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Roseline Godbout - One of the best experts on this subject based on the ideXlab platform.
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Cytoplasmic aggregation of DDX1 in developing embryos: Early embryonic lethality associated with Ddx1 knockout.
Developmental Biology, 2019Co-Authors: Matthew R. Hildebrandt, Yixiong Wang, Lubna Yasmin, Darryl D. Glubrecht, Roseline GodboutAbstract:Abstract Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of Ddx1 causes stalling of mouse embryos at the 2–4 cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2–4 cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2, Zar1, Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development.
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Role for RIF1-interacting partner DDX1 in BLM recruitment to DNA double-strand breaks.
DNA Repair, 2017Co-Authors: Ho-yin Poon, Devon R. Germain, Matthew R. Hildebrandt, Elizabeth A. Monckton, Richard P. Fahlman, Roseline GodboutAbstract:Human Rap1-interacting factor 1 (RIF1) is an important player in the repair of DNA double strand breaks (DSBs). RIF1 acts downstream of 53BP1, with well-documented roles in class switch recombination in B-cells and inhibition of end resection initiation in BRCA1-defective cells. Here, we report that DEAD Box 1 (DDX1), a RNA helicase also implicated in DSB repair, interacts with RIF1, with co-localization of DDX1 and RIF1 observed throughout interphase. Recruitment of DDX1 to DSBs is dependent on RIF1, with RIF1 depletion abolishing DDX1-mediated facilitation of homologous recombination at DSBs. As previously demonstrated for RIF1, DDX1 is also required for chromatin loading of Bloom syndrome helicase (BLM) to ionizing radiation-induced DSBs, a RIF1-related activity that is independent of 53BP1. We show that DDX1 and RIF1 have different nucleic acid requirements for accumulation at DSBs, with RNA-DNA hybrids required for DDX1 accrual at DSBs, and single-strand RNA required for accumulation of RIF1 at these sites. Our data suggest both convergent and divergent roles for DDX1 and RIF1 in DSB repair, and may help explain why RIF1 depletion does not fully mimic 53BP1 ablation in the restoration of homologous recombination defects in BRCA1-deficient cells.
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DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks
Molecular and Cellular Biology, 2016Co-Authors: Devon R. Germain, Matthew R. Hildebrandt, Elizabeth A. Monckton, Ho-yin Poon, Michael J. Hendzel, Darin Mcdonald, Roseline GodboutAbstract:Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR.
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Ddx1 knockout results in transgenerational wild-type lethality in mice
Scientific Reports, 2015Co-Authors: Matthew R. Hildebrandt, Devon R. Germain, Elizabeth A. Monckton, Miranda Brun, Roseline GodboutAbstract:DEAD box 1 (DDX1) is a member of the DEAD box family of RNA helicases which are involved in all aspects of RNA metabolism. DDX1 has been implicated in a variety of biological processes, including 3’-end processing of mRNA, DNA repair, microRNA processing, tRNA maturation and mRNA transport. To study the role of DDX1 during development, we have generated mice carrying a constitutive Ddx1 knock-out allele. Ddx1+/− mice have no obvious phenotype and express similar levels of DDX1 as wild-type mice indicating compensation from the intact Ddx1 allele. Heterozygote matings produce no viable Ddx1−/− progeny, with Ddx1−/− embryos dying prior to embryonic day (E) 3.5. Intriguingly, the number of wild-type progeny is significantly decreased in heterozygote crosses, with two different heterozygote populations identified based on parental genotype: (i) normal Ddx1+/− mice which generate the expected number of wild-type progeny and (ii) Ddx1*/− mice (with * signifying a non-genetically altered allele) which generate a significantly reduced number of wild-type mice. The transgenerational inheritance of wild-type lethality observed upon crossing Ddx1*/− mice is independent of parental sex and occurs in cis through a mechanism that is different from other types of previously reported transgenerational epigenetic inheritance.
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Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis.
Developmental Biology, 2015Co-Authors: Devon R. Germain, Matthew R. Hildebrandt, Andrew J. Simmonds, Sarah C. Hughes, Roseline GodboutAbstract:Mammalian DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. Here, we report phenotypes associated with a null Ddx1 (Ddx1(AX)) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. We demonstrate that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. Our data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing.
David P. Millar - One of the best experts on this subject based on the ideXlab platform.
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A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.
Nucleic Acids Research, 2017Co-Authors: Rajan Lamichhane, John A. Hammond, James R. Williamson, Edwin J. C. Van Der Schans, Raymond F Pauszek, Rae M Anderson, Ingemar Pedron, David P. MillarAbstract:The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.
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Single-Molecule Studies Reveal that DEAD-Box Protein DDX1 Promotes Oligomerization of HIV-1 Rev on the Rev Response Element
Journal of Molecular Biology, 2011Co-Authors: Rae M. Robertson-anderson, James R. Williamson, Stephen P. Edgcomb, Andrew B. Carmel, Jun Wang, David P. MillarAbstract:Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced human immunodeficiency virus type 1 viral mRNA transcripts. Several host factors, including the human DEAD box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev–RRE complexes in the presence or absence of DDX1 were observed in real time via single-molecule total internal reflection fluorescence microscopy. Binding of up to eight fluorescently labeled Rev monomers to a single RRE molecule was visualized, and the event frequencies and corresponding binding and dissociation rates for the different Rev–RRE stoichiometries were determined. The presence of DDX1 eliminated a second kinetic phase present during the initial Rev binding step, attributed to nonproductive nucleation events, resulting in increased occurrence of higher-order Rev–RRE stoichiometries. This effect was further enhanced upon the addition of a non-hydrolyzable ATP analog (adenylyl-imidophosphate), whereas ADP had no effect beyond that of DDX1 alone. Notably, the first three Rev monomer binding events were accelerated in the presence of DDX1 and adenylyl-imidophosphate, while the dissociation rates remained unchanged. Measurements performed across a range of DDX1 concentrations suggest that DDX1 targets Rev rather than the RRE to promote oligomeric assembly. Moreover, DDX1 is able to restore the oligomerization activity of a Rev mutant that is otherwise unable to assemble on the RRE beyond a monomeric complex. Taken together, these results suggest that DDX1 acts as a cellular cofactor by promoting oligomerization of Rev on the RRE.
Anton Meinhart - One of the best experts on this subject based on the ideXlab platform.
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rna helicase ddx1 converts rna g quadruplex structures into r loops to promote igh class switch recombination
Molecular Cell, 2018Co-Authors: Claudia Ribeiro De Almeida, Anton Meinhart, Somdutta Dhir, Ashish Dhir, Amin E Moghaddam, Quentin J Sattentau, Nick J ProudfootAbstract:Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.
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Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein.
Acta crystallographica. Section F Structural biology communications, 2015Co-Authors: Julian N. Kellner, Anton MeinhartAbstract:The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein-protein interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein-protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.
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Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1
Nucleic Acids Research, 2015Co-Authors: Julian N. Kellner, Jochen Reinstein, Anton MeinhartAbstract:RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1′s enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an ‘open’ to a ‘closed’-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.
James R. Williamson - One of the best experts on this subject based on the ideXlab platform.
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A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.
Nucleic Acids Research, 2017Co-Authors: Rajan Lamichhane, John A. Hammond, James R. Williamson, Edwin J. C. Van Der Schans, Raymond F Pauszek, Rae M Anderson, Ingemar Pedron, David P. MillarAbstract:The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.
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DDX1 Is an RNA-Dependent ATPase Involved in HIV-1 Rev Function and Virus Replication
Journal of Molecular Biology, 2012Co-Authors: Stephen P. Edgcomb, Larry Gerace, Andrew B. Carmel, Souad Naji, Geza Ambrus-aikelin, Jason R. Reyes, Andrew C. S. Saphire, James R. WilliamsonAbstract:The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1–Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.
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Single-Molecule Studies Reveal that DEAD-Box Protein DDX1 Promotes Oligomerization of HIV-1 Rev on the Rev Response Element
Journal of Molecular Biology, 2011Co-Authors: Rae M. Robertson-anderson, James R. Williamson, Stephen P. Edgcomb, Andrew B. Carmel, Jun Wang, David P. MillarAbstract:Oligomeric assembly of Rev on the Rev response element (RRE) is essential for the nuclear export of unspliced and singly spliced human immunodeficiency virus type 1 viral mRNA transcripts. Several host factors, including the human DEAD box protein DDX1, are also known to be required for efficient Rev function. In this study, spontaneous assembly and dissociation of individual Rev–RRE complexes in the presence or absence of DDX1 were observed in real time via single-molecule total internal reflection fluorescence microscopy. Binding of up to eight fluorescently labeled Rev monomers to a single RRE molecule was visualized, and the event frequencies and corresponding binding and dissociation rates for the different Rev–RRE stoichiometries were determined. The presence of DDX1 eliminated a second kinetic phase present during the initial Rev binding step, attributed to nonproductive nucleation events, resulting in increased occurrence of higher-order Rev–RRE stoichiometries. This effect was further enhanced upon the addition of a non-hydrolyzable ATP analog (adenylyl-imidophosphate), whereas ADP had no effect beyond that of DDX1 alone. Notably, the first three Rev monomer binding events were accelerated in the presence of DDX1 and adenylyl-imidophosphate, while the dissociation rates remained unchanged. Measurements performed across a range of DDX1 concentrations suggest that DDX1 targets Rev rather than the RRE to promote oligomeric assembly. Moreover, DDX1 is able to restore the oligomerization activity of a Rev mutant that is otherwise unable to assemble on the RRE beyond a monomeric complex. Taken together, these results suggest that DDX1 acts as a cellular cofactor by promoting oligomerization of Rev on the RRE.
Xinna Zhang - One of the best experts on this subject based on the ideXlab platform.
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Abstract A32: The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression
Regulation of Noncoding RNAs in Cancer, 2016Co-Authors: Cecil Han, Xinna ZhangAbstract:MicroRNAs (miRNAs) are small endogenous non-coding RNAs that regulate gene expression by repressing translation and/or promoting degradation of their target mRNAs. Aberrant dysregulation of mature miRNAs is commonly observed in many human cancers and can have a causative function in tumorigenesis. In the nucleus, primary miRNAs (pri-miRNAs) are first processed by the microprocessor containing RNaseIII enzyme Drosha and its cofactor DGCR8. Although the efficiency of Drosha-mediated process is crucial for determining miRNA abundance, the mechanisms for dynamic and specific regulation of miRNA biogenesis in the Drosha microprocessor are poorly understood. A better understanding of miRNA regulation is required to determine how aberrant miRNA activity mechanistically contributes to tumorigenesis and to predict therapeutic target miRNAs. Increasing evidence has shown that regulatory RNA-binding proteins may confer the specificity on the core components (Drosha and DGCR8) in the Microprocessor for recruiting and processing premature pri-miRNAs. Here, we identified a RNA-binding protein, DEAD-box RNA helicase 1 (DDX1) as a key regulatory component in the Drosha complex, which promotes the processing of a subset of miRNAs including miR-200 family (miR-200a, -200b, -200c, -141, -429). A suppressive role of miR-200s in cancer metastasis has been well known in several cancer types such as ovarian, kidney renal and lung cancer. Inhibiting DDX1 promotes tumor burden in the previous syngeneic ovarian mouse models. Consistent with, low level of DDX1 is associated with poor clinical outcome in patients with serous ovarian cancer and kidney renal clear cell carcinoma. Interestingly, DDX1-dependent miR-200a and miR-200b were also induced after DNA damage and this induction is facilitated by the ATM-mediated phosphorylation of DDX1. These findings suggest that DDX1 may be a key modulator in miRNA maturation in response to cellular signaling pathway and that alteration of level/function of DDX1 may play an important role in tumor progression through dysregulated microRNA pathway. Citation Format: Cecil Han, Xiongbin Lu, Xinna Zhang. The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A32.
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The RNA-binding protein DDX1 promotes primary microRNA maturation and inhibits ovarian tumor progression.
Cell Reports, 2014Co-Authors: Cecil Han, Yunhua Liu, Guohui Wan, Hyun Jin Choi, Luqing Zhao, Cristina Ivan, Anil K. Sood, Xinna ZhangAbstract:Posttranscriptional maturation is a critical step in microRNA (miRNA) biogenesis that determines mature miRNA levels. In addition to core components (Drosha and DGCR8 [DiGeorge syndrome critical region gene 8]) in the microprocessor, regulatory RNA-binding proteins may confer the specificity for recruiting and processing of individual primary miRNAs (pri-miRNAs). Here, we identify DDX1 as a regulatory protein that promotes the expression of a subset of miRNAs, including five members in the microRNA-200 (miR-200) family and four miRNAs in an eight-miRNA signature of a mesenchymal ovarian cancer subtype. A majority of DDX1-dependent miRNAs are induced after DNA damage. This induction is facilitated by the ataxia telangiectasia mutated (ATM)-mediated phosphorylation of DDX1. Inhibiting DDX1 promotes ovarian tumor growth and metastasis in a syngeneic mouse model. Analysis of The Cancer Genome Atlas (TCGA) reveals that low DDX1 levels are associated with poor clinical outcome in patients with serous ovarian cancer. These findings suggest that DDX1 is a key modulator in miRNA maturation and ovarian tumor suppression.