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Clin Transl Med ; 12(5): e881, 2022 May.
Article En | MEDLINE | ID: mdl-35604033

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), a difficult-to-treat cancer, is expected to become the second-largest cause of cancer-related deaths by 2030, while colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer deaths. Currently, there is no effective treatment for PDAC patients. The development of novel agents to effectively treat these cancers remains an unmet clinical need. FL118, a novel anticancer small molecule, exhibits high efficacy against cancers; however, the direct biochemical target of FL118 is unknown. METHODS: FL118 affinity purification, mass spectrometry, Nanosep centrifugal device and isothermal titration calorimetry were used for identifying and confirming FL118 binding to DDX5/p68 and its binding affinity. Immunoprecipitation (IP), western blots, real-time reverse transcription PCR, gene silencing, overexpression (OE) and knockout (KO) were used for analysing gene/protein function and expression. Chromatin IP was used for analysing protein-DNA interactions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid assay and human PDAC/CRC cell/tumour models were used for determining PDAC/CRC cell/tumour in vitro and in vivo growth. RESULTS: We discovered that FL118 strongly binds to dephosphorylates and degrades the DDX5 oncoprotein via the proteasome degradation pathway without decreasing DDX5 mRNA. Silencing and OE of DDX5 indicated that DDX5 is a master regulator for controlling the expression of multiple oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras. Genetic manipulation of DDX5 in PDAC cells affects tumour growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Our human tumour animal model studies further indicated that FL118 exhibits high efficacy to eliminate human PDAC and CRC tumours that have a high expression of DDX5, while FL118 exhibits less effectiveness in PDAC and CRC tumours with low DDX5 expression. CONCLUSION: DDX5 is a bona fide FL118 direct target and can act as a biomarker for predicting PDAC and CRC tumour sensitivity to FL118. This would greatly impact FL118 precision medicine for patients with advanced PDAC or advanced CRC in the clinic. FL118 may act as a 'molecular glue degrader' to directly glue DDX5 and ubiquitination regulators together to degrade DDX5.

Carcinoma, Pancreatic Ductal , Colorectal Neoplasms , Pancreatic Neoplasms , Animals , Benzodioxoles , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Humans , Indolizines , Oncogene Proteins/metabolism , Oncogene Proteins/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Survivin/genetics , Survivin/metabolism , Survivin/therapeutic use
Int J Med Sci ; 19(3): 596-608, 2022.
Article En | MEDLINE | ID: mdl-35370459

Purpose: Mutations (K11E or E271K) of DEAD-box RNA helicase 24 (DDX24) were related to multi-organ venous lymphatic malformation syndrome (MOVLD). However, the relationship between these mutations and DDX24-function still remains unknown. Understanding whether K11E and E271K cause "loss-of-function" or "gain-of-function" for DDX24 is significant for related diseases. DDX24 was reported to be related to tumors closely, thus this study aims to explore how K11E and E271K affect DDX24-function in tumor proliferation. Methods: Cell lines stably expressing wild-type DDX24, K11E-DDX24, E271K-DDX24, along with vector only based on Chinese hamster ovary cells (CHO) and Balb/c tumor-bearing mice models were constructed. Then immunofluorescence staining, proliferation assay and colony formation assay in vitro and 18F-FDG PET/CT-scan were performed. Finally, the tumor tissues were collected to perform transcriptome sequencing to predict the potential mechanism. Results: Contrasted with CHO-WT-DDX24, CHO-K11E-DDX24 or CHO-E271K-DDX24 showed a decreased number of nucleoli, a slower proliferation rate and a lower colony formation rate significantly. Moreover, mice, inoculated with CHO-K11E-DDX24 or CHO-E271K-DDX24 cells, showed lower tumor formation rate, slower tumor growth rate, better prognosis, reduced standard uptake value and Ki of glucose in subcutaneous tumors. Sequencing indicated CHO-K11E-DDX24 or CHO-E271K-DDX24 caused increasing expression of TNF or chemokines and alteration in immune-related signal pathways. Conclusion: K11E or E271K mutation could lead to "loss-of-function" of DDX24 in cell proliferation and tumor bearing mice, which may be acted by non-specific immune killing to inhibit tumor growth.

DEAD-box RNA Helicases , Neoplasms , Animals , CHO Cells , Cricetinae , Cricetulus , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Mice , Mutation , Positron Emission Tomography Computed Tomography
Ren Fail ; 44(1): 137-145, 2022 Dec.
Article En | MEDLINE | ID: mdl-35392757

BACKGROUND: Sustained type I interferon (IFN) activation via Toll-like receptor (TLR) 3, 7 and 9 signaling has been reported to play a pivotal role in the development of lupus nephritis (LN). Although type I IFN activation has been shown to induce interferon-stimulated genes (ISGs) expression in systemic lupus erythematosus, the implication of ISGs expression in intrinsic glomerular cells remains largely unknown. METHODS: We treated cultured human glomerular endothelial cells (GECs) with polyinosinic-polycytidylic acid (poly IC), R848, and CpG (TLR3, TLR7, and TLR9 agonists, respectively) and analyzed the expression of DExD/H-Box Helicase 60 (DDX60), a representative ISG, using quantitative reverse transcription-polymerase chain reaction and western blotting. Additionally, RNA interference against IFN-ß or DDX60 was performed. Furthermore, cleavage of caspase 9 and poly (ADP-ribose) polymerase (PARP), markers of cells undergoing apoptosis, was examined using western blotting. We conducted an immunofluorescence study to examine endothelial DDX60 expression in biopsy specimens from patients with LN. RESULTS: We observed that endothelial expression of DDX60 was induced by poly IC but not by R848 or CpG, and RNA interference against IFN-ß inhibited poly IC-induced DDX60 expression. DDX60 knockdown induced cleavage of caspase 9 and PARP. Intense endothelial DDX60 expression was observed in biopsy specimens from patients with diffuse proliferative LN. CONCLUSION: Glomerular endothelial DDX60 expression may prevent apoptosis, which is involved in the pathogenesis of LN. Modulating the upregulation of the regional innate immune system via TLR3 signaling may be a promising treatment target for LN.

DEAD-box RNA Helicases , Lupus Nephritis , Toll-Like Receptor 3 , Antiviral Agents , Caspase 9/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Endothelial Cells/metabolism , Humans , Interferon-beta/pharmacology , Poly I-C/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism
Cell Rep ; 39(1): 110640, 2022 04 05.
Article En | MEDLINE | ID: mdl-35385737

Synthesis of ribosomes begins in the nucleolus with formation of the 90S pre-ribosome, during which the pre-40S and pre-60S pathways diverge by pre-rRNA cleavage. However, it remains unclear how, after this uncoupling, the earliest pre-60S subunit continues to develop. Here, we reveal a large-subunit intermediate at the beginning of its construction when still linked to the 90S, the precursor to the 40S subunit. This primordial pre-60S is characterized by the SPOUT domain methyltransferase Upa1-Upa2, large α-solenoid scaffolds, Mak5, one of several RNA helicases, and two small nucleolar RNA (snoRNAs), C/D box snR190 and H/ACA box snR37. The emerging pre-60S does not efficiently disconnect from the 90S pre-ribosome in a dominant mak5 helicase mutant, allowing a 70-nm 90S-pre-60S bipartite particle to be visualized by electron microscopy. Our study provides insight into the assembly pathway when the still-connected nascent 40S and 60S subunits are beginning to separate.

Ribosome Subunits, Large, Eukaryotic , Ribosomes , Saccharomyces cerevisiae Proteins , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , RNA, Small Nucleolar/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
Cell Rep ; 39(2): 110673, 2022 04 12.
Article En | MEDLINE | ID: mdl-35417682

RNA activation (RNAa) is an uncharacterized mechanism of transcriptional activation mediated by small RNAs, such as microRNAs (miRNAs). A critical issue in RNAa research is that it is difficult to distinguish between changes in gene expression caused indirectly by post-transcriptional regulation and direct induction of gene expression by RNAa. Therefore, in this study, we seek to identify a key factor involved in RNAa, using the induction of ZMYND10 by miR-34a as a system to evaluate RNAa. We identify the positive transcription elongation factors CDK9 and DDX21, which form a complex with nuclear AGO and TNRC6A, as important transcriptional activators of RNAa. In addition, we find that inhibition of DDX21 suppresses RNAa by miR-34a and other miRNAs without inhibiting post-transcriptional regulation. Our findings reveal a strong connection between RNAa and release of paused Pol II, facilitating RNAa research by making it possible to separately analyze post-transcriptional regulation and RNAa.

Cyclin-Dependent Kinase 9 , DEAD-box RNA Helicases , MicroRNAs , RNA Polymerase II , Cell Nucleus/metabolism , Cyclin-Dependent Kinase 9/metabolism , DEAD-box RNA Helicases/metabolism , Gene Expression Regulation , MicroRNAs/genetics , RNA Polymerase II/metabolism , Transcriptional Activation
Cell Rep ; 39(2): 110636, 2022 04 12.
Article En | MEDLINE | ID: mdl-35417719

Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.

Neoplasms , Oncogenes , DEAD-box RNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Male , Minor Histocompatibility Antigens/metabolism , Neoplasms/genetics , RNA-Binding Proteins/genetics , Sex Chromosomes/metabolism , X Chromosome , Y Chromosome
Life Sci Alliance ; 5(8)2022 Aug.
Article En | MEDLINE | ID: mdl-35440492

Spinal muscular atrophy, the leading genetic cause of infant mortality, is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. SMN is a multifunctional protein that is implicated in numerous cytoplasmic and nuclear processes. Recently, increasing attention is being paid to the role of SMN in the maintenance of DNA integrity. DNA damage and genome instability have been linked to a range of neurodegenerative diseases. The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. Instability in rDNA has been associated with cancer, premature ageing syndromes, and a number of neurodegenerative disorders. Here, we report that SMN-deficient cells exhibit increased rDNA damage leading to impaired ribosomal RNA synthesis and translation. We also unravel an interaction between SMN and RNA polymerase I. Moreover, we uncover an spinal muscular atrophy motor neuron-specific deficiency of DDX21 protein, which is required for resolving R-loops in the nucleolus. Taken together, our findings suggest a new role of SMN in rDNA integrity.

Motor Neurons , Muscular Atrophy, Spinal , DEAD-box RNA Helicases/metabolism , DNA Damage/genetics , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Humans , Infant , Motor Neurons/metabolism , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Ribosomes/genetics , Ribosomes/metabolism
Proc Natl Acad Sci U S A ; 119(16): e2112482119, 2022 04 19.
Article En | MEDLINE | ID: mdl-35412895

MiR-126 and miR-155 are key microRNAs (miRNAs) that regulate, respectively, hematopoietic cell quiescence and proliferation. Herein we showed that in acute myeloid leukemia (AML), the biogenesis of these two miRNAs is interconnected through a network of regulatory loops driven by the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). In fact, FLT3-ITD induces the expression of miR-155 through a noncanonical mechanism of miRNA biogenesis that implicates cytoplasmic Drosha ribonuclease III (DROSHA). In turn, miR-155 down-regulates SH2-containing inositol phosphatase 1 (SHIP1), thereby increasing phosphor-protein kinase B (AKT) that in turn serine-phosphorylates, stabilizes, and activates Sprouty related EVH1 domain containing 1 (SPRED1). Activated SPRED1 inhibits the RAN/XPO5 complex and blocks the nucleus-to-cytoplasm transport of pre-miR-126, which cannot then complete the last steps of biogenesis. The net result is aberrantly low levels of mature miR-126 that allow quiescent leukemia blasts to be recruited into the cell cycle and proliferate. Thus, miR-126 down-regulation in proliferating AML blasts is downstream of FLT3-ITD­dependent miR-155 expression that initiates a complex circuit of concatenated regulatory feedback (i.e., miR-126/SPRED1, miR-155/human dead-box protein 3 [DDX3X]) and feed-forward (i.e., miR-155/SHIP1/AKT/miR-126) regulatory loops that eventually converge into an output signal for leukemic growth.

Leukemia, Myeloid, Acute , MicroRNAs , fms-Like Tyrosine Kinase 3 , DEAD-box RNA Helicases/metabolism , Down-Regulation , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , MicroRNAs/metabolism , Mutation , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolism
Nucleic Acids Res ; 50(7): 4042-4053, 2022 04 22.
Article En | MEDLINE | ID: mdl-35380691

Mtr4 is a eukaryotic RNA helicase required for RNA decay by the nuclear exosome. Previous studies have shown how RNA en route to the exosome threads through the highly conserved helicase core of Mtr4. Mtr4 also contains an arch domain, although details of potential interactions between the arch and RNA have been elusive. To understand the interaction of Saccharomyces cerevisiae Mtr4 with various RNAs, we have characterized RNA binding in solution using hydrogen-deuterium exchange mass spectrometry, and affinity and unwinding assays. We have identified RNA interactions within the helicase core that are consistent with existing structures and do not vary between tRNA, single-stranded RNA and double-stranded RNA constructs. We have also identified novel RNA interactions with a region of the arch known as the fist or KOW. These interactions are important for RNA unwinding and vary in strength depending on RNA structure and length. They account for Mtr4 discrimination between different RNAs. These interactions further drive Mtr4 to adopt a closed conformation characterized by reduced dynamics of the arch arm and intra-domain contacts between the fist and helicase core.

DEAD-box RNA Helicases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , DEAD-box RNA Helicases/metabolism , DNA Helicases/metabolism , Deuterium/metabolism , Deuterium Exchange Measurement , Mass Spectrometry , RNA/genetics , RNA/metabolism , RNA Helicases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
Bioengineered ; 13(4): 10123-10140, 2022 Apr.
Article En | MEDLINE | ID: mdl-35435126

Increasing evidence suggests that plasmacytoma variant translocation 1 (PVT1) plays a vital role in the development of multiple tumors including lung adenocarcinoma (LUAD). Eukaryotic initiation factor 4A-3 (EIF4A3) is considered a key factor in human cancers. However, the role and potential mechanism of PVT1 combined with EIF4A3 in LUAD remain unclear. This study investigated the effects and regulatory mechanisms of PVT1, EIF4A3, and circLMNB2 on the growth, migration, invasion, and epithelial-mesenchymal transition (EMT) of LUAD cells (H1299 and HCC827 cells) The expression level, diagnostic value and prognostic significance of PVT1, EIF4A3, and circLMNB2 were assessed, and enrichment analysis was performed using R package. Rescue experiments and a xenograft model were used to validate the PVT1/EIF4A3/circLMNB2 axis in LUAD. PVT1 and EIF4A3 were upregulated and indicated poor prognosis in LUAD. Knockdown of PVT1 and EIF4A3 suppressed LUAD cell proliferation, migration, invasion, and EMT. Mechanistically, PVT1 was stabilized by EIF4A3. PVT1 could recruit EIF4A3 to promote circLMNB2 expression. Rescue experiments indicated that circLMNB2 overexpression could reverse the reduced behavior caused by PVT1 or EIF4A3 knockdown. Enrichment analysis showed that PVT1/EIF4A3/circLMNB2 may regulate LUAD development by participating in ribosome biogenesis and spliceosome formation. Our findings demonstrate that PVT1/EIF4A3/circLMNB2 enhances the malignant behaviors of LUAD cells, providing a novel perspective for the clinical treatment of LUAD.

Adenocarcinoma of Lung , Lung Neoplasms , Adenocarcinoma of Lung/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Eukaryotic Initiation Factor-3/genetics , Eukaryotic Initiation Factor-3/metabolism , Eukaryotic Initiation Factor-4A/genetics , Eukaryotic Initiation Factor-4A/metabolism , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/pathology , RNA, Circular , RNA, Long Noncoding
Mol Biol (Mosk) ; 56(2): 206-226, 2022.
Article Ru | MEDLINE | ID: mdl-35403616

The review discusses the role that proteins interacting with the translation termination factors eRF1 and eRF3 play in the control of protein synthesis and prionization. These proteins interact not only with each other, but also with many other proteins involved in controlling the efficiency of translation termination, and associate translation termination with other cell processes. The termination of translation is directly related not only to translation re-initiation and ribosome recycling, but also to mRNA stability and protein quality control. This connection is ensured by the interaction of eRF1 and eRF3 with proteins participating in various cell metabolic processes, such as mRNA transport from the nucleus into the cytoplasm (Dbp5/DDX19 and Gle1), ribosome recycling (Rli1/ABCE1), mRNA degradation (Upf proteins), and translation initiation (Pab1/PABP). In addition to genetic control, there is epigenetic control of translation termination. This mechanism is associated with prion polymerization of the Sup35 protein to form the [PSI^(+)] prion. The maintenance of the [PSI^(+)] prion, like other yeast prions, requires the operation of a system of molecular chaperones and protein sorting factors. The review considers in detail the interaction of the translation termination factors with proteins involved in various cellular processes.

Prions , Saccharomyces cerevisiae Proteins , DEAD-box RNA Helicases/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Peptide Chain Termination, Translational/genetics , Peptide Termination Factors/genetics , Peptide Termination Factors/metabolism , Poly(A)-Binding Proteins/metabolism , Prions/genetics , Prions/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
Stem Cell Reports ; 17(4): 879-893, 2022 Apr 12.
Article En | MEDLINE | ID: mdl-35303436

DDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms, but whether it affects hematopoiesis is unknown. Using a knockout mouse, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation, particularly of myeloid lineage cells. Transplantation of Ddx41 knockout fetal liver and adult bone marrow (BM) cells was unable to rescue mice from lethal irradiation, and knockout stem cells were also defective in colony formation assays. RNA-seq analysis of Lin-/cKit+/Sca1+Ddx41 knockout cells from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered. Furthermore, differential splicing of genes involved in key biological processes was observed. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through regulating gene expression programs and splicing.

Hematopoiesis , Hematopoietic Stem Cells , Animals , Bone Marrow Cells/metabolism , Cell Differentiation/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Mice , Mice, Knockout
Viruses ; 14(3)2022 02 24.
Article En | MEDLINE | ID: mdl-35336874

The porcine reproductive and respiratory syndrome virus (PRRSV) remains a persistent hazard in the global pig industry. DEAD (Glu-Asp-Ala-Glu) box helicase 21 (DDX21) is a member of the DDX family. In addition to its function of regulating cellular RNA metabolism, DDX21 also regulates innate immunity and is involved in the replication cycle of some viruses. However, the relationship between DDX21 and PRRSV has not yet been explored. Here, we found that a DDX21 overexpression promoted PRRSV replication, whereas knockdown of DDX21 reduced PRRSV proliferation. Mechanistically, DDX21 promoted PRRSV replication independently of its ATPase, RNA helicase, and foldase activities. Furthermore, overexpression of DDX21 stabilized the expressions of PRRSV nsp1α, nsp1ß, and nucleocapsid proteins, three known antagonists of interferon ß (IFN-ß). Knockdown of DDX21 activated the IFN-ß signaling pathway in PRRSV-infected cells, suggesting that the effect of DDX21 on PRRSV-encoded IFN-ß antagonists may be a driving factor for its contribution to viral proliferation. We also found that PRRSV infection enhanced DDX21 expression and promoted its nucleus-to-cytoplasm translocation. Screening PRRSV-encoded proteins showed that nsp1ß interacted with the C-terminus of DDX21 and enhanced the expression of DDX21. Taken together, these findings reveal that DDX21 plays an important role in regulating PRRSV proliferation through multiple mechanisms.

Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Animals , Cell Line , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Interferon-beta/genetics , Porcine Reproductive and Respiratory Syndrome/genetics , Porcine respiratory and reproductive syndrome virus/metabolism , Swine , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/physiology
Sci Rep ; 12(1): 3967, 2022 03 10.
Article En | MEDLINE | ID: mdl-35273248

The human DEAD-box protein 3 (DDX3) has been reported as a positive regulator and functions in the induction of type I interferon signaling. We elucidated the function of DDX3 in the positive regulation of IFNB production in non-pDC cells. We found that DDX3 regulates virus-induced activation of IFNB at the level of IRF-3. However, it does not affect conventional innate signaling, including IRF-3 phosphorylation, dimerization, or nuclear translocation of IRF-3, but has some downstream events after IRF-3 phosphorylation. Co-immunoprecipitation analyses revealed that DDX3 interacts with IRF-3 through its DNA-binding domain and promotes IRF-3-mediated IFNB promoter activation. DDX3 does not affect the formation of the IRF-3/p300/CBP complex. Instead, ChIP and EMSA assay revealed that DDX3 promotes the recruitment of IRF-3 and transcriptional co-activator p300/CBP to the IFNB promoter. The ATP binding pocket of DDX3 is involved in this association and is essential for the transcriptional activation. Taken together, our study demonstrates that DDX3 plays an important role in guiding a transcription factor complex formed by antiviral signaling to the target gene promoter.

DEAD-box RNA Helicases , Interferon Regulatory Factor-3 , Cell Nucleus/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Humans , Immunoprecipitation , Interferon Regulatory Factor-3/metabolism , Promoter Regions, Genetic , Transcriptional Activation
BMC Med Genomics ; 15(1): 72, 2022 03 27.
Article En | MEDLINE | ID: mdl-35346191

BACKGROUND: Heart failure (HF) is the most common potential cause of death, causing a huge health and economic burden all over the world. So far, some impressive progress has been made in the study of pathogenesis. However, the underlying molecular mechanisms leading to this disease remain to be fully elucidated. METHODS: The microarray data sets of GSE76701, GSE21610 and GSE8331 were retrieved from the gene expression comprehensive database (GEO). After merging all microarray data and adjusting batch effects, differentially expressed genes (DEG) were determined. Functional enrichment analysis was performed based on Gene Ontology (GO) resources, Kyoto Encyclopedia of Genes and Genomes (KEGG) resources, gene set enrichment analysis (GSEA), response pathway database and Disease Ontology (DO). Protein protein interaction (PPI) network was constructed using string database. Combined with the above important bioinformatics information, the potential key genes were selected. The comparative toxicological genomics database (CTD) is used to explore the interaction between potential key genes and HF. RESULTS: We identified 38 patients with heart failure and 16 normal controls. There were 315 DEGs among HF samples, including 278 up-regulated genes and 37 down-regulated genes. Pathway enrichment analysis showed that most DEGs were significantly enriched in BMP signal pathway, transmembrane receptor protein serine/threonine kinase signal pathway, extracellular matrix, basement membrane, glycosaminoglycan binding, sulfur compound binding and so on. Similarly, GSEA enrichment analysis showed that DEGs were mainly enriched in extracellular matrix and extracellular matrix related proteins. BBS9, CHRD, BMP4, MYH6, NPPA and CCL5 are central genes in PPI networks and modules. CONCLUSIONS: The enrichment pathway of DEGs and GO may reveal the molecular mechanism of HF. Among them, target genes EIF1AY, RPS4Y1, USP9Y, KDM5D, DDX3Y, NPPA, HBB, TSIX, LOC28556 and XIST are expected to become new targets for heart failure. Our findings provide potential biomarkers or therapeutic targets for the further study of heart failure and contribute to the development of advanced prediction, diagnosis and treatment strategies.

Gene Expression Profiling , Heart Failure , Biomarkers/metabolism , Computational Biology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Gene Ontology , Gene Regulatory Networks , Heart Failure/genetics , Histone Demethylases/genetics , Histone Demethylases/metabolism , Humans , Minor Histocompatibility Antigens , Protein Interaction Maps/genetics
BMB Rep ; 55(3): 125-135, 2022 Mar.
Article En | MEDLINE | ID: mdl-35236544

Continuously renewing the proteome, translation is exquisitely controlled by a number of dedicated factors that interact with the ribosome. The RNA helicase DDX3 belonging to the DEAD box family has emerged as one of the critical regulators of translation, the failure of which is frequently observed in a wide range of proliferative, degenerative, and infectious diseases in humans. DDX3 unwinds double-stranded RNA molecules with coupled ATP hydrolysis and thereby remodels complex RNA structures present in various protein-coding and noncoding RNAs. By interacting with specific features on messenger RNAs (mRNAs) and 18S ribosomal RNA (rRNA), DDX3 facilitates translation, while repressing it under certain conditions. We review recent findings underlying these properties of DDX3 in diverse modes of translation, such as cap-dependent and cap-independent translation initiation, usage of upstream open reading frames, and stress-induced ribonucleoprotein granule formation. We further discuss how disease-associated DDX3 variants alter the translation landscape in the cell. [BMB Reports 2022; 55(3): 125-135].

DEAD-box RNA Helicases , Ribosomes , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Humans , RNA, Messenger , Ribonucleoproteins/genetics , Ribosomes/metabolism
Nucleic Acids Res ; 50(7): 3998-4011, 2022 04 22.
Article En | MEDLINE | ID: mdl-35286399

The DEAD-box protein Dbp5 is essential for RNA export, which involves regulation by the nucleoporins Gle1 and Nup159 at the cytoplasmic face of the nuclear pore complex (NPC). Mechanistic understanding of how these nucleoporins regulate RNA export requires analyses of the intrinsic and activated Dbp5 ATPase cycle. Here, kinetic and equilibrium analyses of the Saccharomyces cerevisiae Gle1-activated Dbp5 ATPase cycle are presented, indicating that Gle1 and ATP, but not ADP-Pi or ADP, binding to Dbp5 are thermodynamically coupled. As a result, Gle1 binds Dbp5-ATP > 100-fold more tightly than Dbp5 in other nucleotide states and Gle1 equilibrium binding of ATP to Dbp5 increases >150-fold via slowed ATP dissociation. Second, Gle1 accelerated Dbp5 ATPase activity by increasing the rate-limiting Pi release rate constant ∼20-fold, which remains rate limiting. These data show that Gle1 activates Dbp5 by modulating ATP binding and Pi release. These Gle1 activities are expected to facilitate ATPase cycling, ensuring a pool of ATP bound Dbp5 at NPCs to engage RNA during export. This work provides a mechanism of Gle1-activation of Dbp5 and a framework to understand the joint roles of Gle1, Nup159, and other nucleoporins in regulating Dbp5 to mediate RNA export and other Dbp5 functions in gene expression.

DEAD-box RNA Helicases , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins , Saccharomyces cerevisiae Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Nuclear Pore Complex Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Phosphates/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
J Tradit Chin Med ; 42(1): 108-115, 2022 02.
Article En | MEDLINE | ID: mdl-35294130

OBJECTIVE: To further clarify the anticancer mechanisms of Liujunzi decoction and provide possible targets for the treatment of advanced-stage nonsmall cell lung cancer (NSCLC) by re-analyzing differential gene expression profile of peripheral blood mononuclear cells (PBMCs) from Liujunzi decoctiontreated NSCLC patients receiving first-line chemotherapy. METHODS: The PBMC gene expression microarray data set GSE61926 was retrieved from a high throughput gene expression database. Differentially expressed genes (DEGs) were screened by paired sample t-test and the multiple ratio method. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed using the DAVID database. The protein-protein interaction (PPI) network was constructed using interaction gene library retrieval tools and Cytoscape software. RESULTS: A total of 162 DEGs were identified, with 67 upregulated genes and 95 downregulated genes. The functional distribution of Gene Oncology (GO) genes showed that DEGs were mostly concentrated in extracellular regions, calcium ion binding, and transcriptase activity. KEGG pathway analysis showed that cytokine-cytokine receptor interactions were significantly enriched. PPI network analysis screened out the top 10 central protein-coding genes with the highest nodal degree: IL2, PIWIL4, DICER1, PIWIL2, SAA1, XCL1, IL22RA1, ARHGAP11A, DCP1A, and GDNF. Among them, the central protein-coding gene with the highest node degree was IL2. In addition, the central protein-coding genes with high node degrees and high molecular complex detection (MCODE) scores were PIWIL4, DICER1, PIWIL2, and DCP1A, all of which are related to tumor development. CONCLUSIONS: One signaling pathway and 10 central protein-coding genes related to anticancer mechanisms were screened by re-analysis of GSE61926 data. IL2, PIWIL4, DICER1, PIWIL2, and DCP1A may have important roles in the mechanism of Liujunzi decoction treatment against NSCLC. Our results suggest that the anticancer mechanism of Liujunzi decoction may be related to gene silencing by RNA and the biological processes of piwi-interacting RNA and other small RNAs.

Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Computational Biology/methods , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Drugs, Chinese Herbal , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Humans , Interleukin-2/genetics , Leukocytes, Mononuclear/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Ribonuclease III/genetics , Ribonuclease III/metabolism
Environ Toxicol ; 37(6): 1483-1494, 2022 Jun.
Article En | MEDLINE | ID: mdl-35343646

BACKGROUND: Triple-negative breast cancer (TNBC) is a common hypotype of breast cancer. Circular RNAs (circRNAs) are burgeoning serve as vital controllers in numerous tumors. Nevertheless, the expression and regulatory mode of circRNAs in TNBC are still indistinct. This paper aimed to reveal the function and molecular mechanism of circular RNA dehydrodolichyl diphosphate synthase (circDHDDS) in TNBC. METHODS: The contents of circDHDDS, DHDDS mRNA, microRNA-362-3p (miR-362-3p) and DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (DDX5) were indicated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were executed to assess cell proliferation. The flow cytometry assay was utilized to detect cell apoptosis. The transwell assay and tube formation assay were applied to measure cell migration, invasion and angiogenesis. The targeted relationships of miR-362-3p and circDHDDS or DDX5 were forecasted and detected by dual-luciferase reporter assay. The in vivo test was implemented to confirm the effect of circDHDDS. RESULTS: The contents of circDHDDS and DDX5 were increased, and miR-362-3p level was decreased in TNBC. CircDHDDS deficiency reserved cell proliferation, migration, invasion and angiogenesis, while facilitated cell apoptosis in TNBC cells. Furthermore, miR-362-3p was validated to exert a tumor repressive effect in TNBC cells by suppressing DDX5. Moreover, DDX5 could regulate the development of TNBC. The experimental data exposed that levels of miR-362-3p presented noteworthy negative correlation with circDHDDS and DDX5, while circDHDDS and DDX5 exhibited significant positive correlation. In mechanism, circDHDDS bound to miR-362-3p to modulate DDX5 expression. In addition, circDHDDS knock-down also attenuated tumor growth. CONCLUSION: CircDHDDS expedited TNBC by swelling DDX5 via adapting miR-362-3p.

MicroRNAs , Triple Negative Breast Neoplasms , Alkyl and Aryl Transferases , Cell Line, Tumor , Cell Movement , Cell Proliferation/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology
Gene ; 823: 146369, 2022 May 20.
Article En | MEDLINE | ID: mdl-35240256

Vasa is the most studied germ cell marker that is indispensable for germ cell development in teleost fishes. Here, a vasa full-length cDNA from Oryzias celebensis was isolated. Analysis of gene expression by reversed transcription polymerase chain reaction and in situ hybridization showed the vasa transcript was maternally inherited and specifically expressed in germ cells during embryogenesis and in adult gonads. During embryogenesis, vasa mRNA was widely distributed in the embryos until the somitogenesis stage and then specifically expressed in primordial germ cells (PGCs). In the testis, vasa expression was highest in spermatogonia and gradually decreased during spermatogenesis. In ovary, vasa expression was present predominantly in immature oocytes and persisted throughout oogenesis. Constructs containing green or red fluorescence proteins and vasa 3' UTR or dnd 3' UTR, confirmed stable vasa expression in the PGCs of O. celebensis and co-expression of the two genes. In summary, the conservation of vasa expression in embryonic and adult germ cells of both sexes compared to other vertebrates suggests its function is also widely conserved.

DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Oocytes/metabolism , Oryzias/embryology , Testis/metabolism , Animals , Cloning, Molecular , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Female , Fish Proteins/genetics , Fish Proteins/metabolism , Gene Expression Regulation, Developmental , In Situ Hybridization, Fluorescence , Male , Maternal Inheritance , Oryzias/genetics , Oryzias/metabolism , Tissue Distribution