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UAP56/DDX39B is a major cotranscriptional RNA-DNA helicase that unwinds harmful R loops genome-wide.

ABSTRACT: Nonscheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R-loop accumulation. One mechanism relies on the conserved THO complex in association with cotranscriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R-loop removal. We show that UAP56 depletion causes R-loop accumulation, R-loop-mediated genome instability, and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and show that its overexpression suppresses R loops and genome instability induced by depleting five different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful cotranscriptional RNA structures that otherwise would block transcription and replication.

SUBMITTER: Perez-Calero C

PROVIDER: S-EPMC7328515 | biostudies-literature |

REPOSITORIES: biostudies-literature

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UAP56/DDX39b is a major co-transcriptional RNA-DNA helicase that unwinds harmful R loops genome-wide

Project description:Non-scheduled R loops represent a major source of DNA damage and replication stress. Cells have different ways to prevent R loop accumulation. One mechanism relies on the conserved THO complex in association with co-transcriptional RNA processing factors including the RNA-dependent ATPase UAP56/DDX39B and histone modifiers such as the SIN3 deacetylase in humans. We investigated the function of UAP56/DDX39B in R loop removal. We show that UAP56 depletion causes R loop accumulation, R loop-mediated genome instability and replication fork stalling. We demonstrate an RNA-DNA helicase activity in UAP56 and that its overexpression suppresses R loops and genome instability induced by depleting 5 different unrelated factors. UAP56/DDX39B localizes to active chromatin and prevents the accumulation of RNA-DNA hybrids over the entire genome. We propose that, in addition to its RNA processing role, UAP56/DDX39B is a key helicase required to eliminate harmful co-transcriptional RNA structures that otherwise would block transcription and replication.

2020-05-25 | GSE127979 | GEO

UAP56/DDX39b is a major co-transcriptional RNA-DNA helicase that unwinds harmful R loops genome-wide

Project description:UAP56/DDX39b is a major co-transcriptional RNA-DNA helicase that unwinds harmful R loops genome-wide

| PRJNA525976 | ENA

Polymerase ?-helicase efficiently unwinds DNA and RNA-DNA hybrids.

Project description:POLQ is a unique multifunctional replication and repair gene that encodes for a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase ?-helicase (Pol?-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Pol?-helicase (residues 1-894) efficiently unwinds DNA with 3'-5' polarity, including DNA with 3' or 5' overhangs, blunt-ended DNA, and replication forks. Pol?-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Pol?-helicase can facilitate strand displacement synthesis by Pol?-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Pol? in replication repair.

| S-EPMC5892577 | biostudies-literature

The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate.

Project description:Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene (IL7R) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

| S-EPMC10234631 | biostudies-literature

A Helicase Unwinds Hexanucleotide Repeat RNA G-Quadruplexes and Facilitates Repeat-Associated Non-AUG Translation.

Project description:The expansion of a hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion leads to repeat-associated non-AUG (RAN) translation and the production of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN translation remain enigmatic. Here, we report that the RNA helicase DHX36 is a robust positive regulator of C9orf72 RAN translation. DHX36 has a high affinity for the G4C2 repeat RNA, preferentially binds to the repeat RNA's G-quadruplex conformation, and efficiently unwinds the G4C2 G-quadruplex structures. Native DHX36 interacts with the G4C2 repeat RNA and is essential for effective RAN translation in the cell. In induced pluripotent stem cells and differentiated motor neurons derived from C9orf72-linked ALS patients, reducing DHX36 significantly decreased the levels of endogenous DPR proteins. DHX36 is also aberrantly upregulated in tissues of C9orf72-linked ALS patients. These results indicate that DHX36 facilitates C9orf72 RAN translation by resolving repeat RNA G-quadruplex structures and may be a potential target for therapeutic intervention.

| S-EPMC8610145 | biostudies-literature

Human DHX9 helicase unwinds triple-helical DNA structures.

Project description:Naturally occurring poly(purine.pyrimidine) rich regions in the human genome are prone to adopting non-canonical DNA structures such as intramolecular triplexes (i.e., H-DNA). Such structure-forming sequences are abundant and can regulate the expression of several disease-linked genes. In addition, the use of triplex-forming oligonucleotides (TFOs) to modulate gene structure and function has potential as an approach to targeted gene therapy. Previously, we found that endogenous H-DNA structures can induce DNA double-strand breaks and promote genomic rearrangements. Herein, we find that the DHX9 helicase co-immunoprecipitates with triplex DNA structures in mammalian cells, suggesting a role in the maintenance of genome stability. We tested this postulate by assessing the helicase activity of purified human DHX9 on various duplex and triplex DNA substrates in vitro. DHX9 displaced the third strand from a specific triplex DNA structure and catalyzed the unwinding with a 3' --> 5' polarity with respect to the displaced third strand. Helicase activity required a 3'-single-stranded overhang on the third strand and was dependent on ATP hydrolysis. The reaction kinetics consisted of a pre-steady-state burst phase followed by a linear, steady-state pseudo-zero-order reaction. In contrast, very little if any helicase activity was detected on blunt triplexes, triplexes with 5'-overhangs, blunt duplexes, duplexes with overhangs, or forked duplex substrates. Thus, triplex structures containing a 3'-overhang represent preferred substrates for DHX9, where it removes the strand with Hoogsteen hydrogen-bonded bases. Our results suggest the involvement of DHX9 in maintaining genome integrity by unwinding mutagenic triplex DNA structures.

| S-EPMC2949957 | biostudies-literature

The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection.

Project description:The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.

| S-EPMC3165823 | biostudies-literature

Arabidopsis DEAD-box RNA helicase UAP56 interacts with both RNA and DNA as well as with mRNA export factors.

Project description:The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.

| S-EPMC3608606 | biostudies-literature

The Cellular DExD/H-Box RNA Helicase UAP56 Co-localizes With the Influenza A Virus NS1 Protein.

Project description:UAP56, a member of the DExD/H-box RNA helicase family, is essential for pre-mRNA splicing and mRNA export in eukaryotic cells. In influenza A virus-infected cells, UAP56 mediates viral mRNA nuclear export, facilitates viral ribonucleoprotein complex formation through direct interaction with the viral nucleoprotein, and may indirectly affect antiviral host responses by binding to and/or facilitating the activation of the antiviral host factors MxA and PKR. Here, we demonstrate that UAP56 also co-localizes with the influenza A viral NS1 protein, which counteracts host cell innate immune responses stimulated by virus infection. The UAP56-NS1 association relies on the RNA-binding residues R38 and K41 in NS1 and may be mediated by single-stranded RNA. UAP56 association with NS1 does not affect the NS1-mediated downregulation of cellular innate immune pathways in reporter gene assays, leaving in question the exact biological role and relevance of the UAP56-NS1 association.

| S-EPMC6144874 | biostudies-literature

Hexameric helicase G40P unwinds DNA in single base pair steps.

Project description:Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

| S-EPMC6370340 | biostudies-literature

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