
Corazón/fisiología , Miocitos Cardíacos/metabolismo , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Citocinesis , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Infarto del Miocardio/veterinaria , Miocitos Cardíacos/citología , Cadenas Pesadas de Miosina/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Ratas , Regeneración , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.
Asunto(s)
Meiosis , Oocitos , Biosíntesis de Proteínas , Factores de Transcripción , Factores de Escisión y Poliadenilación de ARNm , Animales , Femenino , Ratones , Aurora Quinasa A/metabolismo , Aurora Quinasa A/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Oocitos/metabolismo , Oocitos/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Daño del ADN , Replicación del ADN , ADN/biosíntesis , Reordenamiento Génico , Mitosis , Proteínas Quinasas/metabolismo , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , ADN/genética , Reparación del ADN , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo , ADN Polimerasa thetaRESUMEN
Progression through the mitotic and meiotic cell cycle is driven by fluctuations in the levels of cyclins, the regulatory subunits controlling the localization and activity of CDK1 kinases. Cyclin levels are regulated through a precise balance of synthesis and degradation. Here we demonstrate that the synthesis of Cyclin B1 during the oocyte meiotic cell cycle is defined by the selective translation of mRNA variants generated through alternative cleavage and polyadenylation (APA). Using gene editing in mice, we introduced mutations into the proximal and distal polyadenylation elements of the 3' untranslated region (UTR) of the Ccnb1 mRNA. Through in vivo loss-of-function experiments, we demonstrate that the translation of mRNA with a short 3' UTR specifies Cyclin B1 protein levels that set the timing of meiotic re-entry. In contrast, translation directed by a long 3' UTR is necessary to direct Cyclin B1 protein accumulation during the MI/MII transition. These findings establish that the progression through the cell cycle is dependent on the selective translation of multiple mRNA variants generated by APA.
Asunto(s)
Ciclina B1 , Meiosis , Poliadenilación , Animales , Ratones , Regiones no Traducidas 3'/genética , Ciclo Celular/genética , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The final stages of female gamete maturation occur in the virtual absence of transcription, with gene expression driven by a program of selective unmasking, translation, and degradation of maternal mRNAs. Here we demonstrate that the timing of Ccnb1 mRNA translation in mouse oocytes is dependent on the presence of transcripts with different 3' untranslated regions (UTRs). This 3' UTR heterogeneity directs distinct temporal patterns of translational activation or repression. Inclusion or exclusion of cis-acting elements is responsible for these divergent regulations. Our findings reveal an additional layer of translation control through alternative polyadenylation usage required to fine-tune the timing of meiosis progression.
Asunto(s)
Ciclina B1/genética , Regulación del Desarrollo de la Expresión Génica , Meiosis/genética , Oocitos/crecimiento & desarrollo , ARN Mensajero/genética , Regiones no Traducidas 3'/genética , Animales , Ciclina B1/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Oocitos/citología , Poliadenilación , ARN Mensajero/metabolismoRESUMEN
The spindle checkpoint protects against aneuploidy by ensuring that dividing cells only proceed with chromosome segregation once all kinetochores are stably attached to spindle microtubules. The checkpoint protein MAD1 localizes to the corona, a structural expansion of the kinetochore forming in the absence of microtubule attachment, but molecular mechanism or functional significance of this localization remains unknown. Recent results now show that cyclin B1 recruits MAD1 to the corona and that this MAD1 pool is required for robust checkpoint signaling.
Asunto(s)
Cinetocoros , Puntos de Control de la Fase M del Ciclo Celular , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Microtúbulos , Huso Acromático/genéticaRESUMEN
Cyclin B:CDK1 is the master kinase regulator of mitosis. We show here that, in addition to its kinase functions, mammalian Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminal region of MAD1, and point mutations in this sequence abolish MAD1 corona localisation and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona, and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness arises because Cyclin B1:MAD1 localisation loses dependence on MPS1 kinase after the corona has been established, ensuring that corona-localised MAD1 can still be phosphorylated when MPS1 activity is low. Therefore, this study explains how corona-MAD1 generates a robust SAC signal, and it reveals a scaffolding role for the key mitotic kinase, Cyclin B1:CDK1, which ultimately helps to inhibit its own degradation.
Asunto(s)
Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Cinetocoros/metabolismo , Mitosis , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Células HeLa , Humanos , Mutación Puntual , Dominios ProteicosRESUMEN
Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , División Celular , Ciclina B1 , Microtúbulos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras , Ciclina B1/genética , Ciclina B1/metabolismo , Microtúbulos/metabolismo , Mitosis/genéticaRESUMEN
In this study, we investigated the role of serum/glucocorticoid-regulated kinase 1 (SGK1) in varicella-zoster virus (VZV) replication. VZV DNA replication and plaque formation were inhibited by SGK1 knockout and treatment with an SGK1 inhibitor. Furthermore, SGK1 inhibition suppressed the increase in cyclin B1 expression induced by VZV infection. These results suggest that VZV infection induces SGK1 activation, which is required for efficient viral proliferation through the expression of cyclin B1. This is the first study to report that SGK1 is involved in the VZV life cycle.
Asunto(s)
Ciclina B1 , Herpesvirus Humano 3 , Proteínas Inmediatas-Precoces , Proteínas Serina-Treonina Quinasas , Replicación Viral , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Humanos , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ciclina B1/metabolismo , Ciclina B1/genética , Línea Celular , Replicación del ADNRESUMEN
We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.
Asunto(s)
Proliferación Celular , Citocinas , Proteína con Dominio Pirina 3 de la Familia NLR , Psoriasis , Humanos , Ciclo Celular/genética , Proliferación Celular/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Citocinas/metabolismo , Silenciador del Gen , Células HaCaT , Interleucina-17/metabolismo , Interleucina-17/genética , Interleucina-23/metabolismo , Interleucina-23/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Antígeno Ki-67/metabolismo , Antígeno Ki-67/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Psoriasis/genética , Psoriasis/metabolismo , Psoriasis/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The creation of translation-competent mRNA is dependent on RNA polymerase II transcripts being modified by addition of the 7-methylguanosine (m7G) cap. The factors that mediate splicing, nuclear export, and translation initiation are recruited to the transcript via the cap. The cap structure is formed by several activities and completed by RNMT (RNA guanine-7 methyltransferase), which catalyzes N7 methylation of the cap guanosine. We report that CDK1-cyclin B1 phosphorylates the RNMT regulatory domain on T77 during G2/M phase of the cell cycle. RNMT T77 phosphorylation activates the enzyme both directly and indirectly by inhibiting interaction with KPNA2, an RNMT inhibitor. RNMT T77 phosphorylation results in elevated m7G cap methyltransferase activity at the beginning of G1 phase, coordinating mRNA capping with the burst of transcription that occurs following nuclear envelope reformation. RNMT T77 phosphorylation is required for the production of cohort of proteins, and inhibiting T77 phosphorylation reduces the cell proliferation rate.
Asunto(s)
Ciclina B1/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Fase G1 , Metiltransferasas/metabolismo , Caperuzas de ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Transcripción Genética , Proteína Quinasa CDC2 , Proliferación Celular , Ciclina B1/genética , Quinasas Ciclina-Dependientes/genética , Activación Enzimática , Fase G2 , Células HEK293 , Células HeLa , Humanos , Metilación , Metiltransferasas/genética , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Caperuzas de ARN/genética , Interferencia de ARN , ARN Mensajero/genética , Fase S , Transducción de Señal , Factores de Tiempo , Transfección , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMEN
Meox1 is a critical transcription factor that plays a pivotal role in embryogenesis and muscle development. It has been established as a marker gene for growth-specific muscle stem cells in zebrafish. In this study, we identified the SsMeox1 gene in a large teleost fish, Sebastes schlegelii. Through in situ hybridization and histological analysis, we discovered that SsMeox1 can be employed as a specific marker of growth-specific muscle stem cells, which originate from the somite stage and are primarily situated in the external cell layer (ECL) and myosepta, with a minor population distributed among muscle fibers. The knockdown of SsMeox1 resulted in a significant increase in Ccnb1 expression, subsequently promoting cell cycle progression and potentially accelerating the depletion of the stem cell pool, which ultimately led to significant growth retardation. These findings suggest that SsMeox1 arrests the cell cycle of growth-specific muscle stem cells in the G2 phase by suppressing Ccnb1 expression, which is essential for maintaining the stability of the growth-specific muscle stem cell pool. Our study provides significant insights into the molecular mechanisms underlying the indeterminate growth of large teleosts.
Asunto(s)
Proteínas de Peces , Peces , Desarrollo de Músculos , Animales , Ciclo Celular/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Desarrollo de Músculos/genética , Células Madre/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Peces/crecimiento & desarrollo , Peces/metabolismoRESUMEN
Potassium Calcium-Activated Channel Subfamily N1 (KCNN1), an integral membrane protein, is thought to regulate neuronal excitability by contributing to the slow component of synaptic after hyperpolarization. However, the role of KCNN1 in tumorigenesis has been rarely reported, and the underlying molecular mechanism remains unclear. Here, we report that KCNN1 functions as an oncogene in promoting breast cancer cell proliferation and metastasis. KCNN1 was overexpressed in breast cancer tissues and cells. The pro-proliferative and pro-metastatic effects of KCNN1 were demonstrated by CCK8, clone formation, Edu assay, wound healing assay and transwell experiments. Transcriptomic analysis using KCNN1 overexpressing cells revealed that KCNN1 could regulate key signaling pathways affecting the survival of breast cancer cells. KCNN1 interacts with ERLIN2 and enhances the effect of ERLIN2 on Cyclin B1 stability. Overexpression of KCNN1 promoted the protein expression of Cyclin B1, enhanced its stability and promoted its K63 dependent ubiquitination, while knockdown of KCNN1 had the opposite effects on Cyclin B1. Knockdown (or overexpression) ERLNI2 partially restored Cyclin B1 stability and K63 dependent ubiquitination induced by overexpression (or knockdown) of KCNN1. Knockdown (or overexpression) ERLIN2 also partially neutralizes the effects of overexpression (or knockdown) KCNN1-induced breast cancer cell proliferation, migration and invasion. In paired breast cancer clinical samples, we found a positive expression correlations between KCNN1 and ERLIN2, KCNN1 and Cyclin B1, as well as ERLIN2 and Cyclin B1. In conclusion, this study reveals, for the first time, the role of KCNN1 in tumorigenesis and emphasizes the importance of KCNN1/ERLIN2/Cyclin B1 axis in the development and metastasis of breast cancer.
Asunto(s)
Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/patología , Carcinogénesis , Línea Celular Tumoral , Proliferación Celular/genética , Ciclina B1/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Lymphovascular invasion (LVI) is regulated through complex molecular mechanisms. Cyclin B1 (CCNB1) was previously determined as being associated with LVI using large cohorts of breast cancer (BC) and artificial neural network (ANN) technique. In this study, we aimed to assess the association between CCNB1 and LVI, other clinicopathological and other LVI-related biomarkers at the molecular (RNA transcriptomic) and proteomic levels in BC. METHODS: Two transcriptomic BC cohorts (n = 2834) were used to assess the association between the expression of CCNB1 at the mRNA level and clinicopathological characteristics and patient outcome. Tissue microarrays (TMAs) from a well-characterised BC cohort (n = 2480) with long-term outcome were also used to assess the clinical significance of CCNB1 protein expression using immunohistochemistry. RESULTS: High CCNB1 mRNA expression was associated with aggressive tumour behaviour, including LVI, larger size, higher tumour grade, high lymph nodal stage, hormonal receptor negativity, HER2 positivity and poor clinical outcome (all p < 0.0001). Similarly, high CCNB1 protein expression was associated with higher tumour grade, hormonal receptor negativity and HER2 positivity (all p < 0.0001). Additionally, there was a significant association between CCNB1- and LVI-related biomarkers including N-cadherin, P-cadherin and TWIST2 at the transcriptomic and proteomic level. Multivariate analysis revealed that CCNB1 was an independent predictor of shorter BC-specific survival (HR = 1.3; 95% CI 1.2-1.5; p = 0.010). CONCLUSION: CCNB1 is a key gene associated with LVI in BC and has prognostic value. More functional studies are warranted to unravel the mechanistic role of CCNB1 in the development of LVI.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Relevancia Clínica , Ciclina B1/genética , Proteómica , Pronóstico , ARN Mensajero , Invasividad Neoplásica/genética , Invasividad Neoplásica/patologíaRESUMEN
Ring-shaped cohesin keeps sister chromatids paired until cleavage of its Scc1/Rad21 subunit by separase triggers chromosome segregation in anaphase. Vertebrate separase is held inactive by mutually exclusive binding to securin or Cdk1-cyclin B1 and becomes unleashed only upon ubiquitin-dependent degradation of these regulators. Although most separase is usually found in association with securin, this anaphase inhibitor is dispensable for murine life while Cdk1-cyclin B1-dependent control of separase is essential. Here, we show that securin-independent inhibition of separase by Cdk1-cyclin B1 in early mitosis requires the phosphorylation-specific peptidyl-prolyl cis/trans isomerase Pin1. Furthermore, isomerization of previously securin-bound separase at the metaphase-to-anaphase transition renders it resistant to re-inhibition by residual securin. At the same time, isomerization also limits the half-life of separase's proteolytic activity, explaining how cohesin can be reloaded onto telophase chromatin in the absence of securin and cyclin B1 without being cleaved.
Asunto(s)
Segregación Cromosómica/genética , Regulación Enzimológica de la Expresión Génica , Isomerasa de Peptidilprolil/genética , Separasa/genética , Anafase/genética , Proteína Quinasa CDC2 , Cromátides/genética , Ciclina B1/química , Ciclina B1/genética , Ciclina B1/metabolismo , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Células HEK293 , Humanos , Immunoblotting , Metafase/genética , Microscopía Fluorescente , Mitosis/genética , Modelos Genéticos , Modelos Moleculares , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/metabolismo , Unión Proteica , Conformación Proteica , Interferencia de ARN , Securina/genética , Securina/metabolismo , Separasa/química , Separasa/metabolismoRESUMEN
Background/aim: Hepatocellular carcinoma (HCC) is a common type of cancer. We hypothesize that circular RNA-0006091 (circ-0006091) affects the progression of HCC. The study aims to investigate the effect of circ-0006091 in HCC cells. Materials and methods: The levels of circ-0006091, microRNA-622 (miR-622), and cyclin B1 (CCNB1) were assayed using qRT-PCR and western blotting. The metastasis of the HCC cells was measured with wound healing and transwell assays. The protein expression levels of MMP-2 and MMP-9 were assayed with western blotting. Dual-luciferase reporter and RNA-pulldown assays were used to determine the link between miR-622 and circ-0006091 or CCNB1. Mice-based tests were used to determine the effect of circ-0006091 on the proliferation of HCC cells. Results: The levels of circ-0006091 and CCNB1 were increased in the HCC cells, but miR-622 was down-regulated. Deficiency of circ-0006091 reduced the metastasis of the HCC cells, and silencing of circ-0006091 decreased the activities of MMP-2 and MMP-9 in the same cells. Circ-0006091 modulated the CCNB1 level in the HCC cells via miR-622. Silencing of circ-0006091 suppressed the proliferation of the HCC cells in vivo. Conclusion: Circ-0006091 regulated HCC cell metastasis via the miR-622/CCNB1 axis, a possible therapeutic target in managing HCC.
Asunto(s)
Carcinoma Hepatocelular , Proliferación Celular , Ciclina B1 , Neoplasias Hepáticas , MicroARNs , ARN Circular , Animales , Humanos , Ratones , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , ARN Circular/metabolismoRESUMEN
Ascoviruses are double-stranded DNA viruses that are pathogenic to noctuid larvae. In vitro infection causes the cells to fail to replicate and proliferate normally. However, the molecular mechanisms are unclear. In this study, the transmission electron microscopy data of infected-Spodoptera exigua (Hübner) fat body cells (SeFB, IOZCAS-SpexII-A cells) showed that virions were internalized in phagocytic vesicles, but not in the nucleus. FACS of cell-cycle progression was performed in SeFB cells infected with Heliothis virescens ascovirus 3h (HvAV-3h). The cell cycle phase distributions of the SeFB cells were G1 = 29.52 ± 1.10%, S = 30.33 ± 1.19%, and G2 /M = 40.06 ± 0.75%. The cell culture doubling time was approximately 24 h. The G1 , S, and G2 /M phases were each approximately 8 h. The unsynchronized or synchronized cells were arrested at G2 /M phase after infection with HvAV-3h. Our data also showed that cells with more than 4N DNA content appeared in the HvAV-3h-treated group. While the mRNA levels of cyclin B1 , cyclin H, and cyclin-dependent kinase 1 (CDK1) were downregulated after HvAV-3h infection, the mRNA expression levels of cyclin A, cyclin D, and cyclin B2 were not significantly changed. Western blotting results showed that the expression of cyclin B1 and CDK1 in infected SeFB cells within 24 h postinfection (hpi), and HvAV-3h infection inhibited the expression of cyclin B1 and CDK1 at 12-24 hpi. Overall, these data implied that HvAV-3h infection leads to an accumulation of cells in the G2 /M phases by downregulating the expression of cyclin B1 and CDK1.
Asunto(s)
Ascoviridae , Ciclo Celular , Cuerpo Adiposo , Animales , Ascoviridae/patogenicidad , Proteína Quinasa CDC2/genética , División Celular , Ciclina B1/genética , Cuerpo Adiposo/citología , Cuerpo Adiposo/virología , ARN Mensajero , Spodoptera/genética , Spodoptera/virologíaRESUMEN
Oocyte meiotic maturation failure is one of the major causes for female infertility. Meiotic resumption (the G2/M transition) and progression through metaphase I (MI) are two critical stages of oocyte meiotic maturation. Here, we report that centromere protein T (CENP-T), an internal kinetochore protein, plays a critical role in meiotic resumption of mouse oocytes. Depletion of CENP-T by siRNA injection increased the CDH1 (also known as FZR1) level, resulting in increased activity of the anaphase-promoting complex (APC)-CDH1 complex, and further leading to decreased levels of the cyclin protein CCNB1, attenuated maturation-promoting factor (MPF) activity, and finally severely compromised meiotic resumption. The impaired meiotic resumption caused by CENP-T depletion could be rescued by overexpression of exogenous CCNB1 or knockdown of endogenous CDH1. Overexpression of exogenous CENP-T resulted in decreased CDH1 levels, which accelerated the progression of G2/M transition, and accelerated meiotic cell cycle progression after germinal vesicle breakdown (GVBD). Unexpectedly, spindle organization after GVBD was not affected by the overexpression, but the distribution of chromosomes was affected. Our findings reveal a novel role for CENP-T in regulating meiotic progression by acting through CDH1.
Asunto(s)
Anafase , Meiosis , Animales , Cadherinas , Ciclina B1/genética , Femenino , Mesotelina , Metafase , Ratones , OocitosRESUMEN
Temporal and spatial control of mRNA translation has emerged as a major mechanism for promoting diverse biological processes. However, the molecular nature of temporal and spatial control of translation remains unclear. In oocytes, many mRNAs are deposited as a translationally repressed form and are translated at appropriate times to promote the progression of meiosis and development. Here, we show that changes in subcellular structures and states of the RNA-binding protein pumilio 1 (Pum1) regulate the translation of target mRNAs and progression of oocyte maturation. Pum1 was shown to bind to Mad2 (also known as Mad2l1) and cyclin B1 mRNAs, assemble highly clustered aggregates, and surround Mad2 and cyclin B1 RNA granules in mouse oocytes. These Pum1 aggregates were dissolved prior to the translational activation of target mRNAs, possibly through phosphorylation. Stabilization of Pum1 aggregates prevented the translational activation of target mRNAs and progression of oocyte maturation. Together, our results provide an aggregation-dissolution model for the temporal and spatial control of translation.
Asunto(s)
Ciclina B1 , Proteínas Mad2/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/química , Animales , Ciclina B1/genética , Ciclina B1/metabolismo , Meiosis/genética , Ratones , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND & AIMS: Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that form covalently closed circles. Although circRNAs influence many biological processes, little is known about their role in intestinal epithelium homeostasis. We surveyed circRNAs required to maintain intestinal epithelial integrity and identified circular homeodomain-interacting protein kinase 3 (circHIPK3) as a major regulator of intestinal epithelial repair after acute injury. METHODS: Intestinal mucosal tissues were collected from mice exposed to cecal ligation and puncture for 48 hours and patients with inflammatory bowel diseases and sepsis. We isolated primary enterocytes from the small intestine of mice and derived intestinal organoids. The levels of circHIPK3 were silenced in intestinal epithelial cells (IECs) by transfection with small interfering RNAs targeting the circularization junction of circHIPK3 or elevated using a plasmid vector that overexpressed circHIPK3. Intestinal epithelial repair was examined in an in vitro injury model by removing part of the monolayer. The association of circHIPK3 with microRNA 29b (miR-29b) was determined by biotinylated RNA pull-down assays. RESULTS: Genome-wide profile analyses identified â¼300 circRNAs, including circHIPK3, differentially expressed in the intestinal mucosa of mice after cecal ligation and puncture relative to sham mice. Intestinal mucosa from patients with inflammatory bowel diseases and sepsis had reduced levels of circHIPK3. Increasing the levels of circHIPK3 enhanced intestinal epithelium repair after wounding, whereas circHIPK3 silencing repressed epithelial recovery. CircHIPK3 silencing also inhibited growth of IECs and intestinal organoids, and circHIPK3 overexpression promoted intestinal epithelium renewal in mice. Mechanistic studies revealed that circHIPK3 directly bound to miR-29b and inhibited miR-29 activity, thus increasing expression of Rac1, Cdc42, and cyclin B1 in IECs after wounding. CONCLUSIONS: In studies of mice, IECs, and human tissues, our results indicate that circHIPK3 improves repair of the intestinal epithelium at least in part by reducing miR-29b availability.