DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).

The DEAH-box helicase RHAU is an essential gene and critical for mouse hematopoiesis.

The DEAH helicase RHAU (alias DHX36, G4R1) is the only helicase shown to have G-quadruplex (G4)-RNA resolvase activity and the major source of G4-DNA resolvase activity. Previous report showed RHAU mRNA expression to be elevated in human lymphoid and CD34(+) BM cells, suggesting a potential role in hematopoiesis. Here, we generated a conditional knockout of the RHAU gene in mice. Germ line deletion of RHAU led to embryonic lethality. We then targeted the RHAU gene specifically in the hematopoiesis system, using a Cre-inducible system in which an optimized variant of Cre recombinase was expressed under the control of the Vav1 promoter. RHAU deletion in hematopoietic system caused hemolytic anemia and differentiation defect at the proerythroblast stage. The partial differentiation block of proerythroblasts was because of a proliferation defect. Transcriptome analysis of RHAU knockout proerythroblasts showed that a statistically significant portion of the deregulated genes contain G4 motifs in their promoters. This suggests that RHAU may play a role in the regulation of gene expression that relies on its G4 resolvase activity.

Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.

The DEAH-box RNA helicase RHAU binds an intramolecular RNA G-quadruplex in TERC and associates with telomerase holoenzyme.

Guanine-quadruplexes (G4) consist of non-canonical four-stranded helical arrangements of guanine-rich nucleic acid sequences. The bulky and thermodynamically stable features of G4 structures have been shown in many respects to affect normal nucleic acid metabolism. In vivo conversion of G4 structures to single-stranded nucleic acid requires specialized proteins with G4 destabilizing/unwinding activity. RHAU is a human DEAH-box RNA helicase that exhibits G4-RNA binding and resolving activity. In this study, we employed RIP-chip analysis to identify en masse RNAs associated with RHAU in vivo. Approximately 100 RNAs were found to be associated with RHAU and bioinformatics analysis revealed that the majority contained potential G4-forming sequences. Among the most abundant RNAs selectively enriched with RHAU, we identified the human telomerase RNA template TERC as a true target of RHAU. Remarkably, binding of RHAU to TERC depended on the presence of a stable G4 structure in the 5'-region of TERC, both in vivo and in vitro. RHAU was further found to associate with the telomerase holoenzyme via the 5'-region of TERC. Collectively, these results provide the first evidence that intramolecular G4-RNAs serve as physiologically relevant targets for RHAU. Furthermore, our results suggest the existence of alternatively folded forms of TERC in the fully assembled telomerase holoenyzme.

G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K(d)'s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K(d)'s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.

Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU.

Under physiological conditions, guanine-rich sequences of DNA and RNA can adopt stable and atypical four-stranded helical structures called G-quadruplexes (G4). Such G4 structures have been shown to occur in vivo and to play a role in various processes such as transcription, translation and telomere maintenance. Owing to their high-thermodynamic stability, resolution of G4 structures in vivo requires specialized enzymes. RHAU is a human RNA helicase of the DEAH-box family that exhibits a unique ATP-dependent G4-resolvase activity with a high affinity and specificity for its substrate in vitro. How RHAU recognizes G4-RNAs has not yet been established. Here, we show that the amino-terminal region of RHAU is essential for RHAU to bind G4 structures and further identify within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. G4-resolvase activity and strict RSM dependency are also observed with CG9323, the Drosophila orthologue of RHAU, in the amino terminal region of which the RSM is the only conserved motif. Thus, these results reveal a novel motif in RHAU protein that plays an important role in recognizing and resolving G4-RNA structures, properties unique to RHAU among many known RNA helicases.

Translational control by DHX36 binding to 5'UTR G-quadruplex is essential for muscle stem-cell regenerative functions.

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.

p66SHC-mediated mitochondrial dysfunction in renal proximal tubule cells during oxidative injury.

Mitochondrial dysfunction is involved in pathopysiology of ischemia-reperfusion-induced acute kidney injury (AKI). The p66shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction, which might play a role in AKI-induced renal tubular injury. Oxidative stress-mediated Serine36 phosphorylation of p66shc facilitates its transportation to the mitochondria where it oxidizes cytochrome c and generates excessive amount of reactive oxygen species (ROS). The consequence is mitochondrial depolarization and injury. Earlier we determined that p66shc plays an essential role in injury of cultured mouse renal proximal tubule cells during oxidative stress. Here, we studied the role of p66shc in ROS generation and consequent mitochondrial dysfunction during oxidative injury in renal proximal tubule cells. We employed p66shc knockdown renal proximal tubule cells and cells that overexpress wild-type, Serine phosphorylation (S36A), or cytochrome c-binding (W134F) mutants of p66shc. Inhibition of the mitochondrial electron transport chain or the mitochondrial permeability transition revealed that hydrogen peroxide-induced injury is mitochondrial ROS and consequent mitochondrial depolarization dependent. We also found that through Ser36 phosphorylation and mitochondria/cytochrome c binding, p66shc mediates those effects. We propose a similar mechanism in vivo as we demonstrated mitochondrial binding of p66shc as well as its association with cytochrome c in the postischemic kidneys of mice. Thus, manipulating p66shc might offer a new therapeutic modality to ameliorate renal ischemic injury.

Transcriptional regulation of the plasminogen activator inhibitor type 1--with an emphasis on negative regulation.

By inhibiting plasminogen activators uPA and tPA, inducing uPA-uPAR internalization and interfering with the interaction between extracellular matrix protein vitronectin and alphavbeta3 integrin, plasminogen activator inhibitor type 1 (PAI-1) is active in the regulation of various biological processes involving extracellular proteolysis and tissue remodeling. PAI-1 is expressed in many cell types under the control of a variety of signals, depending on cell type. The most prominent and important of these signals are TGFbeta, hypoxia and insulin. Although the signaling pathways were largely elucidated, recent investigations have revealed more complicated aspects. The pathways interact at the level of both transcription factors and regulatory elements on the promoter. Furthermore, the engagement of negative factors in these pathways has been shown to be important, adding complexity and versatility to PAI-1 gene regulation.

Induction of uPA gene expression by the blockage of E-cadherin via Src- and Shc-dependent Erk signaling.

Loss of E-cadherin-mediated cell-cell adhesion and expression of proteolytic enzymes characterize the transition from benign lesions to invasive, metastatic tumor, a rate-limiting step in the progression from adenoma to carcinoma in vivo. A soluble E-cadherin fragment found recently in the serum and urine of cancer patients has been shown to disrupt cell-cell adhesion and to drive cell invasion in a dominant-interfering manner. Physical disruption of cell-cell adhesion can be mimicked by the function-blocking antibody Decma. We have shown previously in MCF7 and T47D cells that urokinase-type plasminogen activator (uPA) activity is up-regulated upon disruption of E-cadherin-dependent cell-cell adhesion. We explored the underlying molecular mechanisms and found that blockage of E-cadherin by Decma elicits a signaling pathway downstream of E-cadherin that leads to Src-dependent Shc and extracellular regulated kinase (Erk) activation and results in uPAgene activation. siRNA-mediated knockdown of endogenous Src-homology collagen protein (Shc) and subsequent expression of single Shc isoforms revealed that p46(Shc) and p52(Shc) but not p66(Shc) were able to mediate Erk activation. A parallel pathway involving PI3K contributed partially to Decma-induced Erk activation. This report describes that disruption of E-cadherin-dependent cell-cell adhesion induces intracellular signaling with the potential to enhance tumorigenesis and, thus, offers new insights into the pathophysiological mechanisms of tumor development.

Stabilization of urokinase and urokinase receptor mRNAs by HuR is linked to its cytoplasmic accumulation induced by activated mitogen-activated protein kinase-activated protein kinase 2.

The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3' untranslated regions. The cellular proteins binding to these RNA sequences (ARE(uPA/uPAR)) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an ARE(uPA)-containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a beta-globin reporter mRNA containing the ARE(uPA). RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steady-state levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the ARE(uPA), which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.

Transcriptional and posttranscriptional regulation of the plasminogen activator system.

The core protein components of the plasminogen activator (PA) system are two plasminogen activators, two plasminogen activator inhibitors and a urokinase type plasminogen activator-specific cell surface receptor. Various types of biological regulation are exerted through the interplay of these components mutually and with extracellular matrix proteins and cell membrane proteins, with or without involving proteolytic activity. Reflecting these diverse biological roles, the level and activity of each component of the PA system is under the control of a variety of regulatory mechanisms. The expression level of a protein reflects the level of the corresponding mRNA, which is essentially the net result of de novo synthesis, i.e. transcription, and degradation. Many recent studies have shown that the regulation of mRNA stability is dynamic and cell specific. Accordingly, we are learning that the mRNAs of the PA system are also the subject of diverse regulatory mechanisms. In this short review, we summarize current understanding of the transcriptional and mRNA-stability regulation of the PA system.

Post-transcriptional Regulation of Nkx2-5 by RHAU in Heart Development.

RNA G-quadruplexes (G4s) play important roles in RNA biology. However, the function and regulation of mRNA G-quadruplexes in embryonic development remain elusive. Previously, we identified RHAU (DHX36, G4R1) as an RNA helicase that resolves mRNA G-quadruplexes. Here, we find that cardiac deletion of Rhau leads to heart defects and embryonic lethality in mice. Gene expression profiling identified Nkx2-5 mRNA as a target of RHAU that associates with its 5' and 3' UTRs and modulates its stability and translation. The 5' UTR of Nkx2-5 mRNA contains a G-quadruplex that requires RHAU for protein translation, while the 3' UTR of Nkx2-5 mRNA possesses an AU-rich element (ARE) that facilitates RHAU-mediated mRNA decay. Thus, we uncovered the mechanisms underlying Nkx2-5 post-transcriptional regulation during heart development. Meanwhile, this study demonstrates the function of mRNA 5' UTR G-quadruplex-mediated protein translation in organogenesis.

Cisplatin induces apoptosis through the ERK-p66shc pathway in renal proximal tubule cells.

The extracellular signal-regulated kinase (ERK) has been shown to mediate cisplatin (CP)-induced toxicity to renal proximal tubule cells. Here, we demonstrate that ERK serves as the kinase that phosphorylates the pro-apoptotic p66shc protein at its Serine36 residue in CP-treated renal proximal tubule cells. Pharmacologic or dominant-negative inhibition of ERK mitigates cisplatin-induced Ser36 phosphorylation of p66shc. Overexpression of p66shc exacerbates while its knockdown or mutation of the Serine36 site to alanine ameliorates CP-induced nephrotoxicity in vitro. Since p66shc is Serine36 phosphorylated in the kidneys of mice after treatment with CP, a similar mechanism might exist in vivo.

The plasminogen activator inhibitor 2 transcript is destabilized via a multi-component 3' UTR localized adenylate and uridylate-rich instability element in an analogous manner to cytokines and oncogenes.

Plasminogen activator inhibitor type 2 (PAI-2; SERPINB2) is a highly-regulated gene that is subject to both transcriptional and post-transcriptional control. For the latter case, inherent PAI-2 mRNA instability was previously shown to require a nonameric adenylate-uridylate element in the 3' UTR. However, mutation of this site was only partially effective at restoring complete mRNA stabilization. In the present study, we have identified additional regulatory motifs within the 3' UTR that cooperate with the nonameric adenylate-uridylate element to promote mRNA destabilization. These elements are located within a 74 nucleotide U-rich stretch (58%) of the 3' UTR that flanks the nonameric motif; deletion or substitution of this entire region results in complete mRNA stabilization. These new elements are conserved between species and optimize the destabilizing capacity with the nonameric element to ensure complete mRNA instability in a manner analogous to some class I and II adenylate-uridylate elements present in transcripts encoding oncogenes and cytokines. Hence, post-transcriptional regulation of the PAI-2 mRNA transcript involves an interaction between closely spaced adenylate-uridylate elements in a manner analogous to the post-transcriptional regulation of oncogenes and cytokines.

p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells.

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.

Transcription-dependent nucleolar cap localization and possible nuclear function of DExH RNA helicase RHAU.

RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments.

Recruitment of the RNA helicase RHAU to stress granules via a unique RNA-binding domain.

In response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAU-specific RNA-binding domain.

G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates.

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures.