Death-associated protein kinase 1 (DAPK) is a calcium/calmodulin kinase that plays a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in hepatocellular carcinoma (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using a proteomics approach, we identified DEAD-box helicase 20 (DDX20) as an important downstream target of DAPK in HCC cells and critical for DAPK-mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion through the CDC42-integrin pathway, which was previously reported as an important downstream pathway of DAPK in cancer. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and identifies new potential therapeutic targets for HCC.

The human gene ddx42 encodes a human DEAD box protein highly homologous to the p68 subfamily of RNA helicases. In HeLa cells, two ddx42 poly(A)+ RNA species were detected both encoding the nuclear localized 938 amino acid Ddx42p polypeptide. Ddx42p has been heterologously expressed and its biochemical properties characterized. It is an RNA binding protein, and ATP and ADP modulate its RNA binding affinity. Ddx42p is an NTPase with a preference for ATP, the hydrolysis of which is enhanced by various RNA substrates. It acts as a non-processive RNA helicase. Interestingly, RNA unwinding by Ddx42p is promoted in the presence of a single-strand (ss) binding protein (T4gp32). Ddx42p, particularly in the ADP-bound form (the state after ATP hydrolysis), also mediates efficient annealing of complementary RNA strands thereby displacing the ss binding protein. Ddx42p therefore represents the first example of a human DEAD box protein possessing RNA helicase, protein displacement and RNA annealing activities. The adenosine nucleotide cofactor bound to Ddx42p apparently acts as a switch that controls the two opposing activities: ATP triggers RNA strand separation, whereas ADP triggers annealing of complementary RNA strands.

Members of the DEAD-box family of RNA helicases contribute to virtually every aspect of RNA metabolism, in organisms from all domains of life. Many of these helicases are constituents of multicomponent assemblies, and their interactions with partner proteins within the complexes underpin their activities and biological function. In Escherichia coli the DEAD-box helicase RhlB is a component of the multienzyme RNA degradosome assembly, and its interaction with the core ribonuclease RNase E boosts the ATP-dependent activity of the helicase. Earlier studies have identified the regulator of ribonuclease activity A (RraA) as a potential interaction partner of both RNase E and RhlB. We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB. Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å. The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases.

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1's enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an 'open' to a 'closed'-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.

Eukaryotic gene expression involves numerous biochemical steps that are dependent on RNA structure and ribonucleoprotein (RNP) complex formation. The DEAD-box class of RNA helicases plays fundamental roles in formation of RNA and RNP structure in every aspect of RNA metabolism. In an effort to explore the diversity of biological roles for DEAD-box proteins, our laboratory recently demonstrated that the DEAD-box protein Dbp2 associates with actively transcribing genes and is required for normal gene expression in Saccharomyces cerevisiae. We now provide evidence that Dbp2 interacts genetically and physically with the mRNA export factor Yra1. In addition, we find that Dbp2 is required for in vivo assembly of mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)+ RNA. Strikingly, we also show that Dbp2 is an efficient RNA helicase in vitro and that Yra1 decreases the efficiency of ATP-dependent duplex unwinding. We provide a model whereby messenger ribonucleoprotein (mRNP) assembly requires Dbp2 unwinding activity and once the mRNP is properly assembled, inhibition by Yra1 prevents further rearrangements. Both Yra1 and Dbp2 are conserved in multicellular eukaryotes, suggesting that this constitutes a broadly conserved mechanism for stepwise assembly of mature mRNPs in the nucleus.

The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.

The CCR4-NOT complex plays an important role in the translational repression and deadenylation of mRNAs. However, little is known about the specific roles of interacting factors. We demonstrate that the DEAD-box helicases eIF4A2 and DDX6 interact directly with the MA3 and MIF domains of CNOT1 and compete for binding. Furthermore, we now show that incorporation of eIF4A2 into the CCR4-NOT complex inhibits CNOT7 deadenylation activity in contrast to DDX6 which enhances CNOT7 activity. Polyadenylation tests (PAT) on endogenous mRNAs determined that eIF4A2 bound mRNAs have longer poly(A) tails than DDX6 bound mRNAs. Immunoprecipitation experiments show that eIF4A2 does not inhibit CNOT7 association with the CCR4-NOT complex but instead inhibits CNOT7 activity. We identified a CCR4-NOT interacting factor, TAB182, that modulates helicase recruitment into the CCR4-NOT complex, potentially affecting the outcome for the targeted mRNA. Together, these data show that the fate of an mRNA is dependent on the specific recruitment of either eIF4A2 or DDX6 to the CCR4-NOT complex which results in different pathways for translational repression and mRNA deadenylation.

Genome sequences are available for the human blood flukes, Schistosoma japonicum, S. mansoni and S. haematobium. Functional genomic approaches could aid in identifying the role and importance of these newly described schistosome genes. Transgenesis is established for functional genomics in model species, which can lead to gain- or loss-of-functions, facilitate vector-based RNA interference, and represents an effective forward genetics tool for insertional mutagenesis screens. Progress toward routine transgenesis in schistosomes might be expedited if germ cells could be reliably localized in cultured schistosomes. Vasa, a member of the ATP-dependent DEAD-box RNA helicase family, is a prototypic marker of primordial germ cells and the germ line in the Metazoa. Using bioinformatics, 33 putative DEAD-box RNA helicases exhibiting conserved motifs that characterize helicases of this family were identified in the S. mansoni genome. Moreover, three of the helicases exhibited vasa-like sequences; phylogenetic analysis confirmed the three vasa-like genes-termed Smvlg1, Smvlg2, and Smvlg3-were members of the Vasa/PL10 DEAD-box subfamily. Transcripts encoding Smvlg1, Smvlg2, and Smvlg3 were cloned from cDNAs from mixed sex adult worms, and quantitative real time PCR revealed their presence in developmental stages of S. mansoni with elevated expression in sporocysts, adult females, eggs, and miracidia, with strikingly high expression in the undeveloped egg. Whole mount in situ hybridization (WISH) analysis revealed that Smvlg1, Smvlg2 and Smvlg3 were transcribed in the posterior ovary where the oocytes mature. Germ cell specific expression of schistosome vasa-like genes should provide an informative landmark for germ line transgenesis of schistosomes, etiologic agents of major neglected tropical diseases.

DEAD-box helicase proteins perform ATP-dependent rearrangements of structured RNAs throughout RNA biology. Short RNA helices are unwound in a single ATPase cycle, but the ATP requirement for more complex RNA structural rearrangements is unknown. Here we measure the amount of ATP used for native refolding of a misfolded group I intron ribozyme by CYT-19, a Neurospora crassa DEAD-box protein that functions as a general chaperone for mitochondrial group I introns. By comparing the rates of ATP hydrolysis and ribozyme refolding, we find that several hundred ATP molecules are hydrolyzed during refolding of each ribozyme molecule. After subtracting nonproductive ATP hydrolysis that occurs in the absence of ribozyme refolding, we find that approximately 100 ATPs are hydrolyzed per refolded RNA as a consequence of interactions specific to the misfolded ribozyme. This value is insensitive to changes in ATP and CYT-19 concentration and decreases with decreasing ribozyme stability. Because of earlier findings that ∼90% of global ribozyme unfolding cycles lead back to the kinetically preferred misfolded conformation and are not observed, we estimate that each global unfolding cycle consumes ∼10 ATPs. Our results indicate that CYT-19 functions as a general RNA chaperone by using a stochastic, energy-intensive mechanism to promote RNA unfolding and refolding, suggesting an evolutionary convergence with protein chaperones.

Cellular transitions and differentiation processes require mRNAs supporting the new phenotype but also the clearance of existing mRNAs for the parental phenotype. Cellular reprogramming from fibroblasts to induced pluripotent stem cells (iPSCs) occurs at the early stage of mesenchymal epithelial transition (MET) and involves drastic morphological changes. We examined the molecular mechanism for MET, focusing on RNA metabolism. DDX6, an RNA helicase, was indispensable for iPSC formation, in addition to RO60 and RNY1, a non-coding RNA, which form complexes involved in intracellular nucleotide sensing. RO60/RNY1/DDX6 complexes formed prior to processing body formation, which is central to RNA metabolism. The abrogation of DDX6 expression inhibited iPSC generation, which was mediated by RNA decay targeting parental mRNAs supporting mesenchymal phenotypes, along with microRNAs, such as miR-302b-3p. These results show that parental mRNA clearance is a prerequisite for cellular reprogramming and that DDX6 plays a central role in this process.

mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors.

DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA-protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome.

DEAD-box RNA helicase Gemin3 is an essential protein since its deficiency is lethal in both vertebrates and invertebrates. In addition to playing a role in transcriptional regulation and RNA silencing, as a core member of the SMN (survival of motor neurons) complex, Gemin3 is required for the biogenesis of spliceosomal snRNPs (small nuclear ribonucleoproteins), and axonal mRNA metabolism. Studies in the mouse and C. elegans revealed that loss of Gemin3 function has a negative impact on ovarian physiology and development.

Patients with cancer frequently develop autoantibodies, and the identification of panels of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. This study aims to exploit the autoantibody repertoire in pancreatic cancer and identify the possible serum marker for pancreatic cancer. Sera from 55 newly diagnosed patients with pancreatic cancer and 52 healthy controls were analyzed for antibody-based reactivity against Hep-2, a human larynx epithelioma cancer cell line, with one-dimensional immunoblot assay. From this analysis, we observed a prominent band with a molecular weight of 47 kDa in 63.64% (35/55) patients, while in only 1.9% normal group (1/52). Using immunoblot analysis after two-dimensional electrophoresis combined with liquid chromatography-electrospray ionization tandem mass spectrometry, this target antigen was identified as DEAD-box protein 48 (DDX48). BLAST analysis showed that it was highly similar to eukaryotic initiation factor 4A and might play a role in pre-mRNA processing. An enzyme-linked immunosorbent assay was performed using recombinant, purified DDX48 as an antigen to detect anti-DDX48 autoantibodies in sera. Reactivity was observed in 20 of 60 (33.33%) pancreatic cancer patients, 3 of 30 (10.00%) colorectal cancer patients, 2 of 30 (6.67%) gastric cancer patients, 2 of 30 (6.67%) hepatocellular cancer patients, while none of the 20 chronic pancreatitis patients, 30 lung cancer patients, and 60 normal individuals. Together, these results demonstrate that the detection of autoantibodies to DDX48 may have clinical utility for the improved diagnosis of pancreatic cancer.

Introduction: DEAD-box helicase 27 (DDX27) belongs to DEAD-Box nucleic acid helicase family. The function of DDX27 in hepatocellular carcinoma (HCC) remain enigmatic. In light of this, we tried to investigate the regulatory role and underlying mechanism of DDX27 in HCC. Materials and methods: DDX27 expression levels were detected by qRT-PCR, Western blot and immunohistochemistry assays in HCC tissues and cells. Colony formation, CCK-8, growth curve, wound healing and transwell assays were conducted to investigate the effect of DDX27 on the proliferation and metastasis of HCC cells. RNA-sequencing was performed to detect the effect of DDX27 on downstream signaling pathway. The effect of DDX27 on HCC progression was evaluated using in vivo murine xenograft model. Results: we found an increased expression of DDX27 in HCC tissues with comparison to its para-tumor tissues. The high expression levels of DDX27 were associated with poor prognosis in HCC patients. DDX27 upregulation promoted cell metastasis. Mechanistic studies suggested that DDX27 overexpression induces the major vault protein (MVP) expression and enhances the phosphorylation levels of ERK1/2. Inhibition of ERK pathway impaired the cellular metastastic abilities induced by DDX27. The induction of DDX27 in HCC progression was further confirmed from tumors in mouse model. Conclusion: our results disclose a novel mechanism by which DDX27 enhances ERK signaling during HCC progression. DDX27 might be used in targeted therapy for HCC patients.

Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex.

The integrated stress response is a network of highly orchestrated pathways activated when cells are exposed to environmental stressors. While global repression of translation is a well-recognized hallmark of the integrated stress response, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD box 1 (DDX1) protein accumulates at DNA double-strand breaks during genotoxic stress and promotes DNA double-strand break repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors, including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing, we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs are increased during stress in a DDX1-dependent manner. Even though DDX1's RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.

Conditional proteolytic degradation is an irreversible and highly regulated process that fulfills crucial regulatory functions in all organisms. As proteolytic targets tend to be critical metabolic or regulatory proteins, substrates are targeted for degradation only under appropriate conditions through the recognition of an amino acid sequence referred to as a "degron". DEAD-box RNA helicases mediate all aspects of RNA metabolism, contributing to cellular fitness. However, the mechanism by which abiotic-stress modulation of protein stability regulates bacterial helicase abundance has not been extensively characterized. Here, we provide in vivo evidence that proteolytic degradation of the cyanobacterial DEAD-box RNA helicase CrhR is conditional, being initiated by a temperature upshift from 20 to 30 °C in the model cyanobacterium, Synechocystis sp. PCC 6803. We show degradation requires a unique, highly conserved, inherently bipartite degron located in the C-terminal extension found only in CrhR-related RNA helicases in the phylum Cyanobacteria. However, although necessary, the degron is not sufficient for proteolysis, as disruption of RNA helicase activity and/or translation inhibits degradation. These results suggest a positive feedback mechanism involving a role for CrhR in expression of a crucial factor required for degradation. Furthermore, AlphaFold structural prediction indicated the C-terminal extension is a homodimerization domain with homology to other bacterial RNA helicases, and mass photometry data confirmed that CrhR exists as a dimer in solution at 22 °C. These structural data suggest a model wherein the CrhR degron is occluded at the dimerization interface but could be exposed if dimerization was disrupted by nonpermissive conditions.

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