RNA damage compartmentalization by DHX9 stress granules.

Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.

The competitive landscape of the dsRNA world.

The ability to sense and respond to infection is essential for life. Viral infection produces double-stranded RNAs (dsRNAs) that are sensed by proteins that recognize the structure of dsRNA. This structure-based recognition of viral dsRNA allows dsRNA sensors to recognize infection by many viruses, but it comes at a cost-the dsRNA sensors cannot always distinguish between "self" and "nonself" dsRNAs. "Self" RNAs often contain dsRNA regions, and not surprisingly, mechanisms have evolved to prevent aberrant activation of dsRNA sensors by "self" RNA. Here, we review current knowledge about the life of endogenous dsRNAs in mammals-the biosynthesis and processing of dsRNAs, the proteins they encounter, and their ultimate degradation. We highlight mechanisms that evolved to prevent aberrant dsRNA sensor activation and the importance of competition in the regulation of dsRNA sensors and other dsRNA-binding proteins.

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Complex interplay between FMRP and DHX9 during DNA replication stress.

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.

The pseudogene GBP1P1 suppresses influenza A virus replication by acting as a protein decoy for DHX9.

Recently, substantial evidence has demonstrated that pseudogene-derived long noncoding RNAs (lncRNAs) as regulatory RNAs have been implicated in basic physiological processes and disease development through multiple modes of functional interaction with DNA, RNA, and proteins. Here, we report an important role for GBP1P1, the pseudogene of guanylate-binding protein 1, in regulating influenza A virus (IAV) replication in A549 cells. GBP1P1 was dramatically upregulated after IAV infection, which is controlled by JAK/STAT signaling. Functionally, ectopic expression of GBP1P1 in A549 cells resulted in significant suppression of IAV replication. Conversely, silencing GBP1P1 facilitated IAV replication and virus production, suggesting that GBP1P1 is one of the interferon-inducible antiviral effectors. Mechanistically, GBP1P1 is localized in the cytoplasm and functions as a sponge to trap DHX9 (DExH-box helicase 9), which subsequently restricts IAV replication. Together, these studies demonstrate that GBP1P1 plays an important role in antagonizing IAV replication.IMPORTANCELong noncoding RNAs (lncRNAs) are extensively expressed in mammalian cells and play a crucial role as regulators in various biological processes. A growing body of evidence suggests that host-encoded lncRNAs are important regulators involved in host-virus interactions. Here, we define a novel function of GBP1P1 as a decoy to compete with viral mRNAs for DHX9 binding. We demonstrate that GBP1P1 induction by IAV is mediated by JAK/STAT activation. In addition, GBP1P1 has the ability to inhibit IAV replication. Importantly, we reveal that GBP1P1 acts as a decoy to bind and titrate DHX9 away from viral mRNAs, thereby attenuating virus production. This study provides new insight into the role of a previously uncharacterized GBP1P1, a pseudogene-derived lncRNA, in the host antiviral process and a further understanding of the complex GBP network.

An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2.

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.

AURKB targets DHX9 to promote hepatocellular carcinoma progression via PI3K/AKT/mTOR pathway.

Aurora kinase B (AURKB) is known to play a carcinogenic role in a variety of cancers, but its underlying mechanism in liver cancer is unknown. This study aimed to investigate the role of AURKB in hepatocellular carcinoma (HCC) and its underlying molecular mechanism. Bioinformatics analysis revealed that AURKB was significantly overexpressed in HCC tissues and cell lines, and its high expression was associated with a poorer prognosis in HCC patients. Furthermore, downregulation of AURKB inhibited HCC cell proliferation, migration, and invasion, induced apoptosis, and caused cell cycle arrest. Moreover, AURKB downregulation also inhibited lung metastasis of HCC. AURKB interacted with DExH-Box helicase 9 (DHX9) and targeted its expression in HCC cells. Rescue experiments further demonstrated that AURKB targeting DHX9 promoted HCC progression through the PI3K/AKT/mTOR pathway. Our results suggest that AURKB is significantly highly expressed in HCC and correlates with patient prognosis. Targeting DHX9 with AURKB promotes HCC progression via the PI3K/AKT/mTOR pathway.

The Identification and Function of Linc01615 on Influenza Virus Infection and Antiviral Response.

Influenza virus infection poses a great threat to human health globally each year. Non-coding RNAs (ncRNAs) in the human genome have been reported to participate in the replication process of the influenza virus, among which there are still many unknowns about Long Intergenic Non-Coding RNAs (LincRNAs) in the cell cycle of viral infections. Here, we observed an increased expression of Linc01615 in A549 cells upon influenza virus PR8 infection, accompanied by the successful activation of the intracellular immune system. The knockdown of Linc01615 using the shRNAs promoted the proliferation of the influenza A virus, and the intracellular immune system was inhibited, in which the expressions of IFN-ß, IL-28A, IL-29, ISG-15, MX1, and MX2 were decreased. Predictions from the catRAPID website suggested a potential interaction between Linc01615 and DHX9. Also, knocking down Linc01615 promoted influenza virus proliferation. The subsequent transcriptome sequencing results indicated a decrease in Linc01615 expression after influenza virus infection when DHX9 was knocked down. Further analysis through cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) in HEK293 cells stably expressing DHX9 confirmed the interaction between DHX9 and Linc01615. We speculate that DHX9 may interact with Linc01615 to partake in influenza virus replication and that Linc01615 helps to activate the intracellular immune system. These findings suggest a deeper connection between DHX9 and Linc01615, which highlights the significant role of Linc01615 in the influenza virus replication process. This research provides valuable insights into understanding influenza virus replication and offers new targets for preventing influenza virus infections.

DHRS4-AS1 regulate gastric cancer apoptosis and cell proliferation by destabilizing DHX9 and inhibited the association between DHX9 and ILF3.

Gastric cancer (GC) causes millions of cancer-related deaths due to anti-apoptosis and rapid proliferation. However, the molecular mechanisms underlying GC cell proliferation and anti-apoptosis remain unclear. The expression levels of DHRS4-AS1 in GC were analyzed based on GEO database and recruited GC patients in our institution. We found that DHRS4-AS1 was significantly downregulated in GC. The expression of DHRS4-AS1 in GC tissues showed a significant correlation with tumor size, advanced pathological stage, and vascular invasion. Moreover, DHRS4-AS1 levels in GC tissues were significantly associated with prognosis. DHRS4-AS1 markedly inhibited GC cell proliferation and promotes apoptosis in vitro and in vivo assays. Mechanically, We found that DHRS4-AS1 bound to pro-oncogenic DHX9 (DExH-box helicase 9) and recruit the E3 ligase MDM2 that contributed to DHX9 degradation. We also confirmed that DHRS4-AS1 inhibited DHX9-mediated cell proliferation and promotes apoptosis. Furthermore, we found DHX9 interact with ILF3 (Interleukin enhancer Binding Factor 3) and activate NF-kB Signaling in a ILF3-dependent Manner. Moreover, DHRS4-AS1 can also inhibit the association between DHX9 and ILF3 thereby interfered the activation of the signaling pathway. Our results reveal new insights into mechanisms underlying GC progression and indicate that LncRNA DHRS4-AS1 could be a future therapeutic target and a biomarker for GC diagnosis.

DHX9/DNA-tandem repeat-dependent downregulation of ciRNA-Fmn1 in the dorsal horn is required for neuropathic pain.

Circular RNAs (ciRNAs) are emerging as new players in the regulation of gene expression. However, how ciRNAs are involved in neuropathic pain is poorly understood. Here, we identify the nervous-tissue-specific ciRNA-Fmn1 and report that changes in ciRNA-Fmn1 expression in spinal cord dorsal horn neurons play a key role in neuropathic pain after nerve injury. ciRNA-Fmn1 was significantly downregulated in ipsilateral dorsal horn neurons after peripheral nerve injury, at least in part because of a decrease in DNA helicase 9 (DHX9), which regulates production of ciRNA-Fmn1 by binding to DNA-tandem repeats. Blocking ciRNA-Fmn1 downregulation reversed nerve-injury-induced reductions in both the binding of ciRNA-Fmn1 to the ubiquitin ligase UBR5 and the level of ubiquitination of albumin (ALB), thereby abrogating the nerve-injury-induced increase of ALB expression in the dorsal horn and attenuating the associated pain hypersensitivities. Conversely, mimicking downregulation of ciRNA-Fmn1 in naïve mice reduced the UBR5-controlled ubiquitination of ALB, leading to increased expression of ALB in the dorsal horn and induction of neuropathic-pain-like behaviors in naïve mice. Thus, ciRNA-Fmn1 downregulation caused by changes in binding of DHX9 to DNA-tandem repeats contributes to the genesis of neuropathic pain by negatively modulating UBR5-controlled ALB expression in the dorsal horn.

NUSAP1 Binds ILF2 to Modulate R-Loop Accumulation and DNA Damage in Prostate Cancer.

Increased expression of NUSAP1 has been identified as a robust prognostic biomarker in prostate cancer and other malignancies. We have previously shown that NUSAP1 is positively regulated by E2F1 and promotes cancer invasion and metastasis. To further understand the biological function of NUSAP1, we used affinity purification and mass spectrometry proteomic analysis to identify NUSAP1 interactors. We identified 85 unique proteins in the NUSAP1 interactome, including ILF2, DHX9, and other RNA-binding proteins. Using proteomic approaches, we uncovered a function for NUSAP1 in maintaining R-loops and in DNA damage response through its interaction with ILF2. Co-immunoprecipitation and colocalization using confocal microscopy verified the interactions of NUSAP1 with ILF2 and DHX9, and RNA/DNA hybrids. We showed that the microtubule and charged helical domains of NUSAP1 were necessary for the protein-protein interactions. Depletion of ILF2 alone further increased camptothecin-induced R-loop accumulation and DNA damage, and NUSAP1 depletion abolished this effect. In human prostate adenocarcinoma, NUSAP1 and ILF2 mRNA expression levels are positively correlated, elevated, and associated with poor clinical outcomes. Our study identifies a novel role for NUSAP1 in regulating R-loop formation and accumulation in response to DNA damage through its interactions with ILF2 and hence provides a potential therapeutic target.

[Diversity of MLE Helicase Functions in the Regulation of Gene Expression in Higher Eukaryotes].

The Drosophila melanogaster Maleless (MLE) protein is a conserved helicase involved in a wide range of gene expression regulation processes. A MLE ortholog, named DHX9, was found in many higher eukaryotes, including humans. DHX9 is involved in diverse processes, such as genome stability maintenance, replication, transcription, splicing, editing and transport of cellular and viral RNAs, and translation regulation. Some of these functions are understood in detail today, while most of them remain uncharacterized. Study of the functions of the MLE ortholog in mammals in vivo is limited by the fact that the loss of function of this protein is lethal at the embryonic stage. In D. melanogaster, helicase MLE was originally discovered and studied for a long time as a participant in dosage compensation. Recent evidence indicates that helicase MLE is involved in the same cell processes in D. melanogaster and mammals and that many of its functions are evolutionarily conserved. Experiments in D. melanogaster revealed new important MLE functions, such as a role in hormone-dependent regulation of transcription and interactions with the SAGA transcription complex, other transcriptional cofactors, and chromatin remodeling complexes. Unlike in mammals, MLE mutations do not cause embryonic lethality in D. melanogaster, and the MLE functions are possible to study in vivo throughout ontogenesis in females and up to the pupal stage in males. The human MLE ortholog is a potential target for anticancer and antiviral therapies. Further investigation of the MLE functions in D. melanogaster is therefore of both basic and applied importance. The review discusses the systematic position, domain structure, and conserved and specific functions of MLE helicase in D. melanogaster.

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.

DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway.

Colorectal cancer (CRC) is the leading cause of cancer-related mortality worldwide, which makes it urgent to identify novel therapeutic targets for CRC treatment. In this study, DHX9 was filtered out as the prominent proliferation promoters of CRC by siRNA screening. Moreover, DHX9 was overexpressed in CRC cell lines, clinical CRC tissues and colitis-associated colorectal cancer (CAC) mouse model. The upregulation of DHX9 was positively correlated with poor prognosis in patients with CRC. Through gain- and loss-of function experiments, we found that DHX9 promoted CRC cell proliferation, colony formation, apoptosis resistance, migration and invasion in vitro. Furthermore, a xenograft mouse model and a hepatic metastasis mouse model were utilized to confirm that forced overexpression of DHX9 enhanced CRC outgrowth and metastasis in vivo, while DHX9 ablation produced the opposite effect. Mechanistically, from one aspect, DHX9 enhances p65 phosphorylation, promotes p65 nuclear translocation to facilitate NF-κB-mediated transcriptional activity. From another aspect, DHX9 interacts with p65 and RNA polymerase II (RNA Pol II) to enhance the downstream targets of NF-κB (e.g., Survivin, Snail) expression to potentiate the malignant phenotypes of CRC. Together, our results suggest that DHX9 may be a potential therapeutic target for prevention and treatment of CRC patients.

SNORA42 promotes oesophageal squamous cell carcinoma development through triggering the DHX9/p65 axis.

Small nucleolar RNAs (snoRNAs) are an important group of non-coding RNAs that have been reported to play a key role in the occurrence and development of various cancers. Here we demonstrate that Small nucleolar RNA 42 (SNORA42) enhanced the proliferation and migration of Oesophageal squamous carcinoma cells (ESCC) via the DHX9/p65 axis. Our results found that SNORA42 was significantly upregulated in ESCC cell lines, tissues and serum of ESCC patients. The high expression level of SNORA42 was positively correlated with malignant characteristics and over survival probability of patients with ESCC. Through in vitro and in vivo approaches, we demonstrated that knockdown of SNORA42 significantly impeded ESCC growth and metastasis whereas overexpression of SNORA42 got opposite effects. Mechanically, SNORA42 promoted DHX9 expression by attenuating DHX9 transports into the cytoplasm, to protect DHX9 from being ubiquitinated and degraded. From the KEGG analysis of Next-Generation Sequencing, the NF-κB pathway was one of the most regulated pathways by SNORA42. SNORA42 enhanced phosphorylation of p65 and this effect could be reversed by NF-κB inhibitor, BAY11-7082. Moreover, SNORA42 activated NF-κB signaling through promoting the transcriptional co-activator DHX9 interacted with p-p65, inducing NF-κB downstream gene expression. In summary, our study highlights the potential of SNORA42 is up-regulated in ESCC and promotes ESCC development partly via interacting with DHX9 and triggering the DHX9/p65 axis.

Hepatitis B virus X protein modulates upregulation of DHX9 to promote viral DNA replication.

Hepatitis B virus (HBV) infection is a major cause of acute and chronic liver diseases. During the HBV life cycle, HBV hijacks various host factors to assist viral replication. In this research, we find that the HBV regulatory protein X (HBx) can induce the upregulation of DExH-box RNA helicase 9 (DHX9) expression by repressing proteasome-dependent degradation mediated by MDM2. Furthermore, we demonstrate that DHX9 contributes to viral DNA replication in dependence on its helicase activity and nuclear localization. In addition, the promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98. Our findings reveal that HBx-mediated DHX9 upregulation is essential for HBV DNA replication.

High expression of DHX9 promotes the growth and metastasis of hepatocellular carcinoma.

BACKGROUND: DHX9, an NTP-dependent RNA helicase, is closely associated with the proliferation and metastasis of some tumor cells and the prognosis of patients, but its role in hepatocellular carcinoma (HCC) is not well-known. This study was performed to explore the expression and role of DHX9 in HCC. METHODS: The expression of DHX9 in HCC tissues and cell lines was detected by TCGA database, qPCR, western blotting, and immunohistochemistry. The relationship between the DHX9 expression level and the prognosis of patients with HCC was accessed. Then, the function of DHX9 knockdown in HCC cells was examined by CCK-8, scratch, Transwell, and apoptosis assays. Epithelial-mesenchymal transition (EMT) was detected by western blotting. RESULTS: DHX9 was highly expressed in HCC tissues by analyzing both TCGA database and clinical samples. High DHX9 expression level was associated with TNM stage, vascular invasion and metastasis of HCC patients, and was an independent adverse prognostic factor. DHX9 knockdown significantly inhibited cell proliferation, migration, invasion and EMT and increased cell apoptosis in HCC cells. CONCLUSION: Our findings suggest that DHX9 participates in the progression of HCC as an oncogene and may be a potential target for the clinical diagnosis and therapy of HCC.

MLE Helicase Is a New Participant in the Transcription Regulation of the ftz-f1 Gene Encoding Nuclear Receptor in Higher Eukaryotes.

MLE helicase is an evolutionarily conserved eukaryotic protein involved in a wide range of processes in the regulation of gene expression. Previously, we studied the properties of MLE on the Drosophila melanogaster model. In the present work we continue studying the functions of MLE and show that MLE interacts with the components of the SWI/SNF chromatin remodelling complex. To clarify the work of MLE, the profile of MLE binding to the regulatory elements of the SWI/SNF-dependent ftz-f1 gene was analyzed. The effect of MLE on the expression of this gene, the transcription of which occurs by the RNA polymerase II pausing mechanism, was investigated. The data obtained indicate the important role of MLE in ensuring timely activation and high level of expression of the ftz-f1 gene in vivo.

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.