RESUMO
The mechanisms underlying stimuli-induced dynamic structural remodeling of RNAs for the maintenance of cellular physiological function and survival remain unclear. Here, we showed that in MGMT promoter-methylated glioblastoma (GBM), the RNA helicase DEAD-box helicase 46 (DDX46) is phosphorylated by temozolomide (TMZ)-activated checkpoint kinase 1 (CHK1), resulting in a dense-to-loose conformational change and an increase in DDX46 helicase activity. DDX46-mediated tertiary structural remodeling of LINC01956 exposes the binding motifs of LINC01956 to the 3' untranslated region of O6-methylguanine DNA methyltransferase (MGMT). This accelerates recruitment of MGMT mRNA to the RNA export machinery and transportation of MGMT mRNA from the nucleus to the cytoplasm, leading to increased MGMT abundance and TMZ resistance. Using patient-derived xenograft (PDX) and tumor organoid models, we found that treatment with the CHK1 inhibitor SRA737abolishes TMZ-induced structural remodeling of LINC01956 and subsequent MGMT up-regulation, resensitizing TMZ-resistant MGMT promoter-methylated GBM to TMZ. In conclusion, these findings highlight a mechanism underlying temozolomide-induced RNA structural remodeling and may represent a potential therapeutic strategy for patients with TMZ-resistant MGMT promoter-methylated GBM.
Assuntos
RNA Helicases DEAD-box , Metilases de Modificação do DNA , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , RNA Longo não Codificante , Temozolomida , Proteínas Supressoras de Tumor , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Regiões Promotoras Genéticas/genética , Metilação de DNA/genética , Metilação de DNA/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/genética , Linhagem Celular Tumoral , Camundongos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fosforilação/efeitos dos fármacosRESUMO
Cancer predisposition syndromes are recognized in about 10% of pediatric malignancies with several genes specifically involved in a subset of pediatric tumors such as DICER1, in pleuropulmonary blastoma, cystic nephroma, and brain sarcomas. By contrast, the role of BRCA1/2 in pediatric cancer predisposition is still under investigation. We present two cases of young first-degree cousins, both carrying a germline BRCA2 variant and developing tumors characterized by somatic DICER1 mutations. Patient 1 presented with a cystic nephroma harboring a somatic DICER1 variant (p.Asp1810Tyr), while patient 2 had a primary intracranial DICER1-mutated sarcoma showing a distinct somatic DICER1 variant (p.Asp1709Glu) as well as biallelic inactivation of TP53 (p.Val173Leu, VAF 91%) and APC (p.Ile1307Lys, VAF 95%) and a pathogenic variant in KRAS (p.Gln61His). Both patients carried the same germline BRCA2 variant (p.Arg2842Cys) of unknown significance. The same variant was found in the mother of patient 2 and in the father of patient 1, who are siblings. A homologous recombination deficiency signature was not identified in any of the two tumors, possibly suggesting a reduction of BRCA2 activity. The association of BRCA2 and DICER1 variants in our cases hints at a potential cooperative role in cancer pathogenesis. Further studies are warranted to elucidate the interplay between BRCA1/2 and DICER1 variants and their implications for cancer predisposition and treatment in pediatric patients.
Assuntos
Proteína BRCA2 , RNA Helicases DEAD-box , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Ribonuclease III , Humanos , RNA Helicases DEAD-box/genética , Ribonuclease III/genética , Proteína BRCA2/genética , Feminino , Masculino , Linhagem , CriançaRESUMO
RNA helicases constitute a large family of proteins that share a catalytic core with high structural similarity. DEAD-box (DDX) proteins belong to the largest RNA helicase subfamily, and DDX members have been implicated in all facets of RNA metabolism, from transcription to translation, miRNA maturation, and RNA delay and degradation. Interestingly, an increasing number of studies have suggested a relationship between DDX proteins and cancer initiation and progression. The expression levels of many DDX proteins are elevated in a majority of cancers, and recent studies have demonstrated that some DDX proteins have a potent positive effect on promoting the metastasis of malignant cells. Metastasis is a complex, multistep cascade process that includes local invasion, intravasation and survival in the circulation, arrest at a distant organ site, extravasation and metastatic colonization; here, we review this process and present the suggested functions and mechanisms of DDX family proteins in particular steps of the invasionâmetastasis cascade.
Assuntos
RNA Helicases DEAD-box , Metástase Neoplásica , Neoplasias , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Animais , Invasividade Neoplásica , Regulação Neoplásica da Expressão GênicaRESUMO
Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including Dhx30 and Llph, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed that RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts.
Assuntos
Macrófagos , Proteínas Ribossômicas , Ribossomos , Animais , Ribossomos/metabolismo , Ribossomos/genética , Camundongos , Humanos , Macrófagos/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Biossíntese de Proteínas , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Regulação da Expressão Gênica no Desenvolvimento , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BLRESUMO
Immune-mediated liver injury is a common characteristic of various liver diseases, including autoimmune and viral hepatitis. Here, we investigated the role of DEAD-box helicase 3, X-linked (DDX3X) in immune-mediated liver injury. Liver injury was induced in C57BL/6J mice via concanavalin A (Con A). DDX3X hepatocyte-specific knockout (DDX3XΔHep) mice and control (DDX3Xfl/fl) mice were utilized to investigate the role of DDX3X in liver injury. Primary hepatocytes were treated with tunicamycin (TM) to induce ER stress in vitro. The expression of DDX3X in patients with various liver diseases was evaluated. Hepatic DDX3X expression increased, and DDX3X translocated from the cytoplasm to the nucleus during Con A-induced liver injury. DDX3X deficiency ameliorated mouse liver injury and reduced ER stress in liver tissue. The inhibition of ER stress with 4-PBA significantly attenuated liver injury while decreasing DDX3X levels in liver tissue. However, the upregulation of hepatic DDX3X expression reversed Con A-induced liver injury and negated the protective effect of 4-PBA. Mechanistically, the nuclear translocation of DDX3X promoted ER stress-induced apoptosis through the transcriptional induction of CHOP. Moreover, DDX3X was elevated and translocated into the nucleus in patients with HBV-LF and AIH. Additionally, serum DDX3X levels markedly increased in patients with HBV-LF, and a consistent decrease in DDX3X was associated with a good prognosis. The cytoplasmic-to-nuclear translocation of DDX3X promotes ER stress-induced apoptosis, which is an obligatory step that drives hepatic necrosis and tissue damage. Notably, DDX3X is a potential therapeutic target for immune-mediated liver injury.
Assuntos
RNA Helicases DEAD-box , Estresse do Retículo Endoplasmático , Hepatócitos , Camundongos Endogâmicos C57BL , Animais , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Camundongos , Hepatócitos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Camundongos Knockout , Fígado/metabolismo , Fígado/patologia , Fígado/lesões , Masculino , Transporte Ativo do Núcleo Celular , Apoptose/efeitos dos fármacos , Concanavalina ARESUMO
We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.
Assuntos
Vírus dos Macacos de Mason-Pfizer , RNA Viral , Montagem de Vírus , Animais , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/virologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Genoma Viral , Células HEK293 , Vírus dos Macacos de Mason-Pfizer/genética , Vírus dos Macacos de Mason-Pfizer/metabolismo , Vírus dos Macacos de Mason-Pfizer/fisiologia , RNA Helicases/metabolismo , RNA Helicases/genética , RNA Viral/metabolismo , RNA Viral/genética , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
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.
Assuntos
RNA Helicases DEAD-box , Fator de Iniciação 4A em Eucariotos , Biossíntese de Proteínas , RNA Mensageiro , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Simulação de Dinâmica Molecular , Ribossomos/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , Células HEK293 , Compostos de Epóxi , Tiazóis , MacrolídeosRESUMO
Although there are several genome-wide association studies available which highlight genetic variants associated with Alzheimer's disease (AD), often the X chromosome is excluded from the analysis. We conducted an X-chromosome-wide association study (XWAS) in three independent studies with a pathologically confirmed phenotype (total 1970 cases and 1113 controls). The XWAS was performed in males and females separately, and these results were then meta-analysed. Four suggestively associated genes were identified which may be of potential interest for further study in AD, these are DDX53 (rs12006935, OR = 0.52, p = 6.9e-05), IL1RAPL1 (rs6628450, OR = 0.36, p = 4.2e-05; rs137983810, OR = 0.52, p = 0.0003), TBX22 (rs5913102, OR = 0.74, p = 0.0003) and SH3BGRL (rs186553004, OR = 0.35, p = 0.0005; rs113157993, OR = 0.52, p = 0.0003), which replicate across at least two studies. The SNP rs5913102 in TBX22 achieves chromosome-wide significance in meta-analysed data. DDX53 shows highest expression in astrocytes, IL1RAPL1 is most highly expressed in oligodendrocytes and neurons and SH3BGRL is most highly expressed in microglia. We have also identified SNPs in the NXF5 gene at chromosome-wide significance in females (rs5944989, OR = 0.62, p = 1.1e-05) but not in males (p = 0.83). The discovery of relevant AD associated genes on the X chromosome may identify AD risk differences and similarities based on sex and lead to the development of sex-stratified therapeutics.
Assuntos
Doença de Alzheimer , Cromossomos Humanos X , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Humanos , Doença de Alzheimer/genética , Masculino , Feminino , Estudos de Casos e Controles , Cromossomos Humanos X/genética , Idoso , População Branca/genética , Predisposição Genética para Doença , Idoso de 80 Anos ou mais , RNA Helicases DEAD-box/genética , Proteína Acessória do Receptor de Interleucina-1/genéticaRESUMO
High invasive capacity and acquired tyrosine kinase inhibitors (TKI) resistance of kidney renal clear cell carcinoma (KIRC) cells remain obstacles to prolonging the survival time of patients with advanced KIRC. In the present study, we reported that sine oculis homeobox 1 (SIX1) was upregulated in sunitinib-resistant KIRC cells and metastatic KIRC tissues. Subsequently, we found that SIX1 mediated metastasis and sunitinib resistance via Focal adhesion (FA) signaling, and knockdown of SIX1 enhanced the antitumor efficiency of sunitinib in KIRC. Mechanistically, Integrin subunit beta 1 (ITGB1), an upstream gene of FA signaling, was a direct transcriptional target of SIX1. In addition, we showed that DExH-box helicase 9 (DHX9) was an important mediator for SIX1-induced ITGB1 transcription, and silencing the subunits of SIX1/DHX9 complex significantly reduced transcription of ITGB1. Downregulation of SIX1 attenuated nuclear translocation of DHX9 and abrogated the binding of DHX9 to ITGB1 promoter. Collectively, our results unveiled a new signal axis SIX1/ITGB1/FAK in KIRC and identified a novel therapeutic strategy for metastatic KIRC patients.
Assuntos
Carcinoma de Células Renais , RNA Helicases DEAD-box , Resistencia a Medicamentos Antineoplásicos , Adesões Focais , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio , Integrina beta1 , Neoplasias Renais , Metástase Neoplásica , Transdução de Sinais , Sunitinibe , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Neoplasias Renais/patologia , Neoplasias Renais/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Linhagem Celular Tumoral , Integrina beta1/genética , Integrina beta1/metabolismo , Animais , Adesões Focais/genética , Adesões Focais/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Camundongos , Transcrição Gênica , Integrinas/metabolismo , Integrinas/genética , Quinase 1 de Adesão FocalRESUMO
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.
Assuntos
Apoptose , Proteínas Relacionadas à Autofagia , Autofagia , Proliferação de Células , Neoplasias Colorretais , RNA Helicases DEAD-box , Células-Tronco Neoplásicas , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Autofagia/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Camundongos , Animais , Proteínas de Transporte VesicularRESUMO
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.
Assuntos
Carcinoma Hepatocelular , RNA Helicases DEAD-box , Progressão da Doença , Neoplasias Hepáticas , Fator 2 Relacionado a NF-E2 , NF-kappa B , Sorafenibe , Sorafenibe/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Animais , Humanos , Camundongos , NF-kappa B/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Via de Sinalização Wnt/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Ferroptose/genéticaRESUMO
Pre-rRNA processing is essential to ribosome biosynthesis. However, the processing mechanism is not fully understood in plants. Here, we report a DEAD-box RNA helicase DEK51 that mediates the 3' end processing of 18S and 5.8S pre-rRNA in maize (Zea mays L.). DEK51 is localized in the nucleolus, and loss of DEK51 arrests maize seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA. DEK51 interacts with putative key factors in nuclear RNA exosome-mediated pre-rRNA processing, including ZmMTR4, ZmSMO4, ZmRRP44A, and ZmRRP6L2. This suggests that DEK51 facilitates pre-rRNA processing by interacting with the exosome. Loss of ZmMTR4 function arrests seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA, similar to dek51. DEK51 also interacts with endonucleases ZmUTP24 and ZmRCL1, suggesting that it may also be involved in the cleavage at site A2. These results show the critical role of DEK51 in promoting 3' end processing of pre-rRNA.
Assuntos
RNA Helicases DEAD-box , Precursores de RNA , Sementes , Zea mays , Nucléolo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Precursores de RNA/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Sementes/metabolismo , Sementes/crescimento & desenvolvimento , Zea mays/enzimologia , Zea mays/genética , Zea mays/metabolismoRESUMO
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.
Assuntos
Elementos de DNA Transponíveis , Infertilidade Masculina , RNA Interferente Pequeno , Espermatogênese , Testículo , Masculino , Humanos , Espermatogênese/genética , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genética , Elementos de DNA Transponíveis/genética , Animais , Testículo/metabolismo , Camundongos , Adulto , Inativação Gênica , Camundongos Knockout , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Espermatogônias/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , RNA de Interação com PiwiRESUMO
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.
Assuntos
RNA Helicases DEAD-box , Hematopoese , Imunidade Inata , Humanos , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Hematopoese/imunologia , AnimaisAssuntos
RNA Helicases DEAD-box , Sementes , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Sementes/fisiologia , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Fertilidade/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genéticaRESUMO
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.
Assuntos
Coturnix , RNA Helicases DEAD-box , Células Germinativas , Animais , Masculino , Feminino , Células Germinativas/fisiologia , Células Germinativas/citologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Caracteres Sexuais , Proliferação de Células/fisiologia , Sistemas CRISPR-CasRESUMO
Current therapy in chronic myeloid leukemia (CML) has improved patient life expectancy close to that of healthy individuals. However, molecular alterations other than BCR::ABL1 fusion gene in CML are barely known. MicroRNAs are important regulators of gene expression, and variants in some of the components of microRNA biosynthesis pathways have been associated with genetic susceptibility to different types of cancer. Thus, the aim of this study was to evaluate the association of variants located in genes involved in the biogenesis of microRNAs with susceptibility to CML. Fifteen variants in eight genes involved in the biogenesis of miRNAs were genotyped in 296 individuals with CML and 485 healthy participants using TaqMan probes. The association of gene variants with CML and clinical variables was evaluated by a Chi-square test, and odds ratios and 95% confidence intervals were estimated by logistic regression. The variant rs13078 in DICER1 was significantly higher among CML individuals than in healthy participants. In addition, the variants rs7813 and rs2740349 were significantly associated with worse prognosis, according to their Hasford scores, whereas the rs2740349 variant was also associated with a later age at diagnosis. These findings suggest that variants in components of the microRNA biogenesis pathway could be involved in CML genetic risk.
Assuntos
RNA Helicases DEAD-box , Leucemia Mielogênica Crônica BCR-ABL Positiva , MicroRNAs , Polimorfismo de Nucleotídeo Único , Ribonuclease III , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Feminino , Masculino , Pessoa de Meia-Idade , Ribonuclease III/genética , Adulto , RNA Helicases DEAD-box/genética , Idoso , Predisposição Genética para Doença , Estudos de Casos e Controles , PrognósticoRESUMO
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.
Assuntos
RNA Helicases DEAD-box , Ferroptose , Vírus da Síndrome Respiratória e Reprodutiva Suína , Replicação Viral , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Ferroptose/fisiologia , Suínos , Síndrome Respiratória e Reprodutiva Suína/virologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Linhagem CelularRESUMO
CircRNA, an essential RNA molecule involved in various biological functions and diseases, often exhibits decreased expression in tumor tissues, playing a role as a tumor suppressor, and suggesting therapeutic potential for cancer. However, current methods for promoting circRNA production are limited. This study introduces a novel approach for enhancing circRNA biogenesis, termed circRNA promoting RNA (cpRNA). CpRNA is designed to complement the flanking sequences of reverse complementary matches (RCMs) within pre-mRNA, thereby facilitating circRNA formation through improved exon circularization. Using a split-GFP reporter system, we demonstrated that cpRNA significantly enhance circGFP production. Optimization identified the best conditions for cpRNA to promote circRNA biogenesis, and these cpRNAs were then used to augment the production of endogenous circRNAs. These results indicate that cpRNAs can specifically increase the production of endogenous circRNAs with RCMs, such as circZKSCAN1 and circSMARCA5 in cancer cells, thereby inhibiting cell proliferation and migration by modulating circRNA-related pathways, showcasing the therapeutic potential of cpRNAs. Mechanistic studies have also shown that cpRNA promotes circRNA biogenesis, in part, by antagonizing the unwinding function of DHX9. Overall, these findings suggest that cpRNA represents a promising strategy for circRNA overexpression, offering a potential treatment for diseases marked by low circRNA levels.
Assuntos
Proliferação de Células , RNA Circular , RNA Circular/genética , RNA Circular/metabolismo , Humanos , Proliferação de Células/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Linhagem Celular Tumoral , RNA/genética , RNA/metabolismo , Movimento Celular/genética , Precursores de RNA/metabolismo , Precursores de RNA/genética , Neoplasias/genética , Neoplasias/metabolismo , Células HEK293 , Proteínas de NeoplasiasRESUMO
Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.