DEAD-box RNA helicase

Top PDF DEAD-box RNA helicase:

Subsite-specific association of DEAD box RNA helicase DDX60 with the development and prognosis of oral squamous cell carcinoma

Subsite-specific association of DEAD box RNA helicase DDX60 with the development and prognosis of oral squamous cell carcinoma

DEAD-box (DDX) proteins, distinguished by the presence of a conserved amino-acid sequence Asp- Glu-Ala-Asp motif, are the largest family of RNA helicases, with 37 members in humans [1]. DDX proteins interact with RNAs including rRNAs and mRNAs to perform many normal cellular functions, such as translation initiation, mRNA synthesis, RNA splicing and modification, ribosome and spliceosome assembly, and the transcriptional regulation of the genes involved in DNA repair and proliferation, cell cycle arrest, and apoptosis, highlighting the potentially involvement of DDX proteins in cancer [2-10]. DDX60, a novel DEAD box RNA helicase, is induced after a virus infection. The helicase domain of DDX60 binds to viral RNA and DNA, and its ATP-binding site is essential for DDX60-activated RIG, leading to type I interferon (IFN) expression [11- 13]. DDX60 also induces RIG-I-independent viral RNA degradation [11, 13]. DDX60 and its highly similar homolog DEAD box polypeptide 60-like (DDX60L) have recently been described as interferon-stimulated products upon a viral infection. However, DDX60L plays a distinct and specific function in restricting hepatitis C virus replication [14]. Although DDX60 is involved in protection against viral infections, the clinical significance and biological function of DDX60 in cancers, particularly oral cancer, remain largely unknown.
Show more

12 Read more

The DEAD box RNA helicase Ddx39ab is essential for myocyte and lens development in zebrafish

The DEAD box RNA helicase Ddx39ab is essential for myocyte and lens development in zebrafish

Members of the DEAD-box RNA helicase family have been shown to be involved in nearly all aspects of RNA metabolism, from transcription to mRNA decay. Recent studies have started to delineate different functions of DEAD-box RNA helicases in a broader context, including animal development. For example, ddx46 is expressed in the digestive organs and brain and has been shown to be required for the development of these organs (Hozumi et al., 2012). Researchers have also reported that ddx18 is essential for hematopoiesis (Payne et al., 2011). The generation of the ddx39ab gene-trapping allele provided the opportunity to study the function of this gene in vertebrate development. Here we demonstrated that ddx39ab is required for normal gene expression and differentiation of cardiomyocyte, myocyte and lens fiber cells. In previous studies, the development and function of the heart was shown to be sensitive to defects in RNA metabolism (Ding et al., 2004; Xu et al., 2005). Our observations corroborate these results and suggest that myocyte and lens fiber differentiation similarly bear this cell type-specific susceptibility. One possible explanation for this observation is that these three cell types exploit a similar, KMT2 family-dependent mechanism to establish epigenetic status during differentiation. An alternative hypothesis is that, compared with other cell types (such as neurons), a larger fraction of splicing undergoes Ddx39ab-related regulation in these three types of cell. FACS followed by RIP-seq might identify cell type-specific
Show more

12 Read more

The DEAD box RNA helicase Vad1 regulates multiple virulence associated genes in Cryptococcus neoformans

The DEAD box RNA helicase Vad1 regulates multiple virulence associated genes in Cryptococcus neoformans

The study of fungal regulatory networks is essential to the understanding of how these pathogens respond to host environmental signals with effective virulence-associated traits. In this study, a virulence-associated DEAD-box RNA helicase–encoding gene (VAD1) was isolated from a mutant defective in the virulence fac- tor laccase. A Δvad1 mutant exhibited a profound reduction in virulence in a mouse model that was restored after reconstitution with WT VAD1. Loss of VAD1 resulted in upregulation of NOT1, a gene encoding a global repressor of transcription. NOT1 was found to act as an intermediary transcriptional repressor of laccase. Vad1 was located within macromolecular complexes that formed cytoplasmic granular bodies in mature cells and during infection of mouse brain. In addition, VAD1 was shown by in situ hybridization to be expressed in the brain of an AIDS patient coinfected with C. neoformans. To understand the role of VAD1 in virulence, a functional genomics approach was used to identify 3 additional virulence determinants dependent on VAD1: PCK1, TUF1, and MPF3, involved in gluconeogenesis, mitochondrial protein synthesis, and cell wall integrity, respectively. These data show that fungal virulence-associated genes are coordinately regulated and that an analysis of such transcriptomes allows for the identification of important new genes involved in the normal growth and virulence of fungal pathogens.
Show more

11 Read more

Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster

Regulation of Notch Signaling by an Evolutionary Conserved DEAD Box RNA Helicase, Maheshvara in Drosophila melanogaster

The putative Mahe protein contains a highly conserved signature motif DEAD, which is the hallmark of the DEAD box RNA helicase family. Swiss model 2.0 was used for prediction of different motifs across two domains (based on crystal structure of PRP5, which shows the highest alignment score with Mahe). Twelve highly conserved motifs constitute the two domains D1 and D2, which cooperate to perform the unwinding of target nucleic acids. D1 includes motifs Q, I, Ia, Ib, Ic, II (DEAD), and III, whereas motifs IV, IVa, V, Va, and VI make up the D2 domain (Figure 1B). Amino acid sequence com- parison of DDX5 (human), DBP2 (yeast) and Mahe (fly) revealed a significant degree of similarity, indicating that all the mem- bers might have a conserved role to play in gene regulation. A phylogenetic tree was generated to show the homology of Mahe within the species (Figure 1D). On the basis of the conserved domain, Mahe was found to be evolutionarily close to DDX5 (human, mouse, and zebra fish homologs), worm DDX17, yeast DBP2, and fly Rm62, thus revealing that it might have an important role to play across taxa.
Show more

21 Read more

Centroid, a novel putative DEAD box RNA helicase maternal mRNA, is localized in the mitochondrial cloud in Xenopus laevis oocytes

Centroid, a novel putative DEAD box RNA helicase maternal mRNA, is localized in the mitochondrial cloud in Xenopus laevis oocytes

ATP-dependent enzymes involved in many aspects of RNA metabolism such as transcription, RNA splicing, ribosome bio- genesis, translation initiation and RNA transport and degradation and are found in all eukaryotes and most prokaryotes (Cordin et al., 2006; Heung and Del Poeta, 2005). Members of this family share conserved motifs that play a role in ATP binding and hydrolysis, RNA binding and RNA-induced conformational changes. Comparison of the amino acid composition of DEAD- box RNA helicase consensus motifs and centroid showed that centroid contains motifs Q, I (Walker A), II (Walker B), which are involved in ATP binding and hydrolysis, motifs Ia, Ib involved in RNA binding and motif III which is involved in RNA-induced conformational changes (Cordin et al., 2006; Heung and Del Poeta, 2005). The presence in the centroid of the motifs Q and I- III suggests that this protein is involved in the ssRNA binding, ATP hydrolysis and it may possess helicase activity. Interestingly, centroid lacks the motif IV, V and VI, which are present in DEAD- box RNA helicase consensus sequence. So far the function of motif IV is poorly understood but it was suggested that it may be involved in ssRNA binding and that has a functional connection to motif V involved in ATP hydrolysis (Cordin et al., 2006). Motif VI has been shown to participate in RNA binding and ATPase activity (Cordin et al., 2006). The DEAD-box RNA helicases are the multifunctional molecules and their activities depend on the communication and interaction between multifunctional motifs. Only future functional studies will be able to show how the lack of motif IV-VI influences the centroid function in comparison with other known DEAD-box helicases. Some of the members of this protein family, such as the DEAD-box RNA helicase p54 (Ladomery et al., 1997; Weston and Sommerville, 2006), are components of the messenger ribonucleoprotein (mRNP) particles stored in the germ plasm in oocytes of Xenopus, Drosophila and Caenorhabditis species and are believed to play a role in translational activation of stored mRNPs and sorting of mRNPs into the germ plasm (Bilinski et al., 2004; Cordin et al., 2006; Weston and Sommerville, 2006).
Show more

7 Read more

The Sole DEAD Box RNA Helicase of the Gastric Pathogen Helicobacter pylori Is Essential for Colonization

The Sole DEAD Box RNA Helicase of the Gastric Pathogen Helicobacter pylori Is Essential for Colonization

Phylogeny of DEAD-box helicases in Epsilonproteobacteria. We previously dis- covered the existence of a minimal RNA degradosome in H. pylori composed of the essential RNase J ribonuclease and of a newly identified DEAD-box RNA helicase that we designated RhpA (4). H. pylori is one of the most prominent species of the Epsilonproteobacteria class, which contains other important pathogens such as Campy- lobacter jejuni. Interestingly, orthologues of RNase J are found in all Epsilonproteobac- teria species, while no RNase E/G orthologues are detected (Fig. 1). The tree obtained with RNase J (see Fig. S1 in the supplemental material) suggests that the rnj genes were vertically transmitted from the last common ancestor of Epsilonproteobacteria species. We thus decided to investigate for the first time the distribution and phylogeny of the DEAD-box helicases among the Epsilonproteobacteria. In contrast to the other organ- isms analyzed to date, the data we obtained revealed the presence of a sole DEAD-box helicase that is conserved in every sequenced H. pylori strain as well as in each non-pylori Helicobacter species (Fig. 1). More surprisingly, we observed that all of the Campylobacter species lack a DEAD-box helicase with the exception of C. fetus, which contains one such helicase that is phylogenetically distant from that of Helicobacter species and presents characteristics of horizontal gene transfer (Fig. S2). Among the Epsilonproteobacteria, we found a correlation between the absence of helicase and a lower GC content of 23S and 16S rRNAs (Fig. 1). As expected, more-distant free-living Epsilonproteobacteria with larger genomes, like Sulfurospirillum, possess multiple heli- cases, except for the thermophilic species Nitratiruptor sp. and Nautilia profundicola (Fig. 1). Finally, with the exception of the majority of the Campylobacter species, every Epsilonproteobacteria species, including the Helicobacter species, possesses a similar helicase that is distantly related to the CsdA cold shock helicase family, suggesting the presence of such a helicase in their common ancestor (Fig. S2). Thus, H. pylori is an excellent model organism to study the role of a sole DEAD-box helicase.
Show more

17 Read more

DDX3 DEAD-Box RNA Helicase Inhibits Hepatitis B Virus Reverse Transcription by Incorporation into Nucleocapsids

DDX3 DEAD-Box RNA Helicase Inhibits Hepatitis B Virus Reverse Transcription by Incorporation into Nucleocapsids

Experimental strategy. To search for host factors that bind to HBV Pol, we utilized affinity purification of HBV Pol fol- lowed by mass spectrometry to identify copurified host factors. A cell line that stably expresses HBV Pol was generated, as detailed in Materials and Methods, using an episomal Epstein- Barr virus vector, which has been employed previously to ex- press a modest level of a bait protein (26). For affinity purifi- cation, HBV Pol was N-terminally tagged with three copies of the Flag epitope (Flag-tagged HBV Pol is hereinafter referred to as Flag-HBV Pol or HBV Pol). The N-terminal Flag tag did not interfere with HBV Pol function because it complemented the HBV Pol null mutation and induced viral genome replica- tion to a level comparable to that occurring with untagged HBV Pol (data not shown). Flag-specific immunoaffinity iso- lation from the HBV Pol-expressing HEK293 cells was per- formed, and copurified HBV Pol-associated proteins were an- alyzed by SDS-PAGE and detected by silver staining. More than a dozen cellular proteins were copurified with HBV Pol but not isolated from parental HEK293 cells (Fig. 1). Protein bands that were specifically copurified by HBV Pol isolation were excised and identified by LC-MS-MS mass spectrometry. The p90 band was identified as HBV Pol. In addition, heat shock protein 90 (Hsp90), heat shock constitutive protein 70 (Hsc70), and Hsp40 were identified as HBV Pol binding pro- teins. These chaperones are well-characterized HBV Pol bind- ing proteins, indicating that our pull-down analysis isolated cellular HBV Pol binding proteins effectively (6, 16). Some of the HBV Pol binding factors, such as the DDX3 DEAD-box RNA helicase (Fig. 1), appeared to be novel. Since DDX3 is important in HIV and HCV infections, we focused on deter- mining the effect of DDX3 on HBV replication. While this report focuses on DDX3, several other cellular proteins were
Show more

10 Read more

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development

Planarian flatworms have an impressive regenerative power. Although their embryonic de- velopment is still poorly studied and is highly derived it still displays some simple character- istics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zy- gote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.
Show more

10 Read more

Conserved requirement for DEAD box RNA helicase Gemin3 in Drosophila oogenesis

Conserved requirement for DEAD box RNA helicase Gemin3 in Drosophila oogenesis

The DEAD-box RNA helicase Gemin3 (also referred to as DP130 or DDX20) is a core member of the SMN (survival of motor neurons) complex [1]. The latter has been linked to the neuromuscular degenerative disease SMA (spinal muscular atrophy) through decreased levels of the epon- ymous SMN protein [2]. Gemin3 exhibits considerable sequence similarity to other DEAD-box family members within the nine motifs that form its ‘helicase’ core. Its flanking C-terminal domain is however highly divergent and is thus thought to provide specificity of function through specific substrate interactions. DEAD-box RNA helicases or unwindases are capable of rearranging inter- or intra-molecular RNA (ribonucleic acid) structures as well as dissociating or associating RNA-protein complexes [3]. In vitro studies revealed that in the case of Gemin3, the RNA helicase activity has a 5’ to 3’ directionality and is
Show more

9 Read more

DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level

DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level

DDX3 downregulates HBV RNA via transcriptional regula- tion. DDX3 has been implicated in the transcriptional regulation of several promoters (14–16). Thus, it is possible that DDX3 in- hibits viral RNA synthesis by suppressing transcription. To ad- dress this issue, an HBV promoter-driven reporter assay was per- formed as previously described (25). Each HBV reporter construct was transfected into HepG2 cells with an increasing amount of the HA-DDX3 construct, and then luciferase activities were measured (Fig. 4A). Results revealed that overexpression of DDX3 inhibited core promoter activity in a dose-dependent manner (Fig. 4B). Similarly, both S1 and S2 promoter activities were inhibited by DDX3 expression (Fig. 4C). In contrast, the inhibition was not observed when luciferase activities from cells transfected with ei- ther pRL-TK or pRL-CMV, where Renilla luciferase expression is driven from either the HSV-1 TK promoter or the CMV pro- moter, respectively, were measured (data not shown). Because transcriptional repression is a nuclear event, we examined the sub- cellular localization of DDX3. Consistent with previous reports FIG 3 ATPase activity is required for inhibition of HBV RNA synthesis by DDX3. (A) A schematic representation of WT DDX3 (DDX3-WT) and its mutants (Mut1 and Mut2). Amino acids both in the ATPase motif and the helicase motif are denoted, along with the DEAD box. Asterisks indicate the replaced amino acid in the ATPase motif of DDX3-Mut1 (Lys to Glu) and in the helicase motif of DDX3-Mut2 (Ser to Leu). All constructs have a HA tag at the N terminus. (B) HepG2 cells were cotransfected with an HBV 1.3-mer replicon (1 ␮ g) and increasing amounts (2 ␮ g or 6 ␮ g) of DDX3-WT or the DDX3 mutants in duplicate. At 48 h posttransfection, cells were harvested and viral RNAs were analyzed by Northern blotting. The average intensity of viral RNA bands obtained from cells transfected with the HBV 1.3-mer replicon (lanes 1 and 2) was set to 100%. Ribosomal RNAs were detected to confirm that equivalent amounts of RNAs were loaded. DDX3 expression was mea- sured by Western blotting using anti-HA antibody.
Show more

10 Read more

A HIV 1 Tat mutant protein disrupts HIV 1 Rev function by targeting the DEAD box RNA helicase DDX1

A HIV 1 Tat mutant protein disrupts HIV 1 Rev function by targeting the DEAD box RNA helicase DDX1

role for Tat in supporting Rev function. To test this hy- pothesis, we used a recombinant infectious HIV-1 pro- viral construct, pGCH, in which the cytomegalovirus (CMV) immediate-early promoter replaces the HIV-1 5′ U3 region but retained the natural HIV-1 transcriptional start site in the R region [35]. The pGCH plasmid ex- presses all accessory and regulatory genes including Tat and Rev. Importantly, RNA polymerase II-directed tran- scription from the CMV promoter in this construct is only minimally affected by Tat and Nullbasic, thereby allowing the segregation of Tat’s role in transactivation from other potential biological roles in the virus life cycle [35]. As shown in Figure 8, HEK293T cells cotrans- fected with pGCH and a plasmid encoding Nullbasic- FLAG had significantly reduced (p = 0.02) amounts of unspliced viral mRNA relative to total viral mRNA when compared to cells transfected with pGCH alone (Figure 8). Significantly, coexpression of wild type Tat with Nullbasic restored the steady state levels of unspliced
Show more

17 Read more

Involvement of a Toxoplasma DEAD-box RNA Helicase in Post-transcriptional Gene Regulation

Involvement of a Toxoplasma DEAD-box RNA Helicase in Post-transcriptional Gene Regulation

DEAD-box RNA helicases were first described by Gorbalenya et al.(1989), when a group of NTPases were defined based on sequence similarity to the eukaryotic initiation factor 4a (eIF4a) to contain several common sequence elements (Fig.1.4) [57]. Since then, DEAD-box RNA helicases have been classified as the largest helicase family of SF2. The mode of RNA binding in DEAD-box RNA helicases is highly conserved throughout the family [58]. The two central core domains make a contact to its RNA substrate in a non-sequence-specific manner spanning over any consecutive five nucleotides [45]. The interaction involves exclusively the sugar backbone of the RNA. Bound to the enzyme, RNA is bent in a conformation which differs from those seen in most SF2 helicases [45]. In this single stranded RNA (ssRNA) conformation, the RNA undergoes a sharp bend due to ionic and hydrogen bond interactions with several motifs within domain 1 (Fig. 1.4) [59, 60]. The resulting ssRNA structure inhibits the formation of a double stranded helix, giving rise to RNA unwinding [60]. This local strand separation is unique to DEAD-box RNA helicases, as it abolishes the need for translocation in order for strand separation to occur [45].
Show more

121 Read more

DDX3 DEAD-Box RNA Helicase Is Required for Hepatitis C Virus RNA Replication

DDX3 DEAD-Box RNA Helicase Is Required for Hepatitis C Virus RNA Replication

Since helicases are motor enzymes that use energy derived from nucleoside triphosphate hydrolysis to unwind double- stranded nucleic acids, the DDX3–core complex might unwind the HCV double-stranded RNA and separate the RNA strands or might contribute to the function of HCV NS3 helicase. Since the replication of subgenomic replicon RNA was also partially suppressed in DDX3 knockdown cells (Fig. 1D), DDX3 might be associated with an HCV nonstructural pro- tein(s) or HCV RNA itself. Indeed, Tingting et al. recently reported that DDX1 bound to both the HCV 3 ⬘ untranslated region (3 ⬘ UTR) and the HCV 5 ⬘ UTR and that short inter- fering RNA-mediated knockdown of DDX1 caused a marked reduction in the replication of subgenomic replicon RNA (22). Furthermore, Goh et al. demonstrated that DDX5/p68 associ- ated with HCV NS5B and that depletion of endogenous DDX5 correlated with a reduction in the transcription of neg- ative-strand HCV RNA (11). However, we failed to observe an interaction between DDX3 and NS5A or NS5B by immuno- precipitation under our experimental conditions in which the core could interact with DDX3 (Fig. 2C). Importantly, our DDX3 knockdown study demonstrated a more significant re- duction in the accumulation of genome-length HCV RNA (95% reduction) than in the accumulation of subgenomic rep- licon RNA (52% reduction) (Fig. 1B and D). To date, it has been demonstrated that the 5 ⬘ UTR, the 3 ⬘ UTR, and the NS3-to-NS5B coding region are sufficient for HCV RNA rep- lication (16); however, the core might be partly involved in the replication of genome-length HCV RNA. Importantly, DDX1 and DDX3 were specifically detected in the lipid droplets of core-expressing Hep39 cells by proteomic analysis (21), sug- gesting that DDX3 might be associated with HCV assembly or might incorporate into the HCV virion through interaction with the core to act as an RNA chaperone.
Show more

5 Read more

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Different levels of interference have been suggested for DDX3 in the Wnt-signaling pathway. In contrast with our findings, Cruciat, et al. found that DDX3 inhibition had no effect on Wnt signaling activity after induction with β-catenin overexpression and that the involvement of DDX3 in this pathway was independent of its helicase activity. [5] It is possible that other mechanisms by which DDX3 is involved in Wnt signaling, like stabilization of β-catenin indirectly through Rac1-signaling [6] or DDX5, or through a direct interaction with DDX3 [4] are more prominent in colorectal cancers. Unfortunately, only a minority of colorectal cancers (23%) falls into the wild-type APC group. However, mutations in CTNNB1 are highly prevalent in hepatocellular carcinoma (24%), sarcoma (44%) and testicular cancer(24%) [29], suggesting that these cancers may potentially have increased sensitivity to RK-33. In contrast to Sun et al. who found DDX3 to be pro-apoptotic in a p53- wildtype breast cancer cell line and anti-apoptotic in cell lines harboring a p53-mutation [30], we found DDX3 dependency not to differ in the presence or absence of p53.
Show more

15 Read more

The DEAD box RNA helicase Vasa functions in embryonic mitotic progression in the sea urchin

The DEAD box RNA helicase Vasa functions in embryonic mitotic progression in the sea urchin

Vasa was among the first germline genes to be identified (Lasko and Ashburner, 1988). Its DEAD-box motif, its physical interaction with eIF5B and the observation that several proteins do not accumulate in Vasa-mutant flies suggests that it functions in translational regulation (Linder, 2003; Johnstone and Lasko, 2001; Carrera et al., 2000). Curiously, Vasa appears to be inactivated by phosphorylation in response to activation of a meiotic checkpoint during Drosophila oogenesis (Ghabrial and Schüpbach, 1999), implying an involvement of Vasa in meiotic cell cycle progression. Furthermore, recent work suggests that Vasa functions in mitotic activity in Drosophila germline stem cells (Pek and Kai, 2011). Although target mRNAs reported for Vasa function so far are limited to the germ line of a few animals, Vasa orthologs are present throughout the animal kingdom. In addition to its role in the germ line, it has been reported to function in multipotent and somatic stem cell lineages in early embryonic cells, and even to function in tumor progression (e.g. Raz, 2000; Linder and Lasko, 2006; Rosner et al., 2009; Pfister et al., 2008; Shibata et al., 1999; Oyama and Shimizu, 2007; Rebscher et al., 2007; Juliano and Wessel, 2010; Janic et al., 2010). Its function outside of the germ line, though, has not been resolved.
Show more

6 Read more

An Essential Role for the Saccharomyces cerevisiae DEAD-Box Helicase DHH1 in G1/S DNA-Damage Checkpoint Recovery

An Essential Role for the Saccharomyces cerevisiae DEAD-Box Helicase DHH1 in G1/S DNA-Damage Checkpoint Recovery

The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses. While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized. Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint. DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest. Dhh1p has been suggested to play a role in partitioning mRNAs between translat- able and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage. Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease.
Show more

14 Read more

The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding.

The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding.

clude the Gly, Gln, and Lys-Gln positions flanking the GxGKT box, the Glu and Asp moieties just downstream of the DExH box, and various constituents of the QRxGRxGRxxxG motif (see Fig. 1). The non-NPH-II-like group includes three vac- cinia virus-encoded DNA-dependent ATPases (2, 3, 18), the bacterial RecG and RecQ DNA helicases (15, 28), the yeast RAD3 DNA helicase (27), the potyvirus C1 RNA helicase (12), and the bovine viral diarrhea virus NS3 RNA helicase (29). The DExH family members are more closely related to each other within these four protein segments than they are to the eponymous DEAD box RNA helicase eIF-4A (see Fig. 1). Membership in the DExH or DEAD box family does not guarantee an associated helicase activity. Furthermore, even among the family members that are helicases, motif conserva- tion has no clear predictive value regarding the key properties of the helicase, e.g., directionality of unwinding, specificity for DNA versus RNA, etc. Insights into the function of the con- served motifs have come primarily from mutational analyses of representative family members. Among the RNA helicases, eIF-4A, the prototypal DEAD box protein, has been mu- tagenized extensively (19–22). Sonenberg et al. have shown clearly that mutations within the GKT box, the DEAD box, and the HRxGRxxR motif can impair RNA unwinding by eIF-4A by inhibiting ATP binding, ATP hydrolysis, and/or RNA binding (19, 20).
Show more

8 Read more

Characterization of Selected Transposon-mediated Mutants of Listeria monocytogenes Regarding Survival and Growth on Cantaloupe.

Characterization of Selected Transposon-mediated Mutants of Listeria monocytogenes Regarding Survival and Growth on Cantaloupe.

DEAD-box RNA helicases are highly conserved with 12 characteristic sequence motifs present in all eukaryotic cells and many bacteria (Markkula et al., 2012, Vakulskas et al., 2014). DEAD-box proteins get their name from the shared amino acids, Aspartic acid (D)-Glutamic acid (E)-Alanine (A)-Aspartic acid (D), abbreviated DEAD for the single letter code of each amino acid. These enzymes unwind secondary RNA structures and are believed to be important during the growth of L. monocytogenes at low temperatures (Markkula et al., 2012 a, b, Netterling et al., 2012). In the L. monocytogenes EGD-e genome, there are four predicted DEAD-box RNA helicase genes (lmo0866, lmo1246, lmo1450, lmo1722) (Glaser et al., 2001, Markkula et al., 2012 a, b, Netterling et al., 2012). All four of the DEAD-box protein-encoding genes have a role in cold-temperature growth and deletion of three of the DEAD-box RNA helicase genes resulted in impaired growth at temperatures below 10°C (Azizoglu et al., 2010, Markkula et al., 2012 a,b, Netterling et al., 2012). The minimum growth temperatures were higher for three of the mutants (Δlmo0866, Δlmo1450, Δlmo1722) compared to the wild-type strain; in addition, motility of the DEAD-box RNA helicase mutants was impaired, indicating that those genes play a significant role both in cold
Show more

114 Read more

Unexpected roles for DEAD box protein 3 in viral RNA sensing pathways

Unexpected roles for DEAD box protein 3 in viral RNA sensing pathways

Detection of viral nucleic acid within infected cells is essential to an effective anti- viral response. The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLR) form part of the virus detection repertoire and are critically important in sensing viral RNA in the cytoplasm. Efforts continue to define the signalling components downstream of RLR that are required to induce type I IFN (IFN-α and IFN-β ) after viral infection. One surprising finding was that the DEAD (Asp-Glu-Ala- Asp) box helicase DDX3 (DEAD/H Box 3), known for some time to have a number of roles in cellular RNA regulation in the nucleus, has a role in the RLR cytoplasmic signalling pathway involved in IFN-β induction. In this issue of the European Journal of Immunology, an article reports an additional distinct
Show more

9 Read more

Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase.

Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase.

The effect of eliminating the histidine side chain in the DExH box of NPH-II is to constitutively activate NTPase, without need for a nucleic acid cofactor. Because wild-type NPH-II does display a low basal rate of NTP hydrolysis without a cofactor (this applies to the native enzyme from virions as well as the recombinant protein), it is axiomatic that the inter- action of NPH-II with polynucleotides must accelerate the rate-limiting step in the NTPase cycle. The rate-limiting steps under single-turnover and steady-state conditions have not been determined for NPH-II (or, to our knowledge, for any other DExH or DEAD protein). The H299A mutant can bind stably to RNA, and yet its ATPase is not stimulated apprecia- bly by RNA. Thus, whatever rate-enhancing conformational effect is elicited in the wild-type enzyme by RNA binding would essentially preexist in the H299A mutant. If RNA bind- ing causes the position of the His-299 residue to change rela- tive to the ground state (in order to relieve a suppressive effect of this moiety on the rate of hydrolysis), the elimination of the side chain by Ala substitution would be expected to mimic the
Show more

10 Read more

Show all 10000 documents...