RESUMEN

Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only a limited number of examples have been reported to date. Here, we show that desmethyl desamino pateamine A (DMDA-PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, onto GNG motifs. By systematically comparing multiple eIF4A inhibitors by ribosome profiling, we found that DMDA-PatA has unique mRNA selectivity for translation repression. Unbiased Bind-n-Seq reveals that DMDA-PatA-targeted eIF4A exhibits a preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. A combination of classical molecular dynamics simulations and quantum chemical calculations, and the subsequent development of an inactive DMDA-PatA derivative reveals that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified that DDX3, another DEAD-box protein, is an alternative DMDA-PatA target with the same effects on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and the mRNA-selective inhibition of protein synthesis by small-molecule compounds.

Asunto(s)

ARN Helicasas DEAD-box , Factor 4A Eucariótico de Iniciación , Biosíntesis de Proteínas , ARN Mensajero , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Simulación de Dinámica Molecular , Ribosomas/metabolismo , Motivos de Nucleótidos , Unión Proteica , Células HEK293 , Compuestos Epoxi , Tiazoles , Macrólidos

Asunto(s)

ARN Helicasas DEAD-box , Semillas , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Semillas/crecimiento & desarrollo , Semillas/genética , Semillas/fisiología , Óvulo Vegetal/genética , Óvulo Vegetal/crecimiento & desarrollo , Óvulo Vegetal/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Fertilidad/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética

RESUMEN

GC (Gastric cancer) is one of the most common malignant tumours, with over 95% of gastric cancer patients being adenocarcinoma and most gastric cancer patients having no apparent symptoms in the early stages. Finding biomarkers for early screening of gastric cancer and exploring new targets for gastric cancer treatment are urgent problems to be solved in the treatment of gastric cancer, with significant clinical outcomes for the survival rate of gastric cancer patients. The AAA+ family ATPase thyroid hormone receptor-interacting protein 13 (TRIP13) has been reported to play an essential role in developing various tumours. However, the biological function and molecular mechanism of TRIP13 in gastric cancer remain unclear. This study confirms that TRIP13 is highly expressed in gastric cancer tissue samples and that TRIP13 participates in the proliferation, migration, invasion in vitro, and tumourigenesis and metastasis in vivo of gastric cancer cells. Mechanistically, this study confirms that TRIP13 directly interacts with DDX21 and stabilises its expression by restraining its ubiquitination degradation, thereby promoting gastric cancer progression. Additionally, histone deacetylase 1 (HDAC1) is an upstream factor of TRIP13, which could target the TRIP13 promoter region to promote the proliferation, migration, and invasion of gastric cancer cells. These results indicate that TRIP13 serve is a promising biomarker for the treating of gastric cancer patients, and the HDAC1-TRIP13/DDX21 axis might provide a solid theoretical basis for clinical treatment of gastric cancer patients.

Asunto(s)

ATPasas Asociadas con Actividades Celulares Diversas , Movimiento Celular , Proliferación Celular , ARN Helicasas DEAD-box , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Neoplasias Gástricas , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 1/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Ubiquitinación

RESUMEN

piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.

Asunto(s)

Elementos Transponibles de ADN , Infertilidad Masculina , ARN Interferente Pequeño , Espermatogénesis , Testículo , Masculino , Humanos , Espermatogénesis/genética , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/genética , Elementos Transponibles de ADN/genética , Animales , Testículo/metabolismo , Ratones , Adulto , Silenciador del Gen , Ratones Noqueados , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Elementos de Nucleótido Esparcido Largo/genética , Espermatogonias/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN de Interacción con Piwi

RESUMEN

In advanced hepatocellular carcinoma (HCC), RNA helicase DDX5 regulates the Wnt/ß-catenin-ferroptosis axis, influencing the efficacy of the multi-tyrosine kinase inhibitor (mTKI) sorafenib. DDX5 inhibits Wnt/ß-catenin signaling, preventing sorafenib-induced ferroptosis escape. Sorafenib/mTKIs reduce DDX5 expression, correlating with poor patient survival post-sorafenib treatment. Notably, DDX5-knockout in HCC cells activates Wnt/ß-catenin signaling persistently. Herein, we investigate the mechanistic impact of Wnt/ß-catenin activation resulting from DDX5 downregulation in the progression and treatment of HCC. RNAseq analyses identified shared genes repressed by DDX5 and upregulated by sorafenib, including Wnt signaling genes, NF-κB-inducing kinase (NIK) essential for non-canonical NF-κB (p52/RelB) activation, and cytoprotective transcription factor NRF2. We demonstrate, Wnt/ß-catenin activation induced NIK transcription, leading to non-canonical NF-κB activation, which subsequently mediated NRF2 transcription. Additionally, DDX5 deficiency extended NRF2 protein half-life by inactivating KEAP1 through p62/SQSTM1 stabilization. In a preclinical HCC mouse model, NRF2 knockdown or DDX5 overexpression restricted tumor growth upon sorafenib treatment, via induction of ferroptosis. Importantly, DDX5-knockout HCC cells exhibited elevated expression of Wnt signaling genes, NIK, p52/RelB, and NRF2-regulated genes, regardless of sorafenib treatment. Transcriptomic analyses of HCCs from TCGA and the Stelic Animal Model (STAM) of non-alcoholic steatohepatitis revealed elevated expression of these interconnected pathways in the context of DDX5 downregulation. In conclusion, DDX5 deficiency triggers Wnt/ß-catenin signaling, promoting p52/RelB and NRF2 activation, thereby enabling ferroptosis evasion upon sorafenib treatment. Similarly, independent of sorafenib, DDX5 deficiency in liver tumors enhances activation and gene expression of these interconnected pathways, underscoring the clinical relevance of DDX5 deficiency in HCC progression and therapeutic response.

Asunto(s)

Carcinoma Hepatocelular , ARN Helicasas DEAD-box , Progresión de la Enfermedad , Neoplasias Hepáticas , Factor 2 Relacionado con NF-E2 , FN-kappa B , Sorafenib , Sorafenib/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Animales , Humanos , Ratones , FN-kappa B/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Línea Celular Tumoral , Vía de Señalización Wnt/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Ferroptosis/genética

RESUMEN

DEAD-box RNA helicase 4 (DDX4) is posited to be a key maternal germ cell factor regulating avian germ cell formation. We herein showed that the DDX4 gene product of zygotic genome activation associated with the nuclear localization of the cyclin D1 protein in presumptive primordial germ cells (PGCs) plays an essential role in the proliferation of PGCs using a CRISPR/Cas9 system approach combined with in vitro fertilization techniques in Japanese quail. A proteome analysis also revealed molecular-based differences in the features of early male and female PGCs.

Asunto(s)

Coturnix , ARN Helicasas DEAD-box , Células Germinativas , Animales , Masculino , Femenino , Células Germinativas/fisiología , Células Germinativas/citología , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Caracteres Sexuales , Proliferación Celular/fisiología , Sistemas CRISPR-Cas

RESUMEN

Colorectal cancer (CRC) remains a highly prevalent gastrointestinal neoplasm, presenting significant prevalence and lethality rate. DEAD/H box RNA helicase 10 (DDX10) has been proposed as a potential oncogene in CRC, the specific action mechanism by which DDX10 modulates the aggressive biological cellular events in CRC remains implicitly elucidated, however. During this study, DDX10 expression was detected via RT-qPCR and Western blotting. Cell proliferation was estimated via EDU staining. TUNEL staining and Western blotting appraised cell apoptosis. Cell stemness was evaluated by sphere formation assay, RT-qPCR, Western blotting as well as immunofluorescence staining. Relevant assay kit examined aldehyde dehydrogenase (ALDH) activity. Western blotting and immunofluorescence staining also detected autophagy. DDX10 was hyper-expressed in CRC cells. Down-regulation of DDX10 hampered cell proliferation, aggravated the apoptosis while eliminated the ability to form spheroid cells in CRC. In addition, DDX10 deletion improved ATG10 expression and therefore activated autophagy in CRC cells. Consequently, ATG10 depletion or treatment with autophagy inhibitor 3-Methyladenine (3-MA) partially compensated the influences of DDX10 silencing on the proliferation, apoptosis and stemness of CRC cells. Accordingly, DDX10 deficiency may aggravate autophagy mediated by ATG10 to impede cell proliferation, stemness and facilitate cell apoptosis, hence blocking the progression of CRC.

Asunto(s)

Apoptosis , Proteínas Relacionadas con la Autofagia , Autofagia , Proliferación Celular , Neoplasias Colorrectales , ARN Helicasas DEAD-box , Células Madre Neoplásicas , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Autofagia/fisiología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismo , Línea Celular Tumoral , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Ratones , Animales , Proteínas de Transporte Vesicular

RESUMEN

Pacific abalone (Haliotis discus hannai) is a marine gastropod mollusc with significant economic importance in both global fisheries and aquaculture. However, studies exploring the gonadal development and regulatory mechanisms of Haliotis discus hannai are limited. This study aimed to explore whether the vasa gene acted as a molecular marker for germ cells. Initially, the vasa gene was successfully cloned using the cDNA-end rapid amplification technique. The cloned gene had a 2478-bp-long open reading frame and encoded 825 amino acids. Then, a recombinant expression vector was constructed based on the Vasa protein, and an 87-kDa recombinant protein was prepared. Subsequently, a polyclonal antibody was prepared using the purified recombinant protein. The enzyme-linked immunosorbent assay (ELISA) confirmed the titer of the antibody to be ≥512 K. The immunohistochemical analysis revealed that Vasa was widely expressed in oogonia, Stage I oocytes, spermatogonia, and primary spermatocytes. The specific expression of Vasa in the hermaphroditic gonads of abalone was assessed using western blotting to investigate the effects of different photoperiods (12 L:12D, 24 L:0D, 18 L:6D, and 6 L:18D) on the gonadal development of abalone (P < 0.05), with higher expression levels observed in the ovarian proliferative and spermary maturing stages compared with other developmental stages (P < 0.05). Additionally, Vasa exhibited the highest expression in the spermary and ovary under a photoperiod of 18 L:6D (P < 0.05). These data demonstrated the key role of Vasa in developing germ cells in abalone. They shed light upon the molecular mechanism through which the photoperiod influenced Vasa expression and regulated gonadal development in abalone. The findings might provide theoretical references for analyzing the differentiation pattern of abalone germ cells and the genetic improvement and conservation of germplasm resources.

Asunto(s)

ARN Helicasas DEAD-box , Gastrópodos , Animales , Femenino , Masculino , Secuencia de Aminoácidos , Clonación Molecular/métodos , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Gametogénesis/genética , Gastrópodos/genética , Gónadas/metabolismo , Fotoperiodo

RESUMEN

DEAD-box helicases are multifunctional proteins participating in many aspects of cellular RNA metabolism. DEAD-box helicase 41 (DDX41) in particular has pivotal roles in innate immune sensing and hematopoietic homeostasis. DDX41 recognizes foreign or self-nucleic acids generated during microbial infection, thereby initiating anti-pathogen responses. DDX41 also binds to RNA (R)-loops, structures consisting of DNA/RNA hybrids and a displaced strand of DNA that occur during transcription, thereby maintaining genome stability by preventing their accumulation. DDX41 deficiency leads to increased R-loop levels, resulting in inflammatory responses that likely influence hematopoietic stem and progenitor cell production and development. Beyond nucleic acid binding, DDX41 associates with proteins involved in RNA splicing as well as cellular proteins involved in innate immunity. DDX41 is also a tumor suppressor in familial and sporadic myelodysplastic syndrome/acute myelogenous leukemia (MDS/AML). In the present review, we summarize the functions of DDX helicases in critical biological processes, particularly focusing on DDX41's association with cellular molecules and the mechanisms underlying its roles in innate immunity, hematopoiesis and the development of myeloid malignancies.

Asunto(s)

ARN Helicasas DEAD-box , Hematopoyesis , Inmunidad Innata , Humanos , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , Hematopoyesis/inmunología , Animales

RESUMEN

Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.

Asunto(s)

Ciclooxigenasa 1 , Complejo IV de Transporte de Electrones , Inflamación , Hígado , Fosforilación Oxidativa , Especies Reactivas de Oxígeno , Animales , Femenino , Humanos , Masculino , Ratones , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Complejo IV de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/genética , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , Hígado/metabolismo , Hígado/patología , Proteínas de la Membrana , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mutación , Biosíntesis de Proteínas , Especies Reactivas de Oxígeno/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo

RESUMEN

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe disease with substantial economic consequences for the swine industry. The DEAD-box helicase 3 (DDX3X) is an RNA helicase that plays a crucial role in regulating RNA metabolism, immunological response, and even RNA virus infection. However, it is unclear whether it contributes to PRRSV infection. Recent studies have found that the expression of DDX3X considerably increases in Marc-145 cells when infected with live PRRSV strains Ch-1R and SD16; however, it was observed that inactivated viruses did not lead to any changes. By using the RK-33 inhibitor or DDX3X-specific siRNAs to reduce DDX3X expression, there was a significant decrease in the production of PRRSV progenies. In contrast, the overexpression of DDX3X in host cells substantially increased the proliferation of PRRSV. A combination of transcriptomics and metabolomics investigations revealed that in PRRSV-infected cells, DDX3X gene silencing severely affected biological processes such as ferroptosis, the FoxO signalling pathway, and glutathione metabolism. The subsequent transmission electron microscopy (TEM) imaging displayed the typical ferroptosis features in PRRSV-infected cells, such as mitochondrial shrinkage, reduction or disappearance of mitochondrial cristae, and cytoplasmic membrane rupture. Conversely, the mitochondrial morphology was unchanged in DDX3X-inhibited cells. Furthermore, silencing of the DDX3X gene changed the expression of ferroptosis-related genes and inhibited the virus proliferation, while the drug-induced ferroptosis inversely promoted PRRSV replication. In summary, these results present an updated perspective of how PRRSV infection uses DDX3X for self-replication, potentially leading to ferroptosis via various mechanisms that promote PRRSV replication.

Asunto(s)

ARN Helicasas DEAD-box , Ferroptosis , Virus del Síndrome Respiratorio y Reproductivo Porcino , Replicación Viral , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Animales , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Ferroptosis/fisiología , Porcinos , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Línea Celular

RESUMEN

mRNA biogenesis in the eukaryotic nucleus is a highly complex process. The numerous RNA processing steps are tightly coordinated to ensure that only fully processed transcripts are released from chromatin for export from the nucleus. Here, we present the hypothesis that fission yeast Dbp2, a ribonucleoprotein complex (RNP) remodelling ATPase of the DEAD-box family, is the key enzyme in an RNP assembly checkpoint at the 3'-end of genes. We show that Dbp2 interacts with the cleavage and polyadenylation complex (CPAC) and localises to cleavage bodies, which are enriched for 3'-end processing factors and proteins involved in nuclear RNA surveillance. Upon loss of Dbp2, 3'-processed, polyadenylated RNAs accumulate on chromatin and in cleavage bodies, and CPAC components are depleted from the soluble pool. Under these conditions, cells display an increased likelihood to skip polyadenylation sites and a delayed transcription termination, suggesting that levels of free CPAC components are insufficient to maintain normal levels of 3'-end processing. Our data support a model in which Dbp2 is the active component of an mRNP remodelling checkpoint that licenses RNA export and is coupled to CPAC release.

Asunto(s)

ARN Helicasas DEAD-box , Ribonucleoproteínas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Poliadenilación , ARN Mensajero/metabolismo , ARN Mensajero/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Cromatina/metabolismo , ARN de Hongos/metabolismo , ARN de Hongos/genética , Núcleo Celular/metabolismo

RESUMEN

Though RNAi and RNA-splicing machineries are involved in regulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, their precise roles in coronavirus disease 2019 (COVID-19) pathogenesis remain unclear. Herein, we show that decreased RNAi component (Dicer and XPO5) and splicing factor (SRSF3 and hnRNPA3) expression correlate with increased COVID-19 severity. SARS-CoV-2 N protein induces the autophagic degradation of Dicer, XPO5, SRSF3, and hnRNPA3, inhibiting miRNA biogenesis and RNA splicing and triggering DNA damage, proteotoxic stress, and pneumonia. Dicer, XPO5, SRSF3, and hnRNPA3 knockdown increases, while their overexpression decreases, N protein-induced pneumonia's severity. Older mice show lower expression of Dicer, XPO5, SRSF3, and hnRNPA3 in their lung tissues and exhibit more severe N protein-induced pneumonia than younger mice. PJ34, a poly(ADP-ribose) polymerase inhibitor, or anastrozole, an aromatase inhibitor, ameliorates N protein- or SARS-CoV-2-induced pneumonia by restoring Dicer, XPO5, SRSF3, and hnRNPA3 expression. These findings will aid in developing improved treatments for SARS-CoV-2-associated pneumonia.

Asunto(s)

COVID-19 , Carioferinas , Ribonucleasa III , SARS-CoV-2 , Factores de Empalme Serina-Arginina , Animales , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/genética , Humanos , Ribonucleasa III/metabolismo , Ribonucleasa III/genética , SARS-CoV-2/genética , COVID-19/metabolismo , COVID-19/virología , COVID-19/genética , Ratones , Carioferinas/metabolismo , Carioferinas/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Regulación hacia Abajo , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Masculino , Femenino , MicroARNs/genética , MicroARNs/metabolismo , Empalme del ARN , Autofagia/genética , Daño del ADN , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B

RESUMEN

Protein synthesis in response to neuronal activity, known as activity-dependent translation, is critical for synaptic plasticity and memory formation. However, the signaling cascades that couple neuronal activity to the translational events remain elusive. In this study, we identified the role of calmodulin (CaM), a conserved Ca2+-binding protein, in ribosomal RNA (rRNA) biogenesis in neurons. We found the CaM-regulated rRNA synthesis is Ca2+-dependent and necessary for nascent protein synthesis and axon growth in hippocampal neurons. Mechanistically, CaM interacts with nucleolar DEAD (Asp-Glu-Ala-Asp) box RNA helicase (DDX21) in a Ca2+-dependent manner to regulate nascent rRNA transcription within nucleoli. We further found CaM alters the conformation of DDX21 to liberate the DDX21-sequestered RPA194, the catalytic subunit of RNA polymerase I, to facilitate transcription of ribosomal DNA. Using high-throughput screening, we identified the small molecules batefenterol and indacaterol that attenuate the CaM-DDX21 interaction and suppress nascent rRNA synthesis and axon growth in hippocampal neurons. These results unveiled the previously unrecognized role of CaM as a messenger to link the activity-induced Ca2+ influx to the nucleolar events essential for protein synthesis. We thus identified the ability of CaM to transmit information to the nucleoli of neurons in response to stimulation.

Asunto(s)

Calmodulina , ARN Helicasas DEAD-box , Hipocampo , ARN Ribosómico , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Animales , ARN Ribosómico/metabolismo , Calmodulina/metabolismo , Hipocampo/metabolismo , Hipocampo/citología , Humanos , Neuronas/metabolismo , Ratas , Nucléolo Celular/metabolismo , Células Cultivadas , Células HEK293 , Ratones , Calcio/metabolismo

RESUMEN

Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.

Asunto(s)

Núcleo Celular , Cromatina , ARN Helicasas DEAD-box , ARN Mensajero , Animales , Humanos , Ratones , ARN Mensajero/metabolismo , ARN Mensajero/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Cromatina/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Citoplasma/genética , Estabilidad del ARN , Transporte Activo de Núcleo Celular , Polirribosomas/metabolismo , Polirribosomas/genética , Aprendizaje Automático , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Exosomas/metabolismo , Exosomas/genética

RESUMEN

Spermatogonia cryopreservation can be a strategy for future conservation actions. The neotropical Siluriformes Pseudopimelodus mangurus was already classified as vulnerable on the Red List of Threatened Species. P. mangurus spermatogonial cells were isolated, assessed, and cryopreserved. Fragments of the testis were enzymatically dissociated, purified using Percoll density gradient, and submitted to differential plating. Fractionated cells were evaluated by microscopy, ddx4 (vasa) relative expression, and alkaline phosphatase activity. Cryopreservation was conducted using ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and propanediol at 1 M, 1.5 M, and 2 M. Cell viability was evaluated and cell concentration was determined. Cell fractions from 20 % and 30 % Percoll gradient bands showed the highest concentrations of spermatogonia. The fraction mix showed 54 % purity and 93 % viability. After differential plating, 60 % purity and 92 % viability were obtained. Spermatogonial cells showed high alkaline phosphatase activity compared to spermatocytes and spermatids. The relative spermatogonial ddx4 expression from the Percoll density gradient was about twice as high as in samples from the testis and the differential plating. The increased ddx4 expression indicated the enrichment of spermatogonial cells by density gradient step and dead cells expressing ddx4 in differential plating, or ddx4 decreasing expression during cell culture. For this reason, cells from the Percoll gradient were chosen for cryopreservation. Propanediol at 1 M demonstrated the best condition for spermatogonial cell cryopreservation, presenting 98 % viability, while dimethylacetamide at 2 M represented the least favorable condition, with approximately 47 % viability. These findings are essential for P. mangurus spermatogonial cell cryopreservation, aiming to generate a spermatogonia cryobank for future conservation efforts.

Asunto(s)

Bagres , Supervivencia Celular , Criopreservación , Espermatogonias , Animales , Masculino , Criopreservación/métodos , Criopreservación/veterinaria , Espermatogonias/citología , Crioprotectores/farmacología , Testículo/citología , Dimetilsulfóxido/farmacología , Acetamidas/farmacología , Acetamidas/química , Glicol de Etileno/farmacología , ARN Helicasas DEAD-box/metabolismo , Glicerol/farmacología , Glicerol/metabolismo , Fosfatasa Alcalina/metabolismo , Propilenglicol/farmacología , Separación Celular/métodos

RESUMEN

RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-ß (IFN-ß), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway.

Asunto(s)

Proteína 58 DEAD Box , ARN Helicasas DEAD-box , Inmunidad Innata , Receptores Inmunológicos , Humanos , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/inmunología , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/inmunología , Receptores Inmunológicos/metabolismo , Poli I-C/inmunología , Poli I-C/farmacología , ARN Helicasas/metabolismo , ARN Helicasas/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Células HEK293

RESUMEN

Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels. Moreover, we found that either selinexor treatment or DDX19A depletion effectively induced apoptosis of T-ALL lines expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.

Asunto(s)

ARN Helicasas DEAD-box , Hidrazinas , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , ARN Mensajero , Triazoles , Triazoles/farmacología , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Hidrazinas/farmacología , ARN Mensajero/genética , Leucemia/metabolismo , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia/patología , Apoptosis/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Antineoplásicos/farmacología

RESUMEN

Dysregulated mitochondrial dynamics and metabolism play important roles in tumorigenesis. Metastasizing tumor cells predominantly utilize mitochondrial metabolism, and regulators of metabolic reprogramming may provide reliable biomarkers for diagnosing cancer metastasis. Here, we identified a type I arginine methyltransferase-DEAD-box polypeptide 3, X-linked (PRMT1-DDX3) axis that promotes breast cancer metastasis by coordinating mitochondrial biogenesis and mitophagy to ensure mitochondrial quality control. Mechanistically, PRMT1 induces arginine methylation of DDX3, which enhances its protein stability and prevents proteasomal degradation. DDX3 mediates mitochondrial homeostasis by translocating to mitochondria where it facilitates phosphatase and tensin homology-induced kinase 1 translation in response to mitochondrial stress. Inhibition of DDX3 suppresses mitochondrial biogenesis and mitophagy, resulting in diminished cancer stemness and metastatic properties. Overall, this study uncovers a mechanism by which the PRMT1-DDX3 axis regulates mitochondrial homeostasis to support breast cancer metastasis, suggesting strategies for targeting metabolic vulnerabilities to treat metastatic breast cancer. Significance: DDX3 is stabilized by PRMT1-mediated arginine methylation and coordinates mitophagy and mitochondrial biogenesis by upregulating PINK1 to facilitate breast cancer progression.

Asunto(s)

Arginina , Neoplasias de la Mama , ARN Helicasas DEAD-box , Mitocondrias , Mitofagia , Proteína-Arginina N-Metiltransferasas , Proteínas Represoras , Humanos , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Femenino , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Ratones , Animales , Arginina/metabolismo , Metilación , Homeostasis , Línea Celular Tumoral , Metástasis de la Neoplasia , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Ratones Desnudos

Asunto(s)

ARN Helicasas DEAD-box , Humanos , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Animales , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Leucemia Mieloide/metabolismo , Ratones