Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

DDX5 inhibits inflammation by modulating m6A levels of TLR2/4 transcripts during bacterial infection.

DExD/H-box helicases are crucial regulators of RNA metabolism and antiviral innate immune responses; however, their role in bacteria-induced inflammation remains unclear. Here, we report that DDX5 interacts with METTL3 and METTL14 to form an m6A writing complex, which adds N6-methyladenosine to transcripts of toll-like receptor (TLR) 2 and TLR4, promoting their decay via YTHDF2-mediated RNA degradation, resulting in reduced expression of TLR2/4. Upon bacterial infection, DDX5 is recruited to Hrd1 at the endoplasmic reticulum in an MyD88-dependent manner and is degraded by the ubiquitin-proteasome pathway. This process disrupts the DDX5 m6A writing complex and halts m6A modification as well as degradation of TLR2/4 mRNAs, thereby promoting the expression of TLR2 and TLR4 and downstream NF-κB activation. The role of DDX5 in regulating inflammation is also validated in vivo, as DDX5- and METTL3-KO mice exhibit enhanced expression of inflammatory cytokines. Our findings show that DDX5 acts as a molecular switch to regulate inflammation during bacterial infection and shed light on mechanisms of quiescent inflammation during homeostasis.

Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5.

Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein-RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

RNA Helicase DDX5 Maintains Cardiac Function by Regulating CamkIIδ Alternative Splicing.

BACKGROUND: Heart failure (HF) is a leading cause of morbidity and mortality worldwide. RNA-binding proteins are identified as regulators of cardiac disease; DDX5 (dead-box helicase 5) is a master regulator of many RNA processes, although its function in heart physiology remains unclear. METHODS: We assessed DDX5 expression in human failing hearts and a mouse HF model. To study the function of DDX5 in heart, we engineered cardiomyocyte-specific Ddx5 knockout mice. We overexpressed DDX5 in cardiomyocytes using adeno-associated virus serotype 9 and performed transverse aortic constriction to establish the murine HF model. The mechanisms underlined were subsequently investigated using immunoprecipitation-mass spectrometry, RNA-sequencing, alternative splicing analysis, and RNA immunoprecipitation sequencing. RESULTS: We screened transcriptome databases of murine HF and human dilated cardiomyopathy samples and found that DDX5 was significantly downregulated in both. Cardiomyocyte-specific deletion of Ddx5 resulted in HF with reduced cardiac function, an enlarged heart chamber, and increased fibrosis in mice. DDX5 overexpression improved cardiac function and protected against adverse cardiac remodeling in mice with transverse aortic constriction-induced HF. Furthermore, proteomics revealed that DDX5 is involved in RNA splicing in cardiomyocytes. We found that DDX5 regulated the aberrant splicing of Ca2+/calmodulin-dependent protein kinase IIδ (CamkIIδ), thus preventing the production of CaMKIIδA, which phosphorylates L-type calcium channel by serine residues of Cacna1c, leading to impaired Ca2+ homeostasis. In line with this, we found increased intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content in DDX5-depleted cardiomyocytes. Using adeno-associated virus serotype 9 knockdown of CaMKIIδA partially rescued the cardiac dysfunction and HF in Ddx5 knockout mice. CONCLUSIONS: These findings reveal a role for DDX5 in maintaining calcium homeostasis and cardiac function by regulating alternative splicing in cardiomyocytes, identifying the DDX5 as a potential target for therapeutic intervention in HF.

DDX5 enhances HIF-1 activity by promoting the interaction of HIF-1α with HIF-1ß and recruiting the resulting heterodimer to its target gene loci.

BACKGROUND INFORMATION: Cancer cells acquire malignant characteristics and therapy resistance by employing the hypoxia-inducible factor 1 (HIF-1)-dependent adaptive response to hypoxic microenvironment in solid tumors. Since the underlying molecular mechanisms remain unclear, difficulties are associated with establishing effective therapeutic strategies. RESULTS: We herein identified DEAD-box helicase 5 (DDX5) as a novel activator of HIF-1 and found that it enhanced the heterodimer formation of HIF-1α and HIF-1ß and facilitated the recruitment of the resulting HIF-1 to its recognition sequence, hypoxia-response element (HRE), leading to the expression of a subset of cancer-related genes under hypoxia. CONCLUSIONS: This study reveals that the regulation of HIF-1 recruitment to HRE is an important regulatory step in the control of HIF-1 activity. SIGNIFICANCE: The present study provides novel insights for the development of strategies to inhibit the HIF-1-dependent expression of cancer-related genes.

Ribosomal protein L24 mediates mammalian microRNA processing in an evolutionarily conserved manner.

To investigate the mechanism(s) underlying the expression of primate-specific microRNAs (miRs), we sought DNA regulatory elements and proteins mediating expression of the primate-specific hsa-miR-608 (miR-608), which is located in the SEMA4G gene and facilitates the cholinergic blockade of inflammation by targeting acetylcholinesterase mRNA. 'Humanized' mice carrying pre-miR-608 flanked by 250 bases of endogenous sequences inserted into the murine Sema4g gene successfully expressed miR-608. Moreover, by flanking miR-608 by shortened fragments of its human genome region we identified an active independent promoter within the 150 nucleotides 5' to pre-miR-608, which elevated mature miR-608 levels by 100-fold in transfected mouse- and human-originated cells. This highlighted a regulatory role of the 5' flank as enabling miR-608 expression. Moreover, pull-down of the 150-base 5' sequence revealed its interaction with ribosomal protein L24 (RPL24), implicating an additional mechanism controlling miR-608 levels. Furthermore, RPL24 knockdown altered the expression of multiple miRs, and RPL24 immunoprecipitation indicated that up- or down-regulation of the mature miRs depended on whether their precursors bind RPL24 directly. Finally, further tests showed that RPL24 interacts directly with DDX5, a component of the large microprocessor complex, to inhibit miR processing. Our findings reveal that RPL24, which has previously been shown to play a role in miR processing in Arabidopsis thaliana, has a similar evolutionarily conserved function in miR biogenesis in mammals. We thus characterize a novel extra-ribosomal role of RPL24 in primate miR regulation.

DEAD box RNA helicase 5 is a new pro-viral host factor for Sindbis virus infection.

BACKGROUND: RNA helicases are emerging as key factors regulating host-virus interactions. The DEAD-box ATP-dependent RNA helicase DDX5, which plays an important role in many aspects of cellular RNA biology, was also found to either promote or inhibit viral replication upon infection with several RNA viruses. Here, our aim is to examine the impact of DDX5 on Sindbis virus (SINV) infection. METHODS: We analysed the interaction between DDX5 and the viral RNA using imaging and RNA-immunoprecipitation approaches. The interactome of DDX5 in mock- and SINV-infected cells was determined by mass spectrometry. We validated the interaction between DDX17 and the viral capsid by co- immunoprecipitation in the presence or absence of an RNase treatment. We determined the subcellular localization of DDX5, its cofactor DDX17 and the viral capsid protein by co-immunofluorescence. Finally, we investigated the impact of DDX5 depletion and overexpression on SINV infection at the viral protein, RNA and infectious particle accumulation level. The contribution of DDX17 was also tested by knockdown experiments. RESULTS: In this study we demonstrate that DDX5 interacts with the SINV RNA during infection. Furthermore, the proteomic analysis of the DDX5 interactome in mock and SINV-infected HCT116 cells identified new cellular and viral partners and confirmed the interaction between DDX5 and DDX17. Both DDX5 and DDX17 re-localize from the nucleus to the cytoplasm upon SINV infection and interact with the viral capsid protein. We also show that DDX5 depletion negatively impacts the viral replication cycle, while its overexpression has a pro-viral effect. Finally, we observed that DDX17 depletion reduces SINV infection, an effect which is even more pronounced in a DDX5-depleted background, suggesting a synergistic pro-viral effect of the DDX5 and DDX17 proteins on SINV. CONCLUSIONS: These results not only shed light on DDX5 as a novel and important host factor to the SINV life cycle, but also expand our understanding of the roles played by DDX5 and DDX17 as regulators of viral infections.

DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer.

The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

DDX5 inhibits type I IFN production by promoting degradation of TBK1 and disrupting formation of TBK1 - TRAF3 complex.

DExD/H-box helicase (DDX) 5 belongs to the DExD/H-box helicase family. DDX family members play differential roles in the regulation of innate antiviral immune response. However, whether DDX5 is involved in antiviral immunity remains unclear. In this study, we found that DDX5 serves as a negative regulator of type I interferon (IFN) response. Overexpression of DDX5 inhibited IFN production induced by Spring viremia of carp virus (SVCV) and poly(I:C) and enhanced virus replication by targeting key elements of the RLR signaling pathway (MAVS, MITA, TBK1, IRF3 and IRF7). Mechanistically, DDX5 directly interacted with TBK1 to promote its autophagy-mediated degradation. Moreover, DDX5 was shown to block the interaction between TRAF3 and TBK1, hence preventing nuclear translocation of IRF3. Together, these data shed light on the roles of DDX5 in regulating IFN response.

FL118 Is a Potent Therapeutic Agent against Chronic Myeloid Leukemia Resistant to BCR-ABL Inhibitors through Targeting RNA Helicase DDX5.

Chronic myeloid leukemia (CML) is induced by the expression of the fused tyrosine kinase BCR-ABL, which is caused by a chromosomal translocation. BCR-ABL inhibitors have been used to treat CML; however, the acquisition of resistance by CML cells during treatment is a serious issue. We herein demonstrated that BCR-ABL induced the expression of the RNA helicase DDX5 in K562 cells derived from CML patients in a manner that was dependent on its kinase activity, which resulted in cell proliferation and survival. The knockout of DDX5 decreased the expression of BIRC5 (survivin) and activated caspase 3, leading to apoptosis in K562 cells. Similar results were obtained in cells treated with FL118, an inhibitor of DDX5 and a derivative compound of camptothecin (CPT). Furthermore, FL118 potently induced apoptosis not only in Ba/F3 cells expressing BCR-ABL, but also in those expressing the BCR-ABL T315I mutant, which is resistant to BCR-ABL inhibitors. Collectively, these results revealed that DDX5 is a critical therapeutic target in CML and that FL118 is an effective candidate compound for the treatment of BCR-ABL inhibitor-resistant CML.

Arginine methylation of the DDX5 helicase RGG/RG motif by PRMT5 regulates resolution of RNA:DNA hybrids.

Aberrant transcription-associated RNA:DNA hybrid (R-loop) formation often causes catastrophic conflicts during replication, resulting in DNA double-strand breaks and genomic instability. Preventing such conflicts requires hybrid dissolution by helicases and/or RNase H. Little is known about how such helicases are regulated. Herein, we identify DDX5, an RGG/RG motif-containing DEAD-box family RNA helicase, as crucial player in R-loop resolution. In vitro, recombinant DDX5 resolves R-loops in an ATP-dependent manner, leading to R-loop degradation by the XRN2 exoribonuclease. DDX5-deficient cells accumulate R-loops at loci with propensity to form such structures based on RNA:DNA immunoprecipitation (DRIP)-qPCR, causing spontaneous DNA double-strand breaks and hypersensitivity to replication stress. DDX5 associates with XRN2 and resolves R-loops at transcriptional termination regions downstream of poly(A) sites, to facilitate RNA polymerase II release associated with transcriptional termination. Protein arginine methyltransferase 5 (PRMT5) binds and methylates DDX5 at its RGG/RG motif. This motif is required for DDX5 interaction with XRN2 and repression of cellular R-loops, but not essential for DDX5 helicase enzymatic activity. PRMT5-deficient cells accumulate R-loops, resulting in increased formation of γH2AX foci. Our findings exemplify a mechanism by which an RNA helicase is modulated by arginine methylation to resolve R-loops, and its potential role in regulating transcription.

Splicing factor SRSF3 promotes the progression of cervical cancer through regulating DDX5.

Aberrant alternative splicing (AS) profoundly affects tumorigenesis and cancer progression. Serine/arginine-rich splicing factor 3 (SRSF3) regulates the AS of precursor mRNAs and acts as a proto-oncogene in many tumors, but its function and potential mechanisms in cervical cancer remain unclear. Here, we found that SRSF3 was highly expressed in cervical cancer tissues and that SRSF3 expression was correlated with prognosis after analyses of the The Cancer Genome Atlas and GEO databases. Furthermore, knockdown of SRSF3 reduced the proliferation, migration, and invasion abilities of HeLa cells, while overexpression of SRSF3 promoted proliferation, migration, and invasion of CaSki cells. Further studies showed that SRSF3 mediated the variable splicing of exon 12 of the transcriptional cofactor DEAD-box helicase 5 (DDX5). Specifically, overexpression of SRSF3 promoted the production of the pro-oncogenic spliceosome DDX5-L and repressed the production of the repressive spliceosome DDX5-S. Ultimately, both SRSF3 and DDX5-L were able to upregulate oncogenic AKT expression, while DDX5-S downregulated AKT expression. In conclusion, we found that SRSF3 increased the production of DDX5-L and decreased the production of DDX5-S by regulating the variable splicing of DDX5. This, in turn promoted the proliferation, migration, and invasion of cervical cancer by upregulating the expression level of AKT. These results reveal the oncogenic role of SRSF3 in cervical cancer and emphasize the importance of the SRSF3-DDX5-AKT axis in tumorigenesis. SRSF3 and DDX5 are new potential biomarkers and therapeutic targets for cervical cancer.

LncPSCA in the 8q24.3 risk locus drives gastric cancer through destabilizing DDX5.

Genome-wide association studies (GWAS) have identified multiple gastric cancer risk loci and several protein-coding susceptibility genes. However, the role of long-noncoding RNAs (lncRNAs) transcribed from these risk loci in gastric cancer development and progression remains to be explored. Here, we functionally characterize a lncRNA, lncPSCA, as a novel tumor suppressor whose expression is fine-regulated by a gastric cancer risk-associated genetic variant. The rs2978980 T > G change in an intronic enhancer of lncPSCA interrupts binding of transcription factor RORA, which down-regulates lncPSCA expression in an allele-specific manner. LncPSCA interacts with DDX5 and promotes DDX5 degradation through ubiquitination. Increased expression of lncPSCA results in low levels of DDX5, less RNA polymerase II (Pol II) binding with DDX5 in the nucleus, thus activating transcription of multiple p53 signaling genes by Pol II. These findings highlight the importance of functionally annotating lncRNAs in GWAS risk loci and the great potential of modulating lncRNAs as innovative cancer therapy.

DDX5 helicase resolves G-quadruplex and is involved in MYC gene transcriptional activation.

G-quadruplexes (G4) are noncanonical secondary structures formed in guanine-rich DNA and RNA sequences. MYC, one of the most critical oncogenes, forms a DNA G4 in its proximal promoter region (MycG4) that functions as a transcriptional silencer. However, MycG4 is highly stable in vitro and its regulatory role would require active unfolding. Here we report that DDX5, one of the founding members of the DEAD-box RNA helicase family, is extremely proficient at unfolding MycG4-DNA. Our results show that DDX5 is a highly active G4-resolvase that does not require a single-stranded overhang and that ATP hydrolysis is not directly coupled to G4-unfolding of DDX5. The chromatin binding sites of DDX5 are G-rich sequences. In cancer cells, DDX5 is enriched at the MYC promoter and activates MYC transcription. The DDX5 interaction with the MYC promoter and DDX5-mediated MYC activation is inhibited by G4-interactive small molecules. Our results uncover a function of DDX5 in resolving DNA and RNA G4s and suggest a molecular target to suppress MYC for cancer intervention.

Circular RNA dehydrodolichyl diphosphate synthase facilitated triple-negative breast cancer progression via miR-362-3p/DDX5 axis.

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.

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.

Camptothecin induced DDX5 degradation increased the camptothecin resistance of osteosarcoma.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Unfortunately, chemo-resistance is a huge obstacle in the treatment of OS. However, the underlying molecular mechanisms of OS chemo-resistance still remain unknown. Here we reported that the resistance to camptothecin (cpt) therapy was driven by degradation of DDX5. DDX5 knockdown decreased cell death and DNA damage and recovered cell proliferation in cpt treated 143B cells. Furthermore, we found that DDX5 bound to NONO, a kind of DNA repairing protein, and regulated NONO functions. Our data verified that cpt-induced degradation of DDX5 following by breaking down the protein bound of NONO, which participated in the resistance of cpt. In the summary, according to our results, DDX5 might be a potential therapeutic target for improving clinical outcomes of cpt in OS.

Knockdown of terminal differentiation induced ncRNA (TINCR) suppresses proliferation and invasion in hepatocellular carcinoma by targeting the miR-218-5p/DEAD-box helicase 5 (DDX5) axis.

Terminal differentiation induced ncRNA (TINCR), a newly identified lncRNA, has been found to be associated with different human cancers including hepatocellular carcinoma (HCC). However, little is known regarding the pathological mechanisms of TINCR in HCC progression. In this study, we confirmed that TINCR expression was upregulated in HCC tumors and cell lines, and high TINCR expression was associated with larger tumor size, advanced tumor node metastasis stage, and poor prognosis. Functionally, knockdown of TINCR facilitated apoptosis and suppressed viability, colony formation and invasion in Huh7 and Hep3B cells. Mechanically, TINCR functioned as competing endogenous RNA (ceRNA) to regulate DEAD-box helicase 5 (DDX5) expression through sponging miR-218-5p. Moreover, the miR-218-5p expression was downregulated and DDX5 expression was upregulated in HCC tumors. The silencing of miR-218-5p or ectopic expression of DDX5 abated the tumor-suppressive effect of TINCR knockdown in vitro. Furthermore, si-TINCR-induced inactivation of AKT signaling was rescued by suppression of miR-218-5p or overexpression of DDX5. Also, the silencing of TINCR resulted in tumor growth inhibition in vivo. In summary, knockdown of TINCR suppressed HCC progression presumably by inactivation of AKT signaling through targeting the miR-218-5p/DDX5 axis, suggesting a novel TINCR/miR-218-5p/DDX5 pathway and therapy target for HCC.

DDX5 potentiates HIV-1 transcription as a co-factor of Tat.

BACKGROUND: HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein-protein interactions and RNA and ATP binding. RESULTS: We demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable. CONCLUSION: DDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1.

Dual role of the RNA helicase DDX5 in post-transcriptional regulation of myelin basic protein in oligodendrocytes.

In the central nervous system, oligodendroglial expression of myelin basic protein (MBP) is crucial for the assembly and structure of the myelin sheath. MBP synthesis is tightly regulated in space and time, particularly at the post-transcriptional level. We have identified the DEAD-box RNA helicase DDX5 (also known as p68) in a complex with Mbp mRNA in oligodendroglial cells. Expression of DDX5 is highest in progenitor cells and immature oligodendrocytes, where it localizes to heterogeneous populations of cytoplasmic ribonucleoprotein (RNP) complexes associated with Mbp mRNA in the cell body and processes. Manipulation of the amount of DDX5 protein inversely affects the level of MBP. We present evidence that DDX5 is involved in post-transcriptional regulation of MBP protein synthesis, with implications for oligodendroglial development. In addition, knockdown of DDX5 results in an increased abundance of MBP isoforms containing exon 2 in immature oligodendrocytes, most likely by regulating alternative splicing of Mbp Our findings contribute to the understanding of the complex nature of MBP post-transcriptional control in immature oligodendrocytes where DDX5 appears to affect the abundance of MBP proteins via distinct but converging mechanisms.