Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

Stress granule and P-body clearance: Seeking coherence in acts of disappearance.

Stress granules and P-bodies are conserved cytoplasmic biomolecular condensates whose assembly and composition are well documented, but whose clearance mechanisms remain controversial or poorly described. Such understanding could provide new insight into how cells regulate biomolecular condensate formation and function, and identify therapeutic strategies in disease states where aberrant persistence of stress granules in particular is implicated. Here, I review and compare the contributions of chaperones, the cytoskeleton, post-translational modifications, RNA helicases, granulophagy and the proteasome to stress granule and P-body clearance. Additionally, I highlight the potentially vital role of RNA regulation, cellular energy, and changes in the interaction networks of stress granules and P-bodies as means of eliciting clearance. Finally, I discuss evidence for interplay of distinct clearance mechanisms, suggest future experimental directions, and suggest a simple working model of stress granule clearance.

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.

A novel reporter for helicase activity in translation uncovers DDX3X interactions.

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5' UTRs to measure DDX3X mediated translational activity in cells. To directly measure RNA structure in DDX3X dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then employed HART to investigate how sequence alterations influence DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the translational machinery as well as its helicase activity are required for its function in promoting the translation of DDX3X sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally-encoded, colorimetric measurement of DDX3X-dependent translation in cells.

DEAH-Box RNA Helicases in Pre-mRNA Splicing.

In eukaryotic cells, pre-mRNA splicing is catalyzed by the spliceosome, a highly dynamic molecular machinery that undergoes dramatic conformational and compositional rearrangements throughout the splicing cycle. These crucial rearrangements are largely driven by eight DExD/H-box RNA helicases. Interestingly, the four helicases participating in the late stages of splicing are all DEAH-box helicases that share structural similarities. This review aims to provide an overview of the structure and function of these DEAH-box helicases, including new information provided by recent cryo-electron microscopy structures of the spliceosomal complexes.

DDX3X syndrome: From clinical phenotypes to biological insights.

DDX3X syndrome is a neurodevelopmental disorder accounting for up to 3% of cases of intellectual disability (ID) and affecting primarily females. Individuals diagnosed with DDX3X syndrome can also present with behavioral challenges, motor delays and movement disorders, epilepsy, and congenital malformations. DDX3X syndrome is caused by mutations in the X-linked gene DDX3X, which encodes a DEAD-box RNA helicase with critical roles in RNA metabolism, including mRNA translation. Emerging discoveries from animal models are unveiling a fundamental role of DDX3X in neuronal differentiation and development, especially in the neocortex. Here, we review the current knowledge of genetic and neurobiological mechanisms underlying DDX3X syndrome and their relationship with clinical phenotypes.

Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes.

BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

DDX24 Is Essential for Cell Cycle Regulation in Vascular Smooth Muscle Cells During Vascular Development via Binding to FANCA mRNA.

BACKGROUND: The DEAD-box family is essential for tumorigenesis and embryogenesis. Previously, we linked the malfunction of DDX (DEAD-box RNA helicase)-24 to a special type of vascular malformation. Here, we aim to investigate the function of DDX24 in vascular smooth muscle cells (VSMCs) and embryonic vascular development. METHODS: Cardiomyocyte (CMC) and VSMC-specific Ddx24 knockout mice were generated by crossing Tagln-Cre mice with Ddx24flox/flox transgenic mice. The development of blood vessels was explored by stereomicroscope photography and immunofluorescence staining. Flow cytometry and cell proliferation assays were used to verify the regulation of DDX24 on the function of VSMCs. RNA sequencing and RNA immunoprecipitation coupled with quantitative real-time polymerase chain reaction were combined to investigate DDX24 downstream regulatory molecules. RNA pull-down and RNA stability experiments were performed to explore the regulation mechanism of DDX24. RESULTS: CMC/VSMC-specific Ddx24 knockout mice died before embryonic day 13.5 with defects in vessel formation and abnormal vascular remodeling in extraembryonic tissues. Ddx24 knockdown suppressed VSMC proliferation via cell cycle arrest, likely due to increased DNA damage. DDX24 protein bound to and stabilized the mRNA of FANCA (FA complementation group A) that responded to DNA damage. Consistent with the function of DDX24, depletion of FANCA also impacted cell cycle and DNA repair of VSMCs. Overexpression of FANCA was able to rescue the alterations caused by DDX24 deficiency. CONCLUSIONS: Our study unveiled a critical role of DDX24 in VSMC-mediated vascular development, highlighting a potential therapeutic target for VSMC-related pathological conditions.

Nonsense-Mediated Decay Restricts LncRNA Levels in Yeast Unless Blocked by Double-Stranded RNA Structure.

Antisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3' single-stranded (3'-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA mapping revealed that 3'-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated, locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts.

DICER1-mutated rhabdomyosarcoma of the ovary with teratoid features.

DICER1-mutated rhabdomyosarcoma is a rare, emerging entity with a predilection for the gynecologic and genitourinary tracts. We report here a case of DICER1-mutated rhabdomyosarcoma of the ovary in a 14 years old girl which displayed interspersed mature teratoid glands, neuroectodermal rosettes and immature blastematous-like tubes. Morphologically the sarcomatous component predominated, corresponding to a high grade spindle cell rhabdomyosarcoma with botryoid features. Islets of cartilage were present. The sarcomatous proliferation encased the teratoid glands, forming cambium layer-like arrangements. The sarcoma cells were Myogenin and MYOD1 positive, the neuroectodermal rosettes expressed SALL4 along with cytokeratins and EMA and were negative for Inhibin; immature blastematous-like tubes were negative for SALL4 and Inhibin. Whole RNA- and targeted DNA-sequencing revealed two DICER1 mutations in exon 26: c.5113G>A: p.(Glu1705Lys) and exon 12: c.1642C>T: p.(Gln548X). The sarcomatous component harbored a complex genetic profile while the teratoid component was diploid, none of the above displayed abnormality of 12p. DICER1-mutated sarcomas display pathological features similar to embryonal rhabdomyosarcomas, botryoid type. They also display heterogeneous features combining cartilage foci, teratoid mature glands, immature blastematous-like tubes and/or neuroectodermal components. Molecular testing remains necessary to confirm the diagnosis. Further studies need to clarify the nosology of DICER1-mutated sarcomas and devise specific therapeutic strategies.

DExH/D-box helicases at the frontline of intrinsic and innate immunity against viral infections.

DExH/D-box helicases are essential nucleic acid and ribonucleoprotein remodelers involved in all aspects of nucleic acid metabolism including replication, gene expression and post-transcriptional modifications. In parallel to their importance in basic cellular functions, DExH/D-box helicases play multiple roles in viral life cycles, with some of them highjacked by viruses or negatively regulating innate immune activation. However, other DExH/D-box helicases have recurrently been highlighted as direct antiviral effectors or as positive regulators of innate immune activation. Innate immunity relies on the ability of Pathogen Recognition Receptors to recognize viral signatures and trigger the production of interferons (IFNs) and pro-inflammatory cytokines. Secreted IFNs interact with their receptors to establish antiviral cellular reprogramming via expression regulation of the interferon-stimulated genes (ISGs). Several DExH/D-box helicases have been reported to act as viral sensors (DDX3, DDX41, DHX9, DDX1/DDX21/DHX36 complex), and others to play roles in innate immune activation (DDX60, DDX60L, DDX23). In contrast, the DDX39A, DDX46, DDX5 and DDX24 helicases act as negative regulators and impede IFN production upon viral infection. Beyond their role in viral sensing, the ISGs DDX60 and DDX60L act as viral inhibitors. Interestingly, the constitutively expressed DEAD-box helicases DDX56, DDX17, DDX42 intrinsically restrict viral replication. Hence, DExH/D-box helicases appear to form a multilayer network of primary and secondary factors involved in both intrinsic and innate antiviral immunity. In this review, we highlight recent findings on the extent of antiviral defences played by helicases and emphasize the need to better understand their immune functions as well as their complex interplay.

SMaRT lncRNA controls translation of a G-quadruplex-containing mRNA antagonizing the DHX36 helicase.

Guanine-quadruplexes (G4) included in RNA molecules exert several functions in controlling gene expression at post-transcriptional level; however, the molecular mechanisms of G4-mediated regulation are still poorly understood. Here, we describe a regulatory circuitry operating in the early phases of murine muscle differentiation in which a long non-coding RNA (SMaRT) base pairs with a G4-containing mRNA (Mlx-γ) and represses its translation by counteracting the activity of the DHX36 RNA helicase. The time-restricted, specific effect of lnc-SMaRT on the translation of Mlx-γ isoform modulates the general subcellular localization of total MLX proteins, impacting on their transcriptional output and promoting proper myogenesis and mature myotube formation. Therefore, the circuitry made of lnc-SMaRT, Mlx-γ, and DHX36 not only plays an important role in the control of myogenesis but also unravels a molecular mechanism where G4 structures and G4 unwinding activities are regulated in living cells.

RNA folding and functions of RNA helicases in ribosome biogenesis.

Eukaryotic ribosome biogenesis involves the synthesis of ribosomal RNA (rRNA) and its stepwise folding into the unique structure present in mature ribosomes. rRNA folding starts already co-transcriptionally in the nucleolus and continues when pre-ribosomal particles further maturate in the nucleolus and upon their transit to the nucleoplasm and cytoplasm. While the approximate order of folding of rRNA subdomains is known, especially from cryo-EM structures of pre-ribosomal particles, the actual mechanisms of rRNA folding are less well understood. Both small nucleolar RNAs (snoRNAs) and proteins have been implicated in rRNA folding. snoRNAs hybridize to precursor rRNAs (pre-rRNAs) and thereby prevent premature folding of the respective rRNA elements. Ribosomal proteins (r-proteins) and ribosome assembly factors might have a similar function by binding to rRNA elements and preventing their premature folding. Besides that, a small group of ribosome assembly factors are thought to play a more active role in rRNA folding. In particular, multiple RNA helicases participate in individual ribosome assembly steps, where they are believed to coordinate RNA folding/unfolding events or the release of proteins from the rRNA. In this review, we summarize the current knowledge on mechanisms of RNA folding and on the specific function of the individual RNA helicases involved. As the yeast Saccharomyces cerevisiae is the organism in which ribosome biogenesis and the role of RNA helicases in this process is best studied, we focused our review on insights from this model organism, but also make comparisons to other organisms where applicable.

Genome-Wide Identification and Characterization of the Soybean DEAD-Box Gene Family and Expression Response to Rhizobia.

DEAD-box proteins are a large family of RNA helicases that play important roles in almost all cellular RNA processes in model plants. However, little is known about this family of proteins in crops such as soybean. Here, we identified 80 DEAD-box family genes in the Glycine max (soybean) genome. These DEAD-box genes were distributed on 19 chromosomes, and some genes were clustered together. The majority of DEAD-box family proteins were highly conserved in Arabidopsis and soybean, but Glyma.08G231300 and Glyma.14G115100 were specific to soybean. The promoters of these DEAD-box genes share cis-acting elements involved in plant responses to MeJA, salicylic acid (SA), low temperature and biotic as well as abiotic stresses; interestingly, half of the genes contain nodulation-related cis elements in their promoters. Microarray data analysis revealed that the DEAD-box genes were differentially expressed in the root and nodule. Notably, 31 genes were induced by rhizobia and/or were highly expressed in the nodule. Real-time quantitative PCR analysis validated the expression patterns of some DEAD-box genes, and among them, Glyma.08G231300 and Glyma.14G115100 were induced by rhizobia in root hair. Thus, we provide a comprehensive view of the DEAD-box family genes in soybean and highlight the crucial role of these genes in symbiotic nodulation.

Sen1 has unique structural features grafted on the architecture of the Upf1-like helicase family.

The superfamily 1B (SF1B) helicase Sen1 is an essential protein that plays a key role in the termination of non-coding transcription in yeast. Here, we identified the ~90 kDa helicase core of Saccharomyces cerevisiae Sen1 as sufficient for transcription termination in vitro and determined the corresponding structure at 1.8 Å resolution. In addition to the catalytic and auxiliary subdomains characteristic of the SF1B family, Sen1 has a distinct and evolutionarily conserved structural feature that "braces" the helicase core. Comparative structural analyses indicate that the "brace" is essential in shaping a favorable conformation for RNA binding and unwinding. We also show that subdomain 1C (the "prong") is an essential element for 5'-3' unwinding and for Sen1-mediated transcription termination in vitro Finally, yeast Sen1 mutant proteins mimicking the disease forms of the human orthologue, senataxin, show lower capacity of RNA unwinding and impairment of transcription termination in vitro The combined biochemical and structural data thus provide a molecular model for the specificity of Sen1 in transcription termination and more generally for the unwinding mechanism of 5'-3' helicases.

DDX52 knockdown inhibits the growth of prostate cancer cells by regulating c-Myc signaling.

BACKGROUND: DDX52 is a type of DEAD/H box RNA helicase that was identified as a novel prostate cancer (PCa) genetic locus and possible causal gene in a European large-scale transcriptome-wide association study. However, the functions of DDX52 in PCa remain undetermined. The c-Myc oncogene plays a crucial role in the development of PCa, but the factors that regulate the activity of c-Myc in PCa are still unknown. METHODS: We determined DDX52 protein levels in PCa tissues using immunohistochemistry (IHC). DDX52 expression and survival outcomes in other PCa cohorts were examined using bioinformatics analysis. The inhibition of DDX52 via RNA interference with shRNA was used to clarify the effects of DDX52 on PCa cell growth in vitro and in vivo. Gene set enrichment analysis and RNA sequencing were used to explore the signaling regulated by DDX52 in PCa. Western blotting and IHC were used to determine the possible DDX52 signaling mechanism in PCa. RESULTS: DDX52 expression was upregulated in PCa tissues. Bioinformatics analysis showed that the level of DDX52 further increased in advanced PCa, with a high DDX52 level indicating a poor outcome. In vitro and in vivo experiments showed that downregulating DDX52 impeded the growth of PCa cells. High DDX52 levels contributed to activating c-Myc signaling in PCa patients and PCa cells. Furthermore, DDX52 expression was regulated by c-Myc and positively correlated with c-Myc expression in PCa. CONCLUSION: DDX52 was overexpressed in PCa tissues in contrast to normal prostate tissues. DDX52 knockdown repressed the growth of PCa cells in vitro and in vivo. Deleting c-Myc inhibited DDX52 expression, which affected the activation of c-Myc signaling.

Protective Role of RNA Helicase DEAD-Box Protein 5 in Smooth Muscle Cell Proliferation and Vascular Remodeling.

RATIONALE: RNA helicases, highly conserved enzymes, are currently believed to be not only involved in RNA modulation, but also in other biological processes. We recently reported that RNA helicase DDX (DEAD-box protein)-5 is required for maintaining the homeostasis of vascular smooth muscle cells (SMCs). However, the expression and function of RNA helicase in vascular physiology and disease is unknown. OBJECTIVE: To investigate the role of RNA helicase in vascular diseases. METHODS AND RESULTS: We showed here that DDX-5 was the most abundant DEAD-box protein expressed in human and rodent artery, which mainly located in SMCs. It was demonstrated that DDX-5 levels were reduced in cytokine-stimulated SMCs and vascular lesions. DDX-5 knocking down or deficiency increased SMC proliferation and migration, whereas overexpression of DDX-5 prevented aberrant proliferation and migration of SMCs. Mechanistic studies revealed transcription factor GATA (GATA-binding protein)-6 as a novel downstream target of DDX-5, which directly interacted with GATA-6 and protected it from MDM (mouse double minute)-2-mediated degradation. Our ChIP assay identified a previously unreported binding of p27Kip1 promoter to GATA-6. DDX-5 increased the recruitment of GATA-6 to p27Kip1 promoter, which enhanced p27Kip1 expression and maintained SMC quiescence. Finally, we showed exacerbated neointima formation in DDX-5 SMC-deficient mice after femoral artery injury, whereas overexpression of DDX-5 potently inhibited vascular remodeling in balloon-injured rat carotid artery. CONCLUSIONS: These findings provide the first evidence for a role of RNA helicase DDX-5 in the protection against SMC proliferation, migration, and neointimal hyperplasia. Our data extend the fundamental role of RNA helicase beyond RNA modulation, which provides the basic information for new therapeutic strategies for vascular diseases.

1-Aminomethyl SAR in a novel series of flavagline-inspired eIF4A inhibitors: Effects of amine substitution on cell potency and in vitro PK properties.

Flavaglines such as silvestrol (1) and rocaglamide (2) constitute an interesting class of natural products with promising anticancer activities. Their mode of action is based on inhibition of eukaryotic initiation factor 4A (eIF4A) dependent translation through formation of a stable ternary complex with eIF4A and mRNA, thus blocking ribosome scanning. Herein we describe initial SAR studies in a novel series of 1-aminomethyl substituted flavagline-inspired eIF4A inhibitors. We discovered that a variety of N-substitutions at the 1-aminomethyl group are tolerated, making this position pertinent for property and ADME profile tuning. The findings presented herein are relevant to future drug design efforts towards novel eIF4A inhibitors with drug-like properties.

The RNA helicase DDX3 is required for ovarian development and oocyte maturation in Locusta migratoria.

DDX3 represents a well-defined subfamily of DEAD-box RNA helicase and exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility. Our previous study has shown that LmDDX3, the ortholog of DDX3 in Locusta migratoria, is ubiquitously expressed, and with a high abundance in testis and ovary. Knockdown of LmDDX3 results in a lethal phenotype in nymph, but it still remains unclear for its role in reproductive process. In this study, we therefore characterized LmDDX3 expression in female adult locust and analyzed its function in oocyte development. LmDDX3 was expressed in all tissues examined with significant more transcripts in ovary and hindgut. In ovary, a strong expression level was detected at the day just after adult eclosion, and a dramatic reduction then occurred during the oocyte development. LmDDX3 RNAi led to a reduced vitellogenin (Vg) expression in fat body via partially at least, the JH signaling pathway, and caused an upregulation of vitellogenin receptor (VgR) in ovary, and thus blocked the ovarian development and oocyte maturation. Sequence and phylogenetic analysis indicated that LmDDX3 was closely related to termite DDX3. Taken together, these data reveal a critical role for LmDDX3 in regulating the transcription of Vg and VgR, two major factors in vitellogenesis that is a key process required for ovary development and oocyte maturation in locust, and contribute thereof a new putative target for locust biological control.