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James R. Williamson - One of the best experts on this subject based on the ideXlab platform.
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A Survey of DDX21 Activity During Rev/RRE Complex Formation
Journal of Molecular Biology, 2018Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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a survey of DDX21 activity during rev rre complex formation
Journal of Molecular Biology, 2017Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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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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A DEAD-Box Helicase Mediates an RNA Structural Transition in the HIV-1 Rev Response Element.
Journal of Molecular Biology, 2017Co-Authors: John A. Hammond, Rajan Lamichhane, David P. Millar, James R. WilliamsonAbstract:Nuclear export of partially spliced or unspliced HIV-1 RNA transcripts requires binding of the viral protein regulator of expression of virion (Rev) to the Rev response element (RRE) and subsequent oligomerization in a cooperative manner. Cellular DEAD-box helicase DEAD-box protein 1 (DDX1) plays a role in HIV replication, interacting with and affecting Rev-containing HIV transcripts in vivo, interacting directly with the RRE and Rev in vitro, and promoting Rev oligomerization in vitro. Binding of DDX1 results in enhancement of Rev oligomerization on the RRE that is correlated with an RNA structural change within the RRE that persists even after dissociation of DDX1. Furthermore, this structural transition is likely located within the three-way junction of stem II of the RRE that is responsible for initial Rev binding. This discovery of the stem II structural transition leads to a model wherein DDX1 can act as an RNA chaperone, folding stem IIB into a proper Rev binding conformation.
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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.
David P. Millar - One of the best experts on this subject based on the ideXlab platform.
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A Survey of DDX21 Activity During Rev/RRE Complex Formation
Journal of Molecular Biology, 2018Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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a survey of DDX21 activity during rev rre complex formation
Journal of Molecular Biology, 2017Co-Authors: John A. Hammond, Li Zhou, Rajan Lamichhane, Hui-yi Chu, David P. Millar, Larry Gerace, James R. WilliamsonAbstract:HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.
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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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A DEAD-Box Helicase Mediates an RNA Structural Transition in the HIV-1 Rev Response Element.
Journal of Molecular Biology, 2017Co-Authors: John A. Hammond, Rajan Lamichhane, David P. Millar, James R. WilliamsonAbstract:Nuclear export of partially spliced or unspliced HIV-1 RNA transcripts requires binding of the viral protein regulator of expression of virion (Rev) to the Rev response element (RRE) and subsequent oligomerization in a cooperative manner. Cellular DEAD-box helicase DEAD-box protein 1 (DDX1) plays a role in HIV replication, interacting with and affecting Rev-containing HIV transcripts in vivo, interacting directly with the RRE and Rev in vitro, and promoting Rev oligomerization in vitro. Binding of DDX1 results in enhancement of Rev oligomerization on the RRE that is correlated with an RNA structural change within the RRE that persists even after dissociation of DDX1. Furthermore, this structural transition is likely located within the three-way junction of stem II of the RRE that is responsible for initial Rev binding. This discovery of the stem II structural transition leads to a model wherein DDX1 can act as an RNA chaperone, folding stem IIB into a proper Rev binding conformation.
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Role of Dead Box Helicases in HIV-1 Rev Function: a Single-Molecule Approach
Biophysical Journal, 2014Co-Authors: Rajan Lamichhane, David P. MillarAbstract:The HIV-1 Rev (Regulator of Expression of Virion) protein activates nuclear export of unspliced and partially spliced viral mRNAs, which encode the viral genome and the genes encoding viral structural proteins. Rev interacts with a highly conserved region, the Rev Response Element (RRE), located within the viral mRNA. Initially, a single Rev monomer binds to stem loop IIB of the RRE, whereupon additional Rev monomers are recruited to the RRE through a combination of RNA-protein and protein-protein interactions, resulting in the formation of a functional nuclear export complex. In addition, several cellular host proteins, such as the DEAD box helicases DDX1 and DDX21 are known to be required for efficient Rev function in vivo, although their precise role is unknown. In this study, a variety of single-molecule fluorescence spectroscopic methods were used to dissect the role of DDX1 and DDX21 during assembly of Rev-RRE complexes. To facilitate these studies, the large DDX1 and DDX21 proteins were enzymatically labeled with bright and photostable dyes, while Rev was labeled at a single cysteine by maleimide chemistry. Single-color TIRF measurements with labeled Rev were used to monitor individual Rev monomer binding steps during oligomeric Rev-RRE assembly. Moreover, single-color experiments with labeled DDX1 or DDX21 reveal that each helicase is capable of binding directly to the RRE. Two-color colocalization and FRET measurements were used to monitor the simultaneous binding of both Rev and DDX1 to the RRE, revealing the temporal correlation between individual protein binding steps. Together, these studies are revealing how DEAD box helicases are able to promote the oligomeric assembly of Rev on the RRE, thereby acting as cellular cofactors of HIV-1. Supported by NIH P50 grant GM082545.
Yong-jun Liu - One of the best experts on this subject based on the ideXlab platform.
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The E3 ubiquitin ligase TRIM21 negatively regulates the innate immune response to intracellular double-stranded DNA
Nature Immunology, 2013Co-Authors: Zhiqiang Zhang, Musheng Bao, Bin Yuan, Leiyun Weng, Yong-jun LiuAbstract:The intracellular sensor DDX41 is important for generating innate responses to DNA viruses. Liu et al . demonstrate that the ubiquitin ligase TRIM21 degrades and thereby regulates DDX41-dependent responses. DDX41 is a sensor of intracellular double-stranded DNA (dsDNA) in myeloid dendritic cells (mDCs) that triggers a type I interferon response via the signaling adaptor STING. We identified the E3 ligase TRIM21 as a DDX41-interacting protein and found that knockdown of or deficiency in TRIM21 resulted in enhanced type I interferon responses to intracellular dsDNA and DNA viruses. Overexpression of TRIM21 resulted in more degradation of DDX41 and less production of interferon-β (IFN-β) in response to intracellular dsDNA. The SPRY-PRY domain of TRIM21 interacted with the DEADc domain of DDX41. Lys9 and Lys115 of DDX41 were the targets of TRIM21-mediated ubiquitination. TRIM21 is therefore an interferon-inducible E3 ligase that induces the Lys48 (K48)-linked ubiquitination and degradation of DDX41 and negatively regulates the innate immune response to intracellular dsDNA.
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The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells
Nature Immunology, 2011Co-Authors: Zhiqiang Zhang, Taeil Kim, Musheng Bao, Bin Yuan, Yong-jun LiuAbstract:The recognition of pathogenic DNA is important to the initiation of antiviral responses. Here we report the identification of DDX41, a member of the DEXDc family of helicases, as an intracellular DNA sensor in myeloid dendritic cells (mDCs). Knockdown of DDX41 expression by short hairpin RNA blocked the ability of mDCs to mount type I interferon and cytokine responses to DNA and DNA viruses. Overexpression of both DDX41 and the membrane-associated adaptor STING together had a synergistic effect in promoting Ifnb promoter activity. DDX41 bound both DNA and STING and localized together with STING in the cytosol. Knockdown of DDX41 expression blocked activation of the mitogen-activated protein kinase TBK1 and the transcription factors NF-κB and IRF3 by B-form DNA. Our results suggest that DDX41 is an additional DNA sensor that depends on STING to sense pathogenic DNA. Sensors of cytosolic nucleic acid can detect the presence of viruses. Liu and colleagues identify the helicase DDX41 as a sensor of double-stranded DNA that initiates upregulation of type I interferon dependent on the adaptor STING in myeloid DCs.
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DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells.
Immunity, 2011Co-Authors: Zhiqiang Zhang, Taeil Kim, Musheng Bao, Valeria Facchinetti, Sung Yun Jung, Amir Ali Ghaffari, Jun Qin, Genhong Cheng, Yong-jun LiuAbstract:The innate immune system detects viral infection predominantly by sensing viral nucleic acids. We report the identification of a viral sensor, consisting of RNA helicases DDX1, DDX21, and DHX36, and the adaptor molecule TRIF, by isolation and sequencing of poly I:C-binding proteins in myeloid dendritic cells (mDCs). Knockdown of each helicase or TRIF by shRNA blocked the ability of mDCs to mount type I interferon (IFN) and cytokine responses to poly I:C, influenza A virus, and reovirus. Although DDX1 bound poly I:C via its Helicase A domain, DHX36 and DDX21 bound the TIR domain of TRIF via their HA2-DUF and PRK domains, respectively. This sensor was localized within the cytosol, independent of the endosomes. Thus, the DDX1-DDX21-DHX36 complex represents a dsRNA sensor that uses the TRIF pathway to activate type I IFN responses in the cytosol of mDCs.
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.
Sean A. Mckenna - One of the best experts on this subject based on the ideXlab platform.
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An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21.
RNA (New York N.Y.), 2019Co-Authors: Ewan K. S. Mcrae, Steven J. Dupas, Evan P. Booy, Ramanaguru S. Piragasam, Richard P. Fahlman, Sean A. MckennaAbstract:DDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we used label-free proteomic MS/MS to identify 26 proteins that are expressed at significantly different levels in cells expressing an rG4-binding deficient DDX21 (M4). MS data are available via ProteomeXchange with identifier PXD013501. From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5'-UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in MCF-7 cells, rendering them sensitive to TRAIL-mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by the potential to form rG4 in their 5'-UTRs.
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An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21
2019Co-Authors: Ewan K. S. Mcrae, Steven J. Dupas, Evan P. Booy, Ramanaguru S. Piragasam, Richard P. Fahlman, Sean A. MckennaAbstract:Abstract DDX21 is a newly discovered RNA G-quadruplex (rG4) binding protein with no known biological rG4 targets. In this study we identified 26 proteins that are expressed at significantly different levels in cells expressing wild type DDX21 relative to an rG4 binding deficient DDX21 (M4). From this list we validate MAGED2 as a protein that is regulated by DDX21 through rG4 in its 5’UTR. MAGED2 protein levels, but not mRNA levels, are reduced by half in cells expressing only DDX21 M4. MAGED2 has a repressive effect on TRAIL-R2 expression that is relieved under these conditions, resulting in elevated TRAIL-R2 mRNA and protein in cells expressing only DDX21 M4, and rendering previously resistant cells sensitive to TRAIL mediated apoptosis. Our work identifies the role of DDX21 in regulation at the translational level through biologically relevant rG4 and shows that MAGED2 protein levels are regulated, at least in part, by a rG4 forming potential in their 5’UTRs.
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Insights into the RNA quadruplex binding specificity of DDX21.
Biochimica et biophysica acta. General subjects, 2018Co-Authors: Ewan K. S. Mcrae, Steven J. Dupas, David E. Davidson, Sean A. MckennaAbstract:Abstract Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2′OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2′OH of loop nucleotides.
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Human DDX21 binds and unwinds RNA guanine quadruplexes.
Nucleic acids research, 2017Co-Authors: Ewan K. S. Mcrae, Evan P. Booy, Aniel Moya-torres, Peyman Ezzati, Jörg Stetefeld, Sean A. MckennaAbstract:Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.