RESUMEN
Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.
Asunto(s)
Neoplasias Hematológicas , Proteínas Musculares , Mutación , Fosfoproteínas , Factores de Empalme de ARN , Animales , Humanos , Ratones , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/metabolismo , Hematopoyesis/genética , Intrones , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Empalme del ARN , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismoRESUMEN
The spliceosomal gene SF3B1 is frequently mutated in cancer. While it is known that SF3B1 hotspot mutations lead to loss of splicing factor SUGP1 from spliceosomes, the cancer-relevant SF3B1-SUGP1 interaction has not been characterized. To address this issue, we show by structural modeling that two regions flanking the SUGP1 G-patch make numerous contacts with the region of SF3B1 harboring hotspot mutations. Experiments confirmed that all the cancer-associated mutations in these regions, as well as mutations affecting other residues in the SF3B1-SUGP1 interface, not only weaken or disrupt the interaction but also alter splicing similarly to SF3B1 cancer mutations. Finally, structural modeling of a trimeric protein complex reveals that the SF3B1-SUGP1 interaction "loops out" the G-patch for interaction with the helicase DHX15. Our study thus provides an unprecedented molecular view of a protein complex essential for accurate splicing and also reveals that numerous cancer-associated mutations disrupt the critical SF3B1-SUGP1 interaction.
Asunto(s)
Neoplasias , Empalmosomas , Humanos , ARN Mensajero/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo , Factores de Empalme de ARN/química , Empalme del ARN/genética , Neoplasias/genética , Neoplasias/metabolismo , Mutación , Fosfoproteínas/metabolismoRESUMEN
RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA complexes remains poorly understood. In this issue of Genes & Development, Zhang and colleagues (pp. 968-983) combine biochemical experiments with AI-based structure prediction methods to generate a model for the interaction between SF3B1, a core splicing factor essential for the recognition of the intron branchpoint, and SUGP1, a protein that bridges SF3B1 with the helicase DHX15. Interaction with SF3B1 exposes the G-patch domain of SUGP1, facilitating binding to and activation of DHX15. The model can explain the activation of cryptic 3' splice sites induced by mutations in SF3B1 or SUGP1 frequently found in cancer.
Asunto(s)
Empalme del ARN , Empalmosomas , Empalme del ARN/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Sitios de Empalme de ARN , Precursores del ARN/genética , Precursores del ARN/metabolismo , Inteligencia Artificial , Mutación , Fosfoproteínas/metabolismoRESUMEN
Benbarche, Pineda, Galvis, et al. delineate an essential role for the G-patch motif-containing protein GPATCH8 in mis-splicing associated with cancer-driving mutations of the splicing factor SF3B1. GPATCH8 cooperates with SF3B1 mutants, affecting the splicing machinery. Targeting GPATCH8 reveals therapeutic opportunities for SF3B1 mutant cancers and other splicing-related diseases.
Asunto(s)
Neoplasias , Factores de Empalme de ARN , Empalme del ARN , Humanos , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genéticaRESUMEN
A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation, and several RNA-dependent ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement, and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNP's engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 may have an expanded role in maintaining splicing fidelity.
Asunto(s)
Ribonucleoproteína Nuclear Pequeña U2 , Empalmosomas , Intrones/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN Nuclear Pequeño/genética , Ribonucleasa H/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/metabolismoRESUMEN
RNA helicases play roles in various essential biological processes such as RNA splicing and editing. Recent in vitro studies show that RNA helicases are involved in immune responses toward viruses, serving as viral RNA sensors or immune signaling adaptors. However, there is still a lack of in vivo data to support the tissue- or cell-specific function of RNA helicases owing to the lethality of mice with complete knockout of RNA helicases; further, there is a lack of evidence about the antibacterial role of helicases. Here, we investigated the in vivo role of Dhx15 in intestinal antibacterial responses by generating mice that were intestinal epithelial cell (IEC)-specific deficient for Dhx15 (Dhx15 f/f Villin1-cre, Dhx15ΔIEC). These mice are susceptible to infection with enteric bacteria Citrobacter rodentium (C. rod), owing to impaired α-defensin production by Paneth cells. Moreover, mice with Paneth cell-specific depletion of Dhx15 (Dhx15 f/f Defensinα6-cre, Dhx15ΔPaneth) are more susceptible to DSS (dextran sodium sulfate)-induced colitis, which phenocopy Dhx15ΔIEC mice, due to the dysbiosis of the intestinal microbiota. In humans, reduced protein levels of Dhx15 are found in ulcerative colitis (UC) patients. Taken together, our findings identify a key regulator of Wnt-induced α-defensins in Paneth cells and offer insights into its role in the antimicrobial response as well as intestinal inflammation.
Asunto(s)
Colitis/inmunología , Defensinas/genética , Infecciones por Enterobacteriaceae/inmunología , Células de Paneth/inmunología , ARN Helicasas/genética , Vía de Señalización Wnt , Animales , Citrobacter rodentium/inmunología , Citrobacter rodentium/patogenicidad , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Defensinas/inmunología , Sulfato de Dextran/administración & dosificación , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/patología , Microbioma Gastrointestinal/inmunología , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Células de Paneth/microbiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , ARN Helicasas/inmunologíaRESUMEN
DEAH-box helicase 15 (DHX15) is ATP-dependent RNA helicase which is known for its role in RNA metabolism. Recent studies reported DHX15 involves in the intestinal immunity. However, the role of DHX15 (or RNA helicase) in intestinal development is poorly understood. Here, we revealed an unidentified role for dhx15 in regulating zebrafish intestinal development. We found the profound intestinal defects in dhx15 knockout zebrafish. Decreased proliferation and increased apoptosis of the intestine cells were observed when dhx15 were deleted. Further RNA genome wide analysis and qRT-PCR analysis showed the Wnt signaling pathway is down-regulated in the dhx15 knockout zebrafish. Thus, we concluded that dhx15 regulates zebrafish intestinal development through the Wnt signaling pathway. Here, we provided new insights into the role of dhx15 in intestinal development beyond its well-characterized role in intestinal immunity.
Asunto(s)
Vía de Señalización Wnt , Pez Cebra , Animales , ARN/metabolismo , ARN Helicasas/genética , Pez Cebra/genéticaRESUMEN
BACKGROUND: Breast cancer (BC) is a common threat to women. The continuous activation of nuclear factor kappa B (NF-κB) signaling pathway contributes to the development of BC. This study aimed to investigate the role of a circular RNA (circRNF10) in BC progression and regulating NF-κB signaling pathway. METHODS: Bioinformatics analysis, RT-qPCR, subcellular fractionation, FISH, RNase R treatment, and actinomycin D assay were used to explore the expression and characteristics of circRNF10 in BC. The biological functions of circRNF10 in BC were analyzed by MTT assay, colony formation assay, wound healing assay, and Transwell assay. RNA pulldown and RIP assay were used to identify the interaction between circRNF10 and DEAH (Asp-Glu-Ala-His) box helicase 15 (DHX15). The impact of circRNF10-DHX15 interaction on NF-κB signaling pathway was explored by western blot, IF, and co-IP. Furthermore, dual-luciferase reporter assay, ChIP, and EMSA were performed to assess the effect of NF-κB p65 on DHX15 transcription. RESULTS: CircRNF10 was downregulated in BC, and lower expression of circRNF10 was related to poor prognosis of patients with BC. CircRNF10 inhibited the proliferation and migration of BC. Mechanically, circRNF10-DHX15 interaction sequestered DHX15 from NF-κB p65, thereby inhibiting the activation of NF-κB signaling pathway. On the other hand, NF-κB p65 enhanced DHX15 transcription by binding to the promoter of DHX15. Altogether, circRNF10 impaired the DHX15-NF-κB p65 positive feedback loop and suppressed the progression of BC. CONCLUSION: CircRNF10-DHX15 interaction suppressed the DHX15-NF-κB p65 positive feedback loop, thereby inhibiting BC progression. These findings provide new insights in the continuous activation of NF-κB signaling pathway and raised potential therapeutic approach for BC treatment.
Asunto(s)
FN-kappa B , Neoplasias , Femenino , Humanos , Línea Celular Tumoral , Retroalimentación , FN-kappa B/metabolismo , Transducción de Señal , Proteínas del Tejido Nervioso/metabolismo , Proteínas Portadoras/metabolismo , ARN Helicasas/metabolismoRESUMEN
BACKGROUND: DHX15 is one of the RNA helicase family members involving in several biological processes. Studies have reported that overexpression of DHX15 is related to cancer progression. However, the role of DHX15 in Burkitt lymphoma (BL) and latent Epstein-Barr virus (EBV) infection remains to be elucidated. METHODS: Expression of DHX15 was measured in BL patient by immunohistochemical staining. In vitro study, a CCK-8 assay was used to analyze cell proliferation and flow cytometry was performed to assess cell cycle, apoptosis and mitochondria membrane potential. Members of NF-κB signaling pathway and apoptotic-related proteins expression were measured by western-blot. EBV latent infection products and RNA polymerase III transcripts expression were determined by quantitative real-time PCR and western-blot. In vivo study, HE, IHC, TUNEL and ISH assays were used to analyze the effect of DHX15 on subcutaneous tumor nodes formation. RESULTS: DHX15 was overexpressed in Burkitt lymphoma patients and tends to be associated with poor progression-free survival and poor overall survival. Knockdown of DHX15 significantly inhibited BL tumor growth, reduced cell proliferation, induced cell cycle arrest and increased cell apoptosis. Further analysis showed that canonical NF-κB signaling and its downstream targets, mitochondria and Caspase were involved in the increased cell apoptosis after DHX15 gene knockdown. Furthermore, knockdown of DHX15 reduced EBV latent infection products expression and inhibited RNA polymerase III activity. CONCLUSION: DHX15 may be an oncogene in the development of BL and a potential therapeutic target for the treatment of BL and latent EBV infection.
RESUMEN
BACKGROUND: DHX15 is a member of the DEAH-box (DHX) RNA helicase family. Our previous study identified it as an AR coactivator which contributes to prostate cancer progression. METHODS: We investigated DHX15 expression in castration resistant prostate cancer specimens and the influence of DHX15 on the responsiveness of prostate cancer cells to DHT stimulation. We also explored the role DHX15 played in enzalutamide resistance and the interacting domain in DHX15 with AR. DHX15 expression level in human CRPC specimens and prostate cancer specimens was detected by tissue microarray (TMA) immunostaining analysis. Colony formation assay was performed to determine the proliferation of cells treated with enzalutamide or DHT. siRNAs were used to knockdown DHX15. The interactions between DHX15 and AR were detected using co-immunoprecipitation assay. RESULTS: The expression level of DHX15 was upregulated in human CRPC specimens compared with hormone naïve prostate cancer specimens. DHX15 knockdown reduced AR sensitivity to low DHT concentrations in C4-2 cells. Inactivation of DHX15 sensitizes the enzalutamide treatment in C4-2 cells. Deletion mutagenesis indicated that DHX1 5 interacts with AR through its N terminal domain. CONCLUSIONS: These findings suggest that DHX15 contributes to prostate cancer progression. DHX15 is required for androgen receptor sensitivity to low DHT concentrations and contributes to enzalutamide resistance in C4-2 cells. Targeting DHX15 may improve the ADT treatment.
Asunto(s)
Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración , ARN Helicasas , Receptores Androgénicos/metabolismo , Antineoplásicos/farmacología , Benzamidas , Línea Celular Tumoral , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoprecipitación/métodos , Masculino , Nitrilos , Feniltiohidantoína/farmacología , Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , ARN Helicasas/genética , ARN Helicasas/metabolismo , Activación Transcripcional , Regulación hacia ArribaRESUMEN
DHX15 plays a role in leukaemogenesis and leukaemia relapse. However, the mechanism underlying the transcriptional regulation of DHX15 in ALL has not been elucidated. Our present study aimed to explore the functional promoter region of DHX15 and to investigate the transcription factors controlling the transcription of this gene. A luciferase assay performed with several truncated constructs identified a 501-bp region as the core promoter region of DHX15. Site-directed mutagenesis, electrophoretic mobility shift and chromatin immunoprecipitation assays showed that ETS1 and SP1 occupied the DHX15 promoter. Furthermore, knockdown of ETS1 and SP1 resulted in suppression of DHX15, whereas the overexpression of these genes led to up-regulation of DHX15. Interestingly, in samples obtained from patients with ALL at diagnosis, both ETS1 and SP1 correlated positively with DHX15 expression. Additionally, differences in methylation of the DHX15 core promoter region were not observed between the patients and controls. In conclusion, we identified the core promoter region of DHX15 and demonstrated that ETS1 and SP1 regulated DHX15 expression in ALL.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , ARN Helicasas/genética , Factor de Transcripción Sp1/metabolismo , Emparejamiento Base/genética , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Islas de CpG/genética , Metilación de ADN/genética , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Regiones Promotoras Genéticas , Unión Proteica , Proteína Proto-Oncogénica c-ets-1/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
BACKGROUND: RNA-binding proteins (RBPs) are crucial factors that function in the posttranscriptional modification process and are significant in cancer. OBJECTIVE: This research aimed for a multigene signature to predict the prognosis and immunotherapy response of patients with colon adenocarcinoma (COAD) based on the expression profile of RNA-binding proteins (RBPs). METHODS: COAD samples retrieved from the TCGA and GEO datasets were utilized for a training dataset and a validation dataset. Totally, 14 shared RBP genes with prognostic significance were identified. Non-negative matrix factorization clusters defined by these RBPs could stratify COAD patients into two molecular subtypes. Cox regression analysis and identification of 8-gene signature categorized COAD patients into high- and low-risk populations with significantly different prognosis and immunotherapy responses. RESULTS: Our prediction signature was superior to another five well-established prediction models. A nomogram was generated to quantificationally predict the overall survival (OS) rate, validated by calibration curves. Our findings also indicated that high-risk populations possessed an enhanced immune evasion capacity and low-risk populations might benefit immunotherapy, especially for the joint combination of PD-1 and CTLA4 immunosuppressants. DHX15 and LARS2 were detected with significantly different expressions in both datasets, which were further confirmed by qRTPCR and immunohistochemical staining. CONCLUSION: Our observations supported an eight-RBP-related signature that could be applied for survival prediction and immunotherapy response of patients with COAD.
Asunto(s)
Adenocarcinoma , Aminoacil-ARNt Sintetasas , Neoplasias del Colon , Humanos , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/genética , Neoplasias del Colon/terapia , Pronóstico , Aprendizaje Automático , Proteínas de Unión al ARN/genética , InmunoterapiaRESUMEN
SARS-CoV-2 causes a spectrum of clinical symptoms from respiratory damage to gastrointestinal disorders. Intestinal infection of SARS-CoV-2 triggers immune response. However, the cellular mechanism that how SARS-CoV-2 initiates and induces intestinal immunity is not understood. Here, we exploited SARS-CoV-2-GFP/ΔN trVLP pseudo-virus system and demonstrated that RIG-I and DHX15 are required for sensing SARS-CoV-2 and inducing cellular immune response through MAVS signaling in intestinal epithelial cells (IECs) upon SARS-CoV-2 infection. NLRP6 also engages in the regulation of SARS-CoV-2 immunity by producing IL-18. Furthermore, primary cellular immune response provoked by SARS-CoV-2 in IECs further cascades activation of MAIT cells and produces cytotoxic cytokines including IFN-γ, granzyme B via an IL-18 dependent mechanism. These findings taken together unveil molecular basis of immune recognition in IECs in response to SARS-CoV-2, and provide insights that intestinal immune cross-talk with other immune cells triggers amplified immunity and probably contributes to immunopathogenesis of COVID-19.
Asunto(s)
COVID-19 , Células Epiteliales , Inmunidad Innata , Intestinos , Humanos , COVID-19/inmunología , Interleucina-18 , SARS-CoV-2 , Transducción de Señal , Células Epiteliales/inmunología , Células Epiteliales/virología , Intestinos/inmunología , Intestinos/virologíaRESUMEN
The RNA helicase DHX15 is widely expressed in immune cells and traditionally thought to be an RNA splicing factor or a viral RNA sensor. However, the role of DHX15 in NK-cell activities has not been studied thus far. Here, we generated Dhx15-floxed mice and found that conditional deletion of Dhx15 in NK cells (Ncr1CreDhx15fl/fl mice) resulted in a marked reduction in NK cells in the periphery and that the remaining Dhx15-deleted NK cells failed to acquire a mature phenotype. As a result, Dhx15-deleted NK cells exhibited profound defects in their cytolytic functions. We also found that deletion of Dhx15 in NK cells abrogated their responsiveness to IL-15, which was associated with inhibition of IL-2/IL-15Rß (CD122) expression and IL-15R signaling. The defects in Dhx15-deleted NK cells were rescued by ectopic expression of a constitutively active form of STAT5. Mechanistically, DHX15 did not affect CD122 mRNA splicing and stability in NK cells but instead facilitated the surface expression of CD122, likely through interaction with its 3'UTR, which was dependent on the ATPase domain of DHX15 rather than its splicing domain. Collectively, our data identify a key role for DHX15 in regulating NK-cell activities and provide novel mechanistic insights into how DHX15 regulates the IL-15 signaling pathway in NK cells.
Asunto(s)
Interleucina-15 , ARN Helicasas , Animales , Homeostasis , Células Asesinas Naturales/metabolismo , Ratones , ARN Helicasas/genética , ARN Helicasas/metabolismo , Transducción de SeñalRESUMEN
DHX15 is an outstanding member of the DEAH-box RNA helicase family. A few studies suggest that DHX15 contributes to carcinogenesis in several tumor cell lines. However, whether DHX15 acts as an oncogene or tumor suppressor and its association with hepatocellular carcinoma (HCC) prognosis are still poorly understood. To address this question, we used immunohistochemistry to evaluate DHX15 expression patterns and their association with clinicopathological factors and the prognosis of patients with HCC. Our results showed that DHX15 expression was significantly higher in cancerous tissues than that in nontumor tissues (Pâ¯<â¯.0001). DHX15 expression in HCC patients was associated with differentiation status (Pâ¯=â¯.018), tumor number (Pâ¯=â¯.048), intrahepatic or extrahepatic metastasis (Pâ¯=â¯.001), serum α-fetoprotein (Pâ¯=â¯.006), hepatitis B virus level (Pâ¯=â¯.018), and recurrence (Pâ¯<â¯.001). In addition, the survival analysis revealed that the DHX15-high group had significantly decreased overall survival time (Pâ¯=â¯.004) and lower 1-year survival rates (Pâ¯=â¯.002) compared with the DHX15-low group. Furthermore, multivariate analysis identified DHX15 expression as an independent factor associated with poor prognosis in HCC (Pâ¯=â¯.036). In summary, these findings demonstrate, for the first time, that DHX15 is significantly upregulated in HCC and its high expression was correlated with poor prognosis, suggesting its pivotal role in the progression of HCC. The present results suggest that DHX15 may serve as a potential prognostic biomarker for HCC patients.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , ARN Helicasas/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/mortalidad , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Regulación hacia Arriba , Adulto JovenRESUMEN
JHDM1D antisense 1 (JHDM1D-AS1), a long non-coding RNA (lncRNA), has been shown to promote pancreatic cancer growth by inducing an angiogenic response. However, its biological and clinical significance in non-small-cell lung cancer (NSCLC) is still unclear. In this study, we examined the expression and prognostic significance of JHDM1D-AS1 in NSCLC. The effects of JHDM1D-AS1 knockdown or overexpression on NSCLC growth and metastasis were investigated. We show that JHDM1D-AS1 is upregulated in NSCLC relative to adjacent normal lung tissues. High JHDM1D-AS1 expression is significantly correlated with advanced tumor, node, and metastasis (TNM) stage and lymph node metastasis. JHDM1D-AS1 expression serves as an independent prognostic factor for overall survival of patients with NSCLC. Functionally, JHDM1D-AS1 knockdown inhibits NSCLC cell aggressiveness both in vitro and in vivo, which is rescued by ectopic expression of JHDM1D-AS1. JHDM1D-AS1 binding stabilizes DHX15 protein in NSCLC cells. DHX15 overexpression enhances NSCLC cell proliferation and invasion, whereas knockdown of DHX15 exerts opposite effects. JHDM1D-AS1-mediated aggressive phenotype is impaired when DHX15 is silenced. Ectopic expression of DHX15 restores the defects in proliferation and invasion of JHDM1D-AS1-depleted NSCLC cells. Collectively, the interaction between JHDM1D-AS1 and DHX15 accounts for NSCLC growth and metastasis. This work provides potential additional therapeutic targets for treatment of NSCLC.
RESUMEN
Genome-wide co-expression analysis is often used for annotating novel gene functions from high-dimensional data. Here, we developed an R package with a Shiny visualization app that creates immuno-networks from RNAseq data using a combination of Weighted Gene Co-expression Network Analysis (WGCNA), xCell immune cell signatures, and Bayesian Network Learning. Using a large publicly available RNAseq dataset we generated a Gene Module-Immune Cell (GMIC) network that predicted causal relationships between DEAH-box RNA helicase (DHX)15 and genes associated with humoral immunity, suggesting that DHX15 may regulate B cell fate. Deletion of DHX15 in mouse B cells led to impaired lymphocyte development, reduced peripheral B cell numbers, and dysregulated expression of genes linked to antibody-mediated immune responses similar to the genes predicted by the GMIC network. Moreover, antigen immunization of mice demonstrated that optimal primary IgG1 responses required DHX15. Intrinsic expression of DHX15 was necessary for proliferation and survival of activated of B cells. Altogether, these results support the use of co-expression networks to elucidate fundamental biological processes.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Inmunomodulación , ARN Helicasas/genética , Animales , Biopsia , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Inmunomodulación/genética , Ratones , ARN Helicasas/metabolismoRESUMEN
RNA helicases play an important role in the response to microbial infection. Retinoic acid inducible gene-I (RIG-I) and members of the RIG-I-like receptor (RLR) family of helicases function as cytoplasmic pattern recognition receptors (PRRs) whose actions are essential for recognition of RNA viruses. RIG-I association with pathogen-associated molecular patterns (PAMPs) within viral RNA leads to its activation and signaling via the mitochondrial antiviral signaling (MAVS) adapter protein. This interaction mediates downstream signaling events that drive the innate immune response to virus infection. Here we identify the DEAH-box RNA helicase DHX15 as a RLR binding partner and signaling cofactor. In human cells, DHX15 is required for virus-induced RLR signaling of innate immune gene expression. Knockdown of DHX15 increased susceptibility to infection by RNA viruses of diverse genera, including Paramyxoviridae, Rhabdoviridae, and Picornaviridae. DHX15 associates with RIG-I caspase activation and recruitment domains (CARDs) through its amino terminus, in which the complex is recruited to MAVS on virus infection. Importantly, although DHX15 cannot substitute for RIG-I in innate immune signaling, DHX15 selectively binds PAMP RNA to promote RIG-I ATP hydrolysis and signaling activation in response to viral RNA. Our results define DHX15 as a coreceptor required for RLR innate immune responses to control RNA virus infection.
Asunto(s)
ARN Helicasas/inmunología , Infecciones por Virus ARN/inmunología , Receptores de Reconocimiento de Patrones/inmunología , Virus Sendai/inmunología , Transducción de Señal/inmunología , Células Cultivadas , Células HEK293 , HumanosRESUMEN
Yeast proteins Ntr1, Ntr2 and Prp43 function in spliceosome disassembly. An Ntr1-Ntr2 protein complex recruits Prp43 to allow the removal of the lariat-intron in late-stage RNA splicing activity. Based on amino-acid sequence similarities across species, TFIP11 and mDEAH9/Dhx15 have been identified as homologues of yeast Ntr1 and Prp43, respectively. The N-terminal region of TFIP11 contains a G-patch, which is a highly conserved domain of many RNA-processing proteins. TFIP11 displays a unique and characteristic subnuclear localization pattern, in close proximity to SC35 nuclear speckles. Transfected GFP-tagged mDEAH9 displays an evenly distributed nuclear localization and is excluded from the nucleoli; however when TFIP11 and mDEAH9 are co-transfected, both proteins colocalize to distinct nuclear speckles. These data show that TFIP11 recruits mDEAH9 suggesting that these two proteins have similar biological activities to their yeast counterparts.
RESUMEN
The role of DHX15, a newly identified DEAH-box RNA helicase, in leukemogenesis remains elusive. Here, we identified a recurrent mutation in DHX15 (NM_001358:c.664C>G: p.(R222G)) in one familial AML patient and 4/240 sporadic AML patients. Additionally, DHX15 was commonly overexpressed in AML patients and associated with poor overall survival (OS) (P=0.019) and relapse-free survival (RFS) (P=0.032). In addition, we found a distinct expression pattern of DHX15. DHX15 was highly expressed in hematopoietic stem cells and leukemia cells but was lowly expressed in mature blood cells. DHX15 was down-regulated when AML patients achieved disease remission or when leukemia cell lines were induced to differentiate. DHX15 silencing greatly inhibited leukemia cell proliferation and induced cell apoptosis and G1-phase arrest. In contrast, the restoration of DHX15 expression rescued cell viability and reduced cell apoptosis. In addition, we found that DHX15 was down-regulated when cell apoptosis was induced by ATO (arsenic trioxide); overexpression of DHX15 caused dramatic resistance to ATO-induced cell apoptosis, suggesting an important role for DHX15 in cell apoptosis. We further explored the mechanism of DHX15 in apoptosis and found that overexpression of DHX15 activated NF-kB transcription. Knockdown of DHX15 inhibited the nuclear translocation and activation of the NF-kB subunit P65 in leukemia cells. Several downstream targets of the NF-kB pathway were also down-regulated, and apoptosis-associated genes CASP3 and PARP were activated. In conclusion, this study represents the first demonstration that DHX15 plays an important role in leukemogenesis via the NF-kB signaling pathway and may serve as an independent prognostic marker for AML.