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RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identified hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma. Show less

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. Show less

G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure and the RHAU helicase has been identified to have high specificity for recognition of parallel-stranded G4s. We have designed and synthesized two stapled peptide analogues of the G4-specfic motif of RHAU, which preserve the G4 binding ability. Characterization of these peptides identified the stapled variants to exhibit higher helical formation propensity in aqueous buffer in comparison to the native RHAU sequence. Moreover, the stapled peptides exhibit superior enzymatic stability towards α-chymotrypsin. Our stapled RHAU peptides can serve as a new tool for targeting G4 nucleic acid structures. Show less

Single-stranded DNA (ssDNA) containing four guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their DNA-binding specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ∼15,000 potential G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4-binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding specificity of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition. Show less

RNA G-quadruplexes (RG4s) appear to be important in post-transcriptional gene regulation, but their pathophysiological functions remain unknown. MicroRNA-26a (miR-26a) is emerging as a therapeutic target for various human diseases, however the mechanisms underlying endogenous miR-26a regulation are poorly understood. Herein, we study the role of RG4 in miR-26a expression and function in vitro and in vivo. Putative RG4s within liver-enriched miRNAs were predicted by bioinformatic analysis, and the presence of an RG4 structure in the miR-26a-1 precursor (pre-miR-26a-1) was further analyzed by biophysical and biochemical methods. RG4 stabilizers, pre-miR-26a-1 overexpression plasmids, and luciferase reporter assays were used to assess the effect of RG4 on pre-miR-26a-1 maturation. Both miR-26a knock-in and knockout mouse models were employed to investigate the influence of this RG4 on miR-26a expression and function. Moreover, the interaction between RG4 in pre-miR-26a-1 and DEAH-box helicase 36 (DHX36) was determined by biophysical and molecular methods. Finally, miR-26a processing and DHX36 expression were quantified in the livers of obese mice. We identify a guanine-rich sequence in pre-miR-26a-1 that can fold into an RG4 structure. This RG4 impairs pre-miR-26a-1 maturation, resulting in a decrease in miR-26a expression and subsequently an increase in miR-26a cognate targets. In line with known miR-26a functions, this RG4 can regulate hepatic insulin sensitivity and lipid metabolism in vitro and in vivo. Furthermore, we reveal that DHX36 can bind and unwind this RG4 structure, thereby enhancing miR-26a maturation. Intriguingly, there is a concordant decrease of miR-26a maturation and DHX36 expression in obese mouse livers. Our findings define a dynamic DHX36/RG4/miR-26a regulatory axis during obesity, highlighting an important role of RG4 in physiology and pathology. Specific RNA sequences called G-quadruplexes (or RG4) appear to be important in post-transcriptional gene regulation. Obesity leads to the formation of these RG4 structures in pre-miR-26a-1 molecules, impairing the maturation and function of miR-26a, which has emerged as a therapeutic target in several diseases. This contributes to hepatic insulin resistance and the dysregulation of liver metabolism. Show less

The DEAH/RHA helicase DHX36 has been linked to cellular RNA and DNA quadruplex structures and to AU-rich RNA elements. In vitro, DHX36 remodels DNA and RNA quadruplex structures and unwinds DNA duplexes in an ATP-dependent manner. DHX36 contains the superfamily 2 helicase core and several auxiliary domains that are conserved in orthologs of the enzyme. The role of these auxiliary domains for the enzymatic function of DHX36 is not well understood. Here, we combine structural and biochemical studies to define the function of three auxiliary domains that contact nucleic acid. We first report the crystal structure of mouse DHX36 bound to ADP. The structure reveals an overall architecture of mouse DHX36 that is similar to previously reported architectures of fly and bovine DHX36. In addition, our structure shows conformational changes that accompany stages of the ATP-binding and hydrolysis cycle. We then examine the roles of the DHX36-specific motif (DSM), the OB-fold, and a conserved β-hairpin (β-HP) in mouse DHX36 in the remodeling of RNA structures. We demonstrate and characterize RNA duplex unwinding for DHX36 and examine the remodeling of inter- and intramolecular RNA quadruplex structures. We find that the DSM not only functions as a quadruplex binding adaptor but also promotes the remodeling of RNA duplex and quadruplex structures. The OB-fold and the β-HP contribute to RNA binding. Both domains are also essential for remodeling RNA quadruplex and duplex structures. Our data reveal roles of auxiliary domains for multiple steps of the nucleic acid remodeling reactions. Show less

The innate immune system recognizes conserved pathogen-associated molecular patterns and produces inflammatory cytokines that direct downstream immune responses. The inappropriate localization of DNA within the cell cytosol or endosomal compartments indicates that a cell may either be infected by a DNA virus or bacterium, or has problems with its own nuclear integrity. This DNA is sensed by certain receptors that mediate cytokine production and, in some cases, initiate an inflammatory and lytic form of cell death called pyroptosis. Dysregulation of these DNA-sensing pathways is thought to contribute to autoimmune diseases and the development of cancer. In this review, we will discuss the DNA sensors Toll-like receptor 9 (TLR9), cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), absent in melanoma 2 (AIM2), and interferon gamma-inducible 16 (IFI16), their ligands, and their physiological significance. We will also examine the less-well-understood DEAH- and DEAD-box helicases DHX9, DHX36, DDX41, and RNA polymerase III, each of which may play an important role in DNA-mediated innate immunity. Show less

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) - a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3'-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5'-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads. Show less

The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays a major role in lung liquid clearance at birth and lung edema resorption in adulthood. We showed previously that αENaC mRNA expression is downregulated in part via posttranscriptional regulation of mRNA stability. In the present work, the role of the αENaC 3' untranslated region (3'UTR) in the regulation of mRNA stability was studied further. Quantitative reverse transcription PCR (qRT-PCR) was performed to investigate the expression of αENaC in alveolar epithelial cells. The role of the αENaC 3'UTR was evaluated through sequential deletions. RNA affinity chromatography and mass spectrometry were achieved to investigate the nature of the proteins that could bind this sequence. The function of these proteins was assessed through knockdown and overexpression in vitro. First, we found that αENaC mRNA half-life was much shorter than expected when using a transcriptionally controlled plasmid expression system compared to Actinomycin D treatment. Sequential deletions of the αENaC 3'UTR revealed that the αENaC 3'UTR plays an important role in the modulation of αENaC mRNA stability, and that there is a complex stabilizing and destabilizing interplay between different regions of the 3'UTR that modulate this process. Finally, we identified RNA-binding proteins that interact with the αENaC 3'UTR and showed that Dhx36 and Tial1 are involved in the decrease in αENaC mRNA stability via the proximal region of its 3'UTR. Taken together, these findings indicate that the αENaC 3'UTR plays an important role in modulating transcript levels, and Dhx36 and Tial1 seem to be involved in posttranscriptional regulation of αENaC expression in alveolar epithelial cells. Show less

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress. Show less

Guanine-quadruplex (G-quadruplex) structures in mRNAs have been shown to modulate gene expression. However, the overall biological relevance of this process is under debate, as cellular helicases unwind G-quadruplex structures. The helicase Rhau (encoded by the DHX36 gene) was reported to be the major source of RNA G-quadruplex resolving activity in lysates of human cells. In the current study, we depleted Rhau by RNAi-mediated silencing and analyzed the effect on proteins whose mRNAs harbor a G-quadruplex motif in their 5'-UTRs. A targeted investigation of the proto-oncogenes Bcl-2 and NRAS, which are well-known examples for the translational repression of G-quadruplex structures, did not reveal effects caused by Rhau silencing. We therefore carried out a global analysis of changes in protein levels by label-free quantification using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Following Rhau knockdown, of all the identified proteins, only 1.9% were significantly downregulated to at least 70%. According to a bioinformatic analysis with the QGRS mapper, 33% of the downregulated proteins were predicted to harbor a G-quadruplex motif in the 5'-UTR of their respective mRNAs, compared to only 11% in the complete dataset. This indicates that in an unexpectedly small set of genes, in which G-quadruplex motifs are unusually common in the 5'-UTR of their mRNAs, Rhau helicase is responsible for the regulation of their expression. Show less

Numerous cytosolic DNA sensors (CDSs), which are very important for recognizing cytosolic dsDNA derived from intracellular viruses and bacteria, exist in mammals. However, teleost CDSs are poorly understood. In this study, four CDSs, including the cyclic GMP-AMP synthase (cGAS), Sm-like protein 14 homolog A (LSm14A), DEAH-box helicase (DHX) 9, and DHX36 genes were identified in Japanese medaka, Oryzias latipes, and their expression patterns were elucidated. The expression of these genes was upregulated in the intestines and kidney of CpG-ODN-stimulated medaka. The cGAS and LSm14A genes were significantly induced in the intestines, kidney, and spleen of formalin-killed Edwardsiella tarda-treated medaka; the DHX9 and DHX36 genes were not. cGAS gene expression was induced only in the intestines of live E. tarda-treated medaka. These results suggest that the transcription of four CDS genes of medaka responds to dsDNA stimulation, and cGAS is probably more important for the immune response against E. tarda infection. Show less

We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence complementary to the target RNA. This RNase:DNA complex targets and efficiently cleaves the single-stranded RNA in a site-specific manner. A major cleavage site occurs at the RNA region that is complementary to the DNA template sequence. The newly designed RNase can serve as a simple tool for RNA manipulation and probing RNA structure. Show less

DHX36 is a DEAH-box helicase that resolves parallel G-quadruplex structures formed in DNA and RNA. The recent co-crystal structure of DHX36 bound G4-DNA revealed an intimate contact, but did not address the role of ATP hydrolysis in G4 resolving activity. Here, we demonstrate that unlike on G4-DNA, DHX36 displays ATP-independent unfolding of G4-RNA followed by ATP-dependent refolding, generating a highly asymmetric pattern of activity. Interestingly, DHX36 refolds G4-RNA in several steps, reflecting the discrete steps in forming the G4 structure. We show that the ATP-dependent activity of DHX36 arises from the RNA tail rather than the G4. Mutations that perturb G4 contact result in quick dissociation of the protein from RNA upon ATP hydrolysis, while mutations that interfere with binding the RNA tail induce dysregulated activity. We propose that the ATP-dependent activity of DHX36 may be useful for dynamically resolving various G4-RNA structures in cells. Show less

Helicase DHX36 plays essential roles in cell development and differentiation at least partially by resolving G-quadruplex (G4) structures. Here we report crystal structures of the Drosophila homolog of DHX36 (DmDHX36) in complex with RNA and a series of DNAs. By combining structural, small-angle X-ray scattering, molecular dynamics simulation, and single-molecule fluorescence studies, we revealed that positively charged amino acids in RecA2 and OB-like domains constitute an elaborate structural pocket at the nucleic acid entrance, in which negatively charged G4 DNA is tightly bound and partially destabilized. The G4 DNA is then completely unfolded through the 3'-5' translocation activity of the helicase. Furthermore, crystal structures and DNA binding assays show that G-rich DNA is preferentially recognized and in the presence of ATP, specifically bound by DmDHX36, which may cooperatively enhance the G-rich DNA translocation and G4 unfolding. On the basis of these results, a conceptual G4 DNA-resolving mechanism is proposed. Show less

Single-stranded DNA (ssDNA) and RNA regions that include at least four closely spaced runs of three or more consecutive guanosines strongly tend to fold into stable G-quadruplexes (G4s). G4s play key roles as DNA regulatory sites and as kinetic traps that can inhibit biological processes, but how G4s are regulated in cells remains largely unknown. Here, we developed a kinetic framework for G4 disruption by DEAH-box helicase 36 (DHX36), the dominant G4 resolvase in human cells. Using tetramolecular DNA and RNA G4s with four to six G-quartets, we found that DHX36-mediated disruption is highly efficient, with rates that depend on G4 length under saturating conditions ( Show less

RNA secondary structures in the 5'-untranslated regions (5'-UTR) of mRNAs are key to the post-transcriptional regulation of gene expression. While it is evident that non-canonical Hoogsteen-paired G-quadruplex (rG4) structures somehow contribute to the regulation of translation initiation, the nature and extent of human mRNAs that are regulated by rG4s is not known. Here, we provide new insights into a mechanism by which rG4 formation modulates translation. Using transcriptome-wide ribosome profiling, we identify rG4-driven mRNAs in HeLa cells and reveal that rG4s in the 5'-UTRs of inefficiently translated mRNAs associate with high ribosome density and the translation of repressive upstream open reading frames (uORF). We demonstrate that depletion of the rG4-unwinding helicases DHX36 and DHX9 promotes translation of rG4-associated uORFs while reducing the translation of coding regions for transcripts that comprise proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites shows that reduced translation is mediated through direct physical interaction between the helicase and its rG4 substrate. This study identifies human mRNAs whose translation efficiency is modulated by the DHX36- and DHX9-dependent folding/unfolding of rG4s within their 5'-UTRs. We reveal a previously unknown mechanism for translation regulation in which unresolved rG4s within 5'-UTRs promote 80S ribosome formation on upstream start codons, causing inhibition of translation of the downstream main open reading frames. Our findings suggest that the interaction of helicases with rG4s could be targeted for future therapeutic intervention. Show less

microRNAs (miRNAs) have emerged as critical regulators of neuronal dendrite development. Specific precursor (pre-)miRNAs are actively transported to dendrites, but whether this process is regulated by neuronal activity and involved in activity-dependent dendritogenesis is unknown. Here we show that BDNF, a neurotrophin that is released in response to increased neuronal activity, promotes dendritic accumulation of pre-miR-134. Dendritic accumulation, but not transcription of pre-miR-134, is abrogated by treatment of neurons with the NMDA receptor (NMDAR) antagonist APV. Furthermore, APV interferes with BDNF-mediated repression of the known miR-134 target Pumilio 2 (Pum2) in a miR-134 binding site-specific manner. At the functional level, both APV treatment and knockdown of the pre-miR-134 transport protein DHX36 antagonize BDNF-induced dendritogenesis. These effects are likely mediated by reduced dendritic miR-134 activity, since both transfection of a synthetic miR-134 duplex or of a dendritically targeted pre-miR-134-181a chimera rescues BDNF-dependent dendritogenesis in the presence of APV. In conclusion, we have identified a novel NMDAR-dependent mechanism involved in the activity-dependent control of miRNA function during neuronal development. Show less

Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than most polymerases can exert Show less

Long non-coding RNAs (lncRNAs) are frequently dysregulated in a variety of human cancers. However, their biological roles in these cancers remain incompletely understood. In this study, we analyze the gene expression profiles of colon cancer tissues and identify a previously unannotated lncRNA, FLJ39051, that we term GSEC (G-quadruplex-forming sequence containing lncRNA), as a lncRNA that is upregulated in colorectal cancer. We further demonstrate that knockdown of GSEC results in the reduction of colon cancer cell motility. We also show that GSEC binds to the DEAH box polypeptide 36 (DHX36) RNA helicase via its G-quadruplex-forming sequence and inhibits DHX36 G-quadruplex unwinding activity. Moreover, knockdown of DHX36 restores the reduced migratory activity of colon cancer cells caused by GSEC knockdown. These results suggest that GSEC plays an important role in colon cancer cell migration by inhibiting the function of DHX36 via its G-quadruplex structure. Show less

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed. Show less

DExD/H-box helicase 36 (DHX36) is known to be an ATP-dependent RNA helicase that unwinds the guanine-quadruplexes DNA or RNA, but emerging data suggest that it also functions as pattern recognition receptor in innate immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Show less

The DEAH-box ATP-dependent RHAU helicases specifically unfold RNA and DNA G-quadruplexes (G4s). However, it remains unclear how the RHAU's G4 unfolding activity is coupled to different stages of the ATPase cycle. Here, using a single-molecule manipulation approach, we show that binding of Drosophila RHAU stabilizes an intramolecularly folded parallel DNA G4 against mechanical unfolding in its nucleotide-free and in its AMP-PNP or ADP bound states, while it destabilizes the G4 when coupled to ATP hydrolysis. Importantly, our results show that the ADP·AlF[Formula: see text]-bound RHAU does not stabilize the G4. We also found that both a single-stranded 3' DNA tail and the RSM domain of RHAU that binds specifically to the G4 structure, are dispensable for the stabilization of the G4, but both are required for G4 destabilization. Our study provides the first evidence that the unfolding kinetics of a G-quadruplex can be modulated by different nucleotide-bound states of the helicase. Show less

G-quadruplexes (G4) are RNA and DNA secondary structures formed by the stacking of guanine quartets in guanine rich sequences. Quadruplex-prone motifs may be found in key genomic regions such as telomeres, ribosomal DNA, transcriptional activators and regulators or oncogene promoters. A number of proteins involved in various biological processes are able to interact with G4s. Among them, proteins dedicated to nucleic acids unwinding such as WRN, BLM, FANCJ or PIF1, can unfold G4 structures. Mutations of these helicases are linked to genome instability and to increases in cancer risks. Here, we present a high-throughput fluorescence-based reliable, inexpensive and fast assay to study G4/RHAU interaction. RHAU is an RNA helicase known as the major source of G4 resolution in HeLa cells. Our assay allows to monitor the unfolding properties of RHAU towards DNA and RNA quadruplexes in parallel and to screen for the optimal conditions for its activity. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Show less

Pre-mRNA 3'-end processing, the process through which almost all eukaryotic mRNAs acquire a poly(A) tail is generally inhibited during the cellular DNA damage response leading to a profound impact on the level of protein expression since unprocessed transcripts at the 3'-end will be degraded or unable to be transported to the cytoplasm. However, a compensatory mechanism involving the binding of the hnRNP H/F family of RNA binding proteins to an RNA G-quadruplex (G4) structure located in the vicinity of a polyadenylation site has previously been described to allow the transcript encoding the p53 tumour suppressor protein to be properly processed during DNA damage and to provide the cells with a way to react to DNA damage. Here we report that the DEAH (Asp-Glu-Ala-His) box RNA helicase DHX36/RHAU/G4R1, which specifically binds to and resolves parallel-stranded G4, is necessary to maintain p53 pre-mRNA 3'-end processing following UV-induced DNA damage. DHX36 binds to the p53 RNA G4, while mutation of the G4 impairs the ability of DHX36 to maintain pre-mRNA 3'-end processing. Stabilization of the p53 RNA G4 with two different G4 ligands ( Show less

Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway. Show less