PARP1, DIDO3, and DHX9 Proteins Mutually Interact in Mouse Fibroblasts, with Effects on DNA Replication Dynamics, Senescence, and Oncogenic Transformation.

The regulated formation and resolution of R-loops is a natural process in physiological gene expression. Defects in R-loop metabolism can lead to DNA replication stress, which is associated with a variety of diseases and, ultimately, with cancer. The proteins PARP1, DIDO3, and DHX9 are important players in R-loop regulation. We previously described the interaction between DIDO3 and DHX9. Here, we show that, in mouse embryonic fibroblasts, the three proteins are physically linked and dependent on PARP1 activity. The C-terminal truncation of DIDO3 leads to the impairment of this interaction; concomitantly, the cells show increased replication stress and senescence. DIDO3 truncation also renders the cells partially resistant to in vitro oncogenic transformation, an effect that can be reversed by immortalization. We propose that PARP1, DIDO3, and DHX9 proteins form a ternary complex that regulates R-loop metabolism, preventing DNA replication stress and subsequent senescence.

G-Quadruplexes and the DNA/RNA helicase DHX36 in health, disease, and aging.

G-Quadruplex (G4) DNA (G4 DNA) and RNA (G4 RNA) are secondary nucleic acid structures that have multiple roles in vital cellular processes. G4 DNA- and RNA-binding proteins and unwinding helicases associate with and regulate G4s during virtually all processes that involve DNA and RNA. DEAH-Box helicase 36 (DHX36), a member of the large DExD/H box helicase family, enzymatically unwinds both G4 DNA and G4 RNA. By exerting its G4 helicase function, DHX36 regulates transcription, genomic stability, telomere maintenance, translation and RNA metabolism. This review will provide an overview of G4s and DHX36, including DHX36's potential role in neuronal development and neurodegeneration. We conclude with a discussion of the possible functions of G4s and DHX36 in the aging brain.

DDX46 accelerates the proliferation of glioblastoma by activating the MAPK-p38 signaling.

PURPOSE: To analyze the influence of DDX46 on the proliferative and migratory potentials of glioblastoma (GBM). METHODS: Differential levels of DDX46 in GBM cases and controls were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. By intervening DDX46 in U87 and U251 cells, proliferative and migratory changes were determined by colony formation assay, 5-Ethynyl-2'- deoxyuridine (EdU) assay and Transwell assay, respectively. Protein levels of p-p38, p38, cyclin D1 and MMP7 in GBM cells intervened by DDX46 or the inhibitor of p38 MAPK were detected. RESULTS: DDX46 was upregulated in GBM cases. Knockdown of DDX46 attenuated the proliferative capacity of GBM cells, and its overexpression enhanced the proliferative rate. The migratory capacity of GBM was not affected by DDX46. Overexpression of DDX46 upregulated p-p38 and cyclin D1 in GBM cells. The regulatory effect of DDX46 on GBM proliferation could be partially reversed by the treatment of doramapimod. CONCLUSIONS: DDX46 is upregulated in GBM, which strengthens the proliferative capacity of GBM by activating the MAPK-p38 signaling.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.

RNA Helicase A/DHX9 Forms Unique Cytoplasmic Antiviral Granules That Restrict Oncolytic Myxoma Virus Replication in Human Cancer Cells.

RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions and antiviral responses and is hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the double-stranded DNA (dsDNA) poxvirus family that is being developed as an oncolytic virus, DHX9, forms unique granular cytoplasmic structures, which we named "DHX9 antiviral granules." These DHX9 antiviral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 antiviral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene transcription and translation were also significantly compromised, particularly in nonpermissive or semipermissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 antiviral granules are induced by MYXV, and other poxviruses, in human cells and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 antiviral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel antiviral function for DHX9 that is recruited from the nucleus into the cytoplasm, and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV. IMPORTANCE The cellular DHX9 has both proviral and antiviral roles against diverse RNA and DNA viruses. In this article, we demonstrate that DHX9 can form unique antiviral granules in the cytoplasm during myxoma virus (MYXV) replication in human cancer cells. These antiviral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9, the formation of DHX9 antiviral granules can be inhibited, which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 antiviral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has antiviral activity in human cancer cells, and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.

The RNA helicase DDX5 promotes viral infection via regulating N6-methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity.

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.

LncRNA DDX11-AS1: a novel oncogene in human cancer.

Long noncoding RNA (lncRNA) is a newly identified type of noncoding RNA with a length of more than 200 nucleotides. The latest research shows that lncRNAs play important roles in the occurrence and development of human tumours by acting both as carcinogenic genes and as tumour suppressor genes. LncRNAs plays a role in various biological processes, such as cell growth, apoptosis, migration and invasion. The newly discovered lncRNA DDX11-AS1 is abnormally highly expressed in various malignant tumours, such as hepatocellular carcinoma, colorectal cancer, osteosarcoma, bladder cancer, NSCLC and gastric cancer. DDX11-AS1 mainly regulates the expression of related genes through direct or indirect ways to perform its functions in carcinogenicity. These results indicate that DDX11-AS1 may be a marker or therapeutic target of tumours. This review summarizes the biological function and mechanism of DDX11-AS1 in the process of tumour development.

DDX5 promotes oncogene C3 and FABP1 expressions and drives intestinal inflammation and tumorigenesis.

Tumorigenesis in different segments of the intestinal tract involves tissue-specific oncogenic drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation and malignancies. By contrast, tumorigenesis in the small intestine involves fatty acid-binding protein 1 (FABP1). However, little is known of the upstream mechanisms driving their expressions in different segments of the intestinal tract. Here, we report that the RNA-binding protein DDX5 binds to the mRNA transcripts of C3 and Fabp1 to augment their expressions posttranscriptionally. Knocking out DDX5 in epithelial cells protected mice from intestinal tumorigenesis and dextran sodium sulfate (DSS)-induced colitis. Identification of DDX5 as a common upstream regulator of tissue-specific oncogenic molecules provides an excellent therapeutic target for intestinal diseases.

The RNA helicase DDX5 supports mitochondrial function in small cell lung cancer.

DEAD-box helicase 5 (DDX5) is a founding member of the DEAD-box RNA helicase family, a group of enzymes that regulate ribonucleoprotein formation and function in every aspect of RNA metabolism, ranging from synthesis to decay. Our laboratory previously found that DDX5 is involved in energy homeostasis, a process that is altered in many cancers. Small cell lung cancer (SCLC) is an understudied cancer type for which effective treatments are currently unavailable. Using an array of methods, including short hairpin RNA-mediated gene silencing, RNA and ChIP sequencing analyses, and metabolite profiling, we show here that DDX5 is overexpressed in SCLC cell lines and that its down-regulation results in various metabolic and cellular alterations. Depletion of DDX5 resulted in reduced growth and mitochondrial dysfunction in the chemoresistant SCLC cell line H69AR. The latter was evidenced by down-regulation of genes involved in oxidative phosphorylation and by impaired oxygen consumption. Interestingly, DDX5 depletion specifically reduced intracellular succinate, a TCA cycle intermediate that serves as a direct electron donor to mitochondrial complex II. We propose that the oncogenic role of DDX5, at least in part, manifests as up-regulation of respiration supporting the energy demands of cancer cells.

Hypoxia-induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase-1.

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia-induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl2 ). Both the hypoxic gas mixture (1% O2 ) and chemical hypoxia-induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia-mediated activation of caspase-1. Active caspase-1 induced the classical caspase-dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase-3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase-1/classical caspase-dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase-3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.

Dicer1 facilitates liver regeneration in a manner dependent on the inhibitory effect of miR-21 on Pten and Rhob expression.

AIMS: Tamoxifen-induced liver-specific Dicer1 deletion (iDicer1-/-) in mature mice may provide clues demonstrating the genuine effects of acute loss of Dicer1 and miRNAs in the liver regeneration process. MAIN METHODS: In this study, mice with tamoxifen-induced Dicer1 deletion through the Cre/LoxP system were constructed and then underwent classic 70% partial hepatectomy or CCl4-induced liver injury. To rescue the inhibitory effect of Dicer1 ablation on liver regeneration, miR-21 agomir was injected into the tail vein of iDicer1-/- mice. KEY FINDINGS: Unlike constitutive embryonic deletion of Dicer1, tamoxifen-induced Dicer1 deletion did not result in severe liver injury or lesions, providing an ideal model for investigating acute loss of Dicer1 and miRNAs in liver regeneration. Dicer1 deletion led to impaired liver regeneration through the inhibitory effect of miR-21 on PTEN and Rhob expression. SIGNIFICANCE: In our previous study, we found that embryonic loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation, while the role of Dicer1 in liver regeneration remains largely unknown. We clearly identified the promotion effect of Dicer1 on liver regeneration by increasing miR-21 expression, which inhibits the expression of two negative cell proliferation regulators, Pten and Rhob.

Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA.

DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNALys3-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.

Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathways.

White bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptor-signaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females' WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.

Bidirectional roles of Dhh1 in regulating autophagy.

Macroautophagy/autophagy activity is carefully modulated to allow cells to adapt to changing environmental conditions and maintain energy homeostasis. This control notably occurs in part through the regulation of autophagy-related (ATG) gene expression. Others and we have jointly shown that under nutrient-rich conditions Dhh1 mediates the degradation of certain ATG mRNAs, most significantly that of ATG8, through a Dcp2-dependent decapping pathway to maintain gene expression and autophagy activity at a basal level. More recently, we illustrated that under nitrogen-starvation conditions Dhh1 switches its role to become a positive regulator of autophagy, and promotes the translation of ATG1 and ATG13 mRNAs to meet the increased demand for autophagy activity. This regulation helps selected ATG mRNAs to escape the general repression in translation that occurs when nutrients are limited and TOR is inhibited. Our studies also suggest that Dhh1's nutrient-dependent bidirectional regulation of auto-phagy is conserved in more complex eukaryotes. Abbreviations: ATG: autophagy related; EIF4EBP: EIF4E binding protein; UTR: untranslated region.

MiR-34 and MiR-200: Regulator of Cell Fate Plasticity and Neural Development.

Studies from last two decades have established microRNAs (miRNAs) as the most influential regulator of gene expression, especially at the post-transcriptional stage. The family of small RNA molecules including miRNAs is highly conserved and expressed throughout the multicellular organism. MiRNAs regulate gene expression by binding to 3' UTR of protein-coding mRNAs and initiating either decay or movement of mRNAs to stress granules. Tissues or cells, which go through cell fate transformation like stem cells, brain cells, iPSCs, or cancer cells show very dynamic expression profile of miRNAs. Inability to pass the developmental stages of Dicer (miRNA maturation enzyme) knockout animals has confirmed that expression of mature and functional miRNAs is essential for proper development of different organs and tissues. Studies from our laboratory and elsewhere have demonstrated the role of miR-200 and miR-34 families in neural development and have shown higher expression of both families in mature and differentiated neurons. In present review, we have provided a general overview of miRNAs and focused on the role of miR-34 and miR-200, two miRNA families, which have the capability to change the phenotype and fate of a cell in different tissues and situations.

Cyclic Dimeric Guanosine Monophosphate: Activation and Inhibition of Innate Immune Response.

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a universally conserved second messenger that contributes to the pathogenicity of numerous bacterial species. In recent years, growing evidence has shown that bacterial extracellular c-di-GMP can interact with the innate immune system and regulate host immune responses. This review summarizes our current understanding on the dual roles of bacterial c-di-GMP in pathogen-host interaction: activation of the antibacterial innate immune response through the cytosolic surveillance pathway and inhibition of innate immune defense for iron restriction.

DEAD Box Protein 5 Inhibits Liver Tumorigenesis by Stimulating Autophagy via Interaction with p62/SQSTM1.

In hepatocellular carcinoma (HCC), dysregulated expression of DDX5 (DEAD box protein 5) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. Here we present evidence to show that, by interacting with autophagic receptor p62, DDX5 promotes autophagy and suppresses tumorigenesis. DDX5 inversely correlated with p62/sequestosome 1 (SQSTM1) expression in hepatitis B virus (HBV)-associated and non-HBV-associated HCCs. Patients with low DDX5 expression showed poor prognosis after tumor resection. We found that DDX5 overexpression induced, while DDX5 knockdown attenuated, autophagic flux in HepG2 and Huh7 cells. DDX5 promoted p62 degradation and markedly reduced the half-life of p62. Moreover, DDX5 overexpression dramatically reduced, while DDX5 knockdown promoted, cancer cell growth and tumorigenesis in vitro and in vivo. We found that DDX5 bound to p62 and interfered with p62/TRAF6 (tumor necrosis factor receptor-associated factor 6) interaction. Further findings revealed that the N-terminal domain of DDX5, involved in the interaction with p62, was sufficient to induce autophagy independent of its RNA binding and helicase activity. DDX5 overexpression decreased p62/TRAF6-mediated lysine 63-linked ubiquitination of mammalian target of rapamycin (mTOR) and subsequently inhibited the mTOR signaling pathway. Knockdown of TRAF6 blocked DDX5-induced autophagy. Furthermore, we showed that miR-17-5p downregulated DDX5 and impaired autophagy. Inhibition of miR-17-5p promoted autophagic flux and suppressed tumor growth in HCC xenograft models. Conclusion: Our findings define a noncanonical pathway that links miR-17-5p, DDX5, p62/TRAF6, autophagy, and HCC. These findings open an avenue for the treatment of HCC.

The RNA helicase DHX33 is required for cancer cell proliferation in human glioblastoma and confers resistance to PI3K/mTOR inhibition.

Human Glioblastoma is one deadly disease; the median survival time is reported to be 13.9 months after treatment. In the present study, we discovered that DHX33 is highly expressed in 84% of all Glioblastoma multiforme (GBM). Knockdown of DHX33 led to significant reduced proliferation and migration in glioblastoma cells in vitro and in vivo. Mechanistically, DHX33 regulated a set of critical genes involved in cell cycle and cell migration to promote glioblastoma development. Additionally, DHX33 was found to be induced by inhibitors of PI3K and mTOR whose activation has been detected in 50% of glioblastoma. Overexpression of wild type DHX33 protein, but not the helicase dead mutant, confers resistance to mTOR inhibitors in glioblastoma cells. DHX33 probably functions as a critical regulator to promote GBM development. Our results highlight its therapeutic potential in treating GBM.

DHX9 helicase promotes R-loop formation in cells with impaired RNA splicing.

R-loops are stable nucleic acid structures that have important physiological functions, but which also pose a significant threat to genomic stability. Increased R-loops cause replication stress and chromosome fragility and have been associated with diseases such as neurodegeneration and cancer. Although excessive R-loops are a feature of cells that are defective in RNA processing, what causes them to form is unclear. Here, we demonstrate that DHX9 (RNA helicase A) promotes the formation of pathological and non-pathological R-loops. In the absence of splicing factors, formation of R-loops correlates with the prolonged association of DHX9 with RNA Polymerase II (RNA Pol II). This leads to the production of DNA-RNA hybrid, which traps RNA Pol II on chromatin with the potential to block DNA replication. Our data provide a molecular mechanism for the formation of R-loops that is relevant to neurodegenerative diseases and cancers in which deregulated RNA processing is a feature.

DDX21 promotes gastric cancer proliferation by regulating cell cycle.

DEAD (Asp-Glu-Ala-Asp) cassette helicase 21 (DDX21) is an ATP-dependent RNA helicase that is overexpressed in various malignancies. There is increasing evidence that DDX21 is involved in carcinogenesis and cancer progression by promoting cell proliferation. However, the functional role of DDX21 in gastric cancer is largely unknown. In this study, we observed that DDX21 was significantly up-regulated in gastric cancer tissues compared to paired adjacent normal tissues. The expression of DDX21 was closely related to the pathological stage of gastric cancer. In vitro and in vivo studies had shown that knockdown of DDX21 inhibited gastric cancer cell proliferation, colony formation, G1/S cell cycle transition and xenograft growth, while ectopic expression of DDX21 promoted these cell functions. Mechanically, DDX21 induced gastric cancer cell growth by up-regulating levels of Cyclin D1 and CDK2. Taken together, these results revealed a novel role for DDX21 in the proliferation of gastric cancer cells via the Cyclin D1 and CDK2 pathways. Therefore, DDX21 can be used as a therapeutic target for gastric cancer.