Helicase Research Articles

Molecular basis for the activation of human spliceosome

The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5′-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.

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

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

Cdt1 Self-associates via the Winged-Helix Domain of the Central Region during the Licensing Reaction, Which Is Inhibited by Geminin.

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two β-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

The RNA helicase DHX35 functions as a co-sensor for RIG-I-mediated innate immunity.

RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-β (IFN-β), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway.

CMG helicase disassembly is essential and driven by two pathways in budding yeast

The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.

Eukaryotic Pif1 helicase unwinds G-quadruplex and dsDNA using a conserved wedge

G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5′ single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5′ most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.

DHX9 SUMOylation is required for the suppression of R-loop-associated genome instability

RNA helicase DHX9 is essential for genome stability by resolving aberrant R-loops. However, its regulatory mechanisms remain unclear. Here we show that SUMOylation at lysine 120 (K120) is crucial for DHX9 function. Preventing SUMOylation at K120 leads to R-loop dysregulation, increased DNA damage, and cell death. Cells expressing DHX9 K120R mutant which cannot be SUMOylated are more sensitive to genotoxic agents and this sensitivity is mitigated by RNase H overexpression. Unlike the mutant, wild-type DHX9 interacts with R-loop-associated proteins such as PARP1 and DDX21 via SUMO-interacting motifs. Fusion of SUMO2 to the DHX9 K120R mutant enhances its association with these proteins, reduces R-loop accumulation, and alleviates survival defects of DHX9 K120R. Our findings highlight the critical role of DHX9 SUMOylation in maintaining genome stability by regulating protein interactions necessary for R-loop balance.

The RNA helicase DDX39 contributes to the nuclear export of spliceosomal U snRNA by loading of PHAX onto RNA.

RNA helicases are involved in RNA metabolism in an ATP-dependent manner. Although many RNA helicases unwind the RNA structure and/or remove proteins from the RNA, some can load their interacting proteins onto RNAs. Here, we developed an in vitro strategy to identify the ATP-dependent factors involved in spliceosomal uridine-rich small nuclear RNA (U snRNA) export. We identified the RNA helicase UAP56/DDX39B, a component of the mRNA export complex named the transcription-export (TREX) complex, and its closely related RNA helicase URH49/DDX39A as the factors that stimulated RNA binding of PHAX, an adapter protein for U snRNA export. ALYREF, another TREX component, acted as a bridge between PHAX and UAP56/DDX39B. We also showed that UAP56/DDX39B and ALYREF participate in U snRNA export through a mechanism distinct from that of mRNA export. This study describes a novel aspect of the TREX components for U snRNP biogenesis and highlights the loading activity of RNA helicases.

The RNA helicases DDX19A/B modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export in leukemia cells.

Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels. Moreover, we found that either selinexor treatment or DDX19A depletion effectively induced apoptosis of T-ALL lines expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.

DEAD box RNA helicases are pervasive protein kinase interactors and activators.

DEAD box (DDX) RNA helicases are a large family of ATPases, many of which have unknown functions. There is emerging evidence that besides their role in RNA biology, DDX proteins may stimulate protein kinases. To investigate if protein kinase-DDX interaction is a more widespread phenomenon, we conducted three orthogonal large-scale screens, including proteomics analysis with 32 RNA helicases, protein array profiling, and kinome-wide in vitro kinase assays. We retrieved Ser/Thr protein kinases as prominent interactors of RNA helicases and report hundreds of binary interactions. We identified members of ten protein kinase families, which bind to, and are stimulated by, DDX proteins, including CDK, CK1, CK2, DYRK, MARK, NEK, PRKC, SRPK, STE7/MAP2K, and STE20/PAK family members. We identified MARK1 in all screens and validated that DDX proteins accelerate the MARK1 catalytic rate. These findings indicate pervasive interactions between protein kinases and DEAD box RNA helicases, and provide a rich resource to explore their regulatory relationships.

Post-transcriptional cross- and auto-regulation buffer expression of the human RNA helicases DDX3X and DDX3Y.

The Y-linked gene DDX3Y and its X-linked homolog DDX3X survived the evolution of the human sex chromosomes from ordinary autosomes. DDX3X encodes a multi-functional RNA helicase, with mutations causing developmental disorders and cancers. We find that, among X-linked genes with surviving Y homologs, DDX3X is extraordinarily dosage-sensitive. Studying cells of individuals with sex chromosome aneuploidy, we observe that when the number of Y chromosomes increases, DDX3X transcript levels fall; conversely, when the number of X chromosomes increases, DDX3Y transcript levels fall. In 46,XY cells, CRISPRi knockdown of either DDX3X or DDX3Y causes transcript levels of the homologous gene to rise. In 46,XX cells, chemical inhibition of DDX3X protein activity elicits an increase in DDX3X transcript levels. Thus, perturbation of either DDX3X or DDX3Y expression is buffered - by negative cross-regulation of DDX3X and DDX3Y in 46,XY cells, and by negative auto-regulation of DDX3X in 46,XX cells. DDX3X-DDX3Y cross-regulation is mediated through mRNA destabilization - as shown by metabolic labeling of newly transcribed RNA - and buffers total levels of DDX3X and DDX3Y protein in human cells. We infer that post-transcriptional auto-regulation of the ancestral (autosomal) DDX3 gene transmuted into auto- and cross-regulation of DDX3X and DDX3Y as these sex-linked genes evolved from ordinary alleles of their autosomal precursor.

Substrate diversity of NSUN enzymes and links of 5-methylcytosine to mRNA translation and turnover.

Maps of the RNA modification 5-methylcytosine (m5C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m5C site calling pipeline. With the resulting union list of 6,393 m5C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m5C methyltransferases NSUN2 and NSUN6 as the main mRNA m5C "writers," but further showed that the rRNA:m5C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m5C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m5C writers and readers and their effect on mRNA function.

UPF1 plays critical roles in early B cell development

The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for VH-DHJH recombination, without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development.

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.

Werner helicase interacting protein 1 contributes to G-quadruplex processing in human cells

Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase.

The Role of the RNA Helicase DDX3X in Medulloblastoma Progression.

Medulloblastoma is the most common pediatric brain cancer, with about five cases per million in the pediatric population. Current treatment strategies have a 5-year survival rate of 70% or more but frequently lead to long-term neurocognitive defects, and recurrence is relatively high. Genomic sequencing of medulloblastoma patients has shown that DDX3X, which encodes an RNA helicase involved in the process of translation initiation, is among the most commonly mutated genes in medulloblastoma. The identified mutations are 42 single-point amino acid substitutions and are mostly not complete loss-of-function mutations. The pathological mechanism of DDX3X mutations in the causation of medulloblastoma is poorly understood, but several studies have examined their role in promoting cancer progression. This review first discusses the known roles of DDX3X and its yeast ortholog Ded1 in translation initiation, cellular stress responses, viral replication, innate immunity, inflammatory programmed cell death, Wnt signaling, and brain development. It then examines our current understanding of the oncogenic mechanism of the DDX3X mutations in medulloblastoma, including the effect of these DDX3X mutations on growth, biochemical functions, translation, and stress responses. Further research on DDX3X's mechanism and targets is required to therapeutically target DDX3X and/or its downstream effects in medulloblastoma progression.

RNA HELICASE 32 is essential for female gametophyte development in Arabidopsis

The normal progression of mitotic cycles and synchronized development within female reproductive organs are pivotal for sexual reproduction in plants. Nevertheless, our understanding of the genetic regulation governing mitotic cycles during the haploid phase of higher plants remains limited. In this study, we characterized RNA HELICASE 32 (RH32), which plays an essential role in female gametogenesis in Arabidopsis. The rh32 heterozygous mutant was semi-sterile, whereas the homozygous mutant was nonviable. The rh32 mutant allele could be transmitted through the male gametophyte, but not the female gametophyte. Phenotypic analysis revealed impaired mitotic progression, synchronization, and cell specification in rh32 female gametophytes, causing the arrest of embryo sacs. In the delayed pollination test, none of the retarded embryo sacs developed into functional female gametophytes, and the vast majority of rh32 female gametophytes were defective in the formation of the large central vacuole. RH32 is strongly expressed in the embryo sac. Knock-down of RH32 resulted in the accumulation of unprocessed 18 S pre-rRNA, implying that RH32 is involved in ribosome synthesis. Based on these findings, we propose that RH32 plays a role in ribosome synthesis, which is critical for multiple processes in female gametophyte development.

Dual genetic level modification engineering accelerate genome evolution of Corynebacterium glutamicum.

High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the 'dock' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02×104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.

RH20, a phase-separated RNA helicase protein, facilitates plant resistance to viruses

RNA silencing, a conserved gene regulatory mechanism, is critical for host resistance to viruses. Liquid-liquid phase separation (LLPS) is an important mechanism in regulating various biological processes. Emerging studies suggest RNA helicases play important roles in microRNA (miRNA) production through LLPS. In this study, we investigated the functional role of RNA helicase 20 (RH20), a DDX5 homolog in Arabidopsis thaliana, in RNA silencing and plant resistance to viruses. Our findings reveal that RH20 localizes in both the cytoplasm and nucleus, with puncta formation in the cytoplasm exhibiting liquid–liquid phase separation behavior. We demonstrate that RH20 plays positive roles in plant immunity against viruses. Further study showed that RH20 interacts with Argonaute 2 (AGO2), a key component of the RNA silencing pathway. Moreover, RH20 promotes the accumulation of both endogenous and exogenous small RNAs (sRNAs). Overall, our study identifies RH20 as a novel phase separation protein that interacting with AGO2, influencing sRNAs accumulation, and enhancing plant resistance to viruses.

Role of senataxin in R-loop-mediated neurodegeneration.

Senataxin is an RNA:DNA helicase that plays an important role in the resolution of RNA:DNA hybrids (R-loops) formed during transcription. R-loops are involved in the regulation of biological processes such as immunoglobulin class switching, gene expression and DNA repair. Excessive accumulation of R-loops results in DNA damage and loss of genomic integrity. Senataxin is critical for maintaining optimal levels of R-loops to prevent DNA damage and acts as a genome guardian. Within the nucleus, senataxin interacts with various RNA processing factors and DNA damage response and repair proteins. Senataxin interactors include survival motor neuron and zinc finger protein 1, with whom it co-localizes in sub-nuclear bodies. Despite its ubiquitous expression, mutations in senataxin specifically affect neurons and result in distinct neurodegenerative diseases such as amyotrophic lateral sclerosis type 4 and ataxia with oculomotor apraxia type 2, which are attributed to the gain-of-function and the loss-of-function mutations in senataxin, respectively. In addition, low levels of senataxin (loss-of-function) in spinal muscular atrophy result in the accumulation of R-loops causing DNA damage and motor neuron degeneration. Senataxin may play multiple functions in diverse cellular processes; however, its emerging role in R-loop resolution and maintenance of genomic integrity is gaining attention in the field of neurodegenerative diseases. In this review, we highlight the role of senataxin in R-loop resolution and its potential as a therapeutic target to treat neurodegenerative diseases.