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Sample records for dead box rna

  1. DEAD-box protein facilitated RNA folding in vivo

    PubMed Central

    Liebeg, Andreas; Mayer, Oliver

    2010-01-01

    In yeast mitochondria the DEAD-box helicase Mss116p is essential for respiratory growth by acting as group I and group II intron splicing factor. Here we provide the first structure-based insights into how Mss116p assists RNA folding in vivo. Employing an in vivo chemical probing technique, we mapped the structure of the ai5γ group II intron in different genetic backgrounds to characterize its intracellular fold. While the intron adopts the native conformation in the wt yeast strain, we found that the intron is able to form most of its secondary structure, but lacks its tertiary fold in the absence of Mss116p. This suggests that ai5γ is largely unfolded in the mss116-knockout strain and requires the protein at an early step of folding. Notably, in this unfolded state misfolded substructures have not been observed. As most of the protein-induced conformational changes are located within domain D1, Mss116p appears to facilitate the formation of this largest domain, which is the scaffold for docking of other intron domains. These findings suggest that Mss116p assists the ordered assembly of the ai5γ intron in vivo. PMID:21045551

  2. DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

    PubMed Central

    Pan, Cynthia; Potratz, Jeffrey P.; Cannon, Brian; Simpson, Zachary B.; Ziehr, Jessica L.; Tijerina, Pilar; Russell, Rick

    2014-01-01

    DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome. PMID:25350280

  3. RNA Remodeling Activity of DEAD Box Proteins Tuned by Protein Concentration, RNA Length, and ATP.

    PubMed

    Kim, Younghoon; Myong, Sua

    2016-09-01

    DEAD box RNA helicases play central roles in RNP biogenesis. We reported earlier that LAF-1, a DEAD box RNA helicase in C. elegans, dynamically interacts with RNA and that the interaction likely contributes to the fluidity of RNP droplets. Here we investigate the molecular basis of the interaction of RNA with LAF-1 and its human homolog, DDX3X. We show that both LAF-1 and DDX3X, at low concentrations, are monomers that induce tight compaction of single-stranded RNA. At high concentrations, the proteins are multimeric and dynamically interact with RNA in an RNA length-dependent manner. The dynamic LAF-1-RNA interaction stimulates RNA annealing activity. ATP adversely affects the RNA remodeling ability of LAF-1 by suppressing the affinity, dynamics, and annealing activity of LAF-1, suggesting that ATP may promote disassembly of the RNP complex. Based on our results, we postulate a plausible molecular mechanism underlying the dynamic equilibrium of the LAF-1 RNP complex. PMID:27546789

  4. DEAD/DExH-Box RNA Helicases in Selected Human Parasites

    PubMed Central

    Marchat, Laurence A.; Arzola-Rodríguez, Silvia I.; Hernandez-de la Cruz, Olga; Lopez-Rosas, Itzel; Lopez-Camarillo, Cesar

    2015-01-01

    DEAD/DExH-box RNA helicases catalyze the folding and remodeling of RNA molecules in prokaryotic and eukaryotic cells, as well as in many viruses. They are characterized by the presence of the helicase domain with conserved motifs that are essential for ATP binding and hydrolysis, RNA interaction, and unwinding activities. Large families of DEAD/DExH-box proteins have been described in different organisms, and their role in all molecular processes involving RNA, from transcriptional regulation to mRNA decay, have been described. This review aims to summarize the current knowledge about DEAD/DExH-box proteins in selected protozoan and nematode parasites of medical importance worldwide, such as Plasmodium falciparum, Leishmania spp., Trypanosoma spp., Giardia lamblia, Entamoeba histolytica, and Brugia malayi. We discuss the functional characterization of several proteins in an attempt to understand better the molecular mechanisms involving RNA in these pathogens. The current data also highlight that DEAD/DExH-box RNA helicases might represent feasible drug targets due to their vital role in parasite growth and development. PMID:26537038

  5. Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2010-01-12

    The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF{sub 3}, or ADP-AlF{sub 4}{sup -}. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge {alpha} helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

  6. SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato.

    PubMed

    Zhu, Mingku; Chen, Guoping; Dong, Tingting; Wang, Lingling; Zhang, Jianling; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The DEAD-box RNA helicases are involved in almost every aspect of RNA metabolism, associated with diverse cellular functions including plant growth and development, and their importance in response to biotic and abiotic stresses is only beginning to emerge. However, none of DEAD-box genes was well characterized in tomato so far. In this study, we reported on the identification and characterization of two putative DEAD-box RNA helicase genes, SlDEAD30 and SlDEAD31 from tomato, which were classified into stress-related DEAD-box proteins by phylogenetic analysis. Expression analysis indicated that SlDEAD30 was highly expressed in roots and mature leaves, while SlDEAD31 was constantly expressed in various tissues. Furthermore, the expression of both genes was induced mainly in roots under NaCl stress, and SlDEAD31 mRNA was also increased by heat, cold, and dehydration. In stress assays, transgenic tomato plants overexpressing SlDEAD31 exhibited dramatically enhanced salt tolerance and slightly improved drought resistance, which were simultaneously demonstrated by significantly enhanced expression of multiple biotic and abiotic stress-related genes, higher survival rate, relative water content (RWC) and chlorophyll content, and lower water loss rate and malondialdehyde (MDA) production compared to wild-type plants. Collectively, these results provide a preliminary characterization of SlDEAD30 and SlDEAD31 genes in tomato, and suggest that stress-responsive SlDEAD31 is essential for salt and drought tolerance and stress-related gene regulation in plants. PMID:26241658

  7. The embryonic RNA helicase gene (ERH): a new member of the DEAD box family of RNA helicases.

    PubMed Central

    Sowden, J; Putt, W; Morrison, K; Beddington, R; Edwards, Y

    1995-01-01

    DEAD box proteins share several highly conserved motifs including the characteristic Asp-Glu-Ala-Asp (D-E-A-D in the amino acid single-letter code) motif and have established or putative ATP-dependent RNA helicase activity. These proteins are implicated in a range of cellular processes that involve regulation of RNA function, including translation initiation, RNA splicing and ribosome assembly. Here we describe the isolation and characterization of an embryonic RNA helicase gene, ERH, which maps to mouse chromosome 1 and encodes a new member of the DEAD box family of proteins. The predicted ERH protein shows high sequence similarity to the testes-specific mouse PL10 and to the maternally acting Xenopus An3 helicase proteins. The ERH expression profile is similar, to that of An3, which localizes to the animal hemisphere of oocytes and is abundantly expressed in the embryo. ERH is expressed in oocytes and is a ubiquitous mRNA in the 9 days-post-conception embryo, and at later stages of development shows a more restricted pattern of expression in brain and kidney. The similarities in sequence and in expression profile suggest that ERH is the murine equivalent of the Xenopus An3 gene, and we propose that ERH plays a role in translational activation of mRNA in the oocyte and early embryo. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8948440

  8. Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana.

    PubMed

    Okanami, M; Meshi, T; Iwabuchi, M

    1998-06-01

    We have isolated cDNAs encoding a novel member of the DEAD box RNA helicase family from Arabidopsis. The protein, named AtDRH1, is composed of 619 amino acids and the central portion has high similarity with the helicase core region of a prototypic RNA helicase, the human nuclear protein p68. The N- and C-terminal regions are considerably diverged from the animal and yeast p68 homologs at the amino acid sequence level, but like the p68 subfamily members, an RGG box-like domain is present near the C-terminus. RNA blot analysis showed that the AtDRH1 transcript accumulates at a high level and almost equally in every part of the Arabidopsis plant. The purified, recombinant AtDRH1 was capable of unwinding double-stranded RNA in the presence of ATP or dATP and of hydrolyzing ATP. The ATPase activity was stimulated by some single-stranded RNAs and DNAs, including poly(A) and poly(dT), but not by poly(dA). The ability of the polynucleotides to stimulate the ATPase activity was largely consistent with their affinity for AtDRH1. These results show that AtDRH1 is a novel type of ATP/dATP-dependent RNA helicase and polynucleotide-dependent ATPase. PMID:9592148

  9. Nucleolar DEAD-Box RNA Helicase TOGR1 Regulates Thermotolerant Growth as a Pre-rRNA Chaperone in Rice

    PubMed Central

    Tang, Ding; Zhang, Yu’e; Cheng, Zhukuan; Xue, Yongbiao

    2016-01-01

    Plants have evolved a considerable number of intrinsic tolerance strategies to acclimate to ambient temperature increase. However, their molecular mechanisms remain largely obscure. Here we report a DEAD-box RNA helicase, TOGR1 (Thermotolerant Growth Required1), prerequisite for rice growth themotolerance. Regulated by both temperature and the circadian clock, its expression is tightly coupled to daily temperature fluctuations and its helicase activities directly promoted by temperature increase. Located in the nucleolus and associated with the small subunit (SSU) pre-rRNA processome, TOGR1 maintains a normal rRNA homeostasis at high temperature. Natural variation in its transcript level is positively correlated with plant height and its overexpression significantly improves rice growth under hot conditions. Our findings reveal a novel molecular mechanism of RNA helicase as a key chaperone for rRNA homeostasis required for rice thermotolerant growth and provide a potential strategy to breed heat-tolerant crops by modulating the expression of TOGR1 and its orthologs. PMID:26848586

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

    PubMed

    Surabhi, Satya; Tripathi, Bipin K; Maurya, Bhawana; Bhaskar, Pradeep K; Mukherjee, Ashim; Mutsuddi, Mousumi

    2015-11-01

    Notch signaling is an evolutionary conserved process that influences cell fate determination, cell proliferation, and cell death in a context-dependent manner. Notch signaling is fine-tuned at multiple levels and misregulation of Notch has been implicated in a variety of human diseases. We have characterized maheshvara (mahe), a novel gene in Drosophila melanogaster that encodes a putative DEAD box protein that is highly conserved across taxa and belongs to the largest group of RNA helicase. A dynamic pattern of mahe expression along with the maternal accumulation of its transcripts is seen during early stages of embryogenesis. In addition, a strong expression is also seen in the developing nervous system. Ectopic expression of mahe in a wide range of tissues during development results in a variety of defects, many of which resemble a typical Notch loss-of-function phenotype. We illustrate that ectopic expression of mahe in the wing imaginal discs leads to loss of Notch targets, Cut and Wingless. Interestingly, Notch protein levels are also lowered, whereas no obvious change is seen in the levels of Notch transcripts. In addition, mahe overexpression can significantly rescue ectopic Notch-mediated proliferation of eye tissue. Further, we illustrate that mahe genetically interacts with Notch and its cytoplasmic regulator deltex in trans-heterozygous combination. Coexpression of Deltex and Mahe at the dorso-ventral boundary results in a wing-nicking phenotype and a more pronounced loss of Notch target Cut. Taken together we report identification of a novel evolutionary conserved RNA helicase mahe, which plays a vital role in regulation of Notch signaling. PMID:26400611

  11. Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures

    PubMed Central

    Vakulskas, Christopher A.; Pannuri, Archana; Cortés-Selva, Diana; Zere, Tesfalem R.; Ahmer, Brian M.; Babitzke, Paul; Romeo, Tony

    2014-01-01

    Summary In Escherichia coli, activity of the global regulatory RNA binding protein CsrA is antagonized by two noncoding sRNAs, CsrB and CsrC, which sequester it away from its lower affinity mRNA targets. Transcription of csrB/C requires the BarA-UvrY two component signal transduction system, which responds to short chain carboxylates. We show that two DEAD-box RNA helicases, DeaD and SrmB, activate csrB/C expression by different pathways. DeaD facilitates uvrY translation by counteracting the inhibitory effect of long distance basepairing between the uvrY mRNA leader and coding region, while SrmB does not affect UvrY or UvrY-phosphate levels. Contrary to the prevailing notion that these helicases act primarily at low temperatures, DeaD and SrmB activated csrB expression over a wide temperature range. High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) revealed in vivo interactions of DeaD with 39 mRNAs, including those of uvrY and 9 other regulatory genes. Studies on the expression of several of the identified genes revealed regulatory effects of DeaD in all cases and diverse temperature response patterns. Our findings uncover an expanded regulatory role for DeaD, which is mediated through novel mRNA targets, important global regulators and under physiological conditions that were considered to be incompatible with its function. PMID:24708042

  12. DEAD-Box RNA helicases in Bacillus subtilis have multiple functions and act independently from each other.

    PubMed

    Lehnik-Habrink, Martin; Rempeters, Leonie; Kovács, Ákos T; Wrede, Christoph; Baierlein, Claudia; Krebber, Heike; Kuipers, Oscar P; Stülke, Jörg

    2013-02-01

    DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the importance of RNA, our knowledge about RNA helicases is limited. In this study, we investigated the role of the four DEAD-box RNA helicases in the Gram-positive model organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. The deletion of cshA, cshB, or yfmL in particular leads to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis, suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype, and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis, we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis. PMID:23175651

  13. A Cold-Inducible DEAD-Box RNA Helicase from Arabidopsis thaliana Regulates Plant Growth and Development under Low Temperature

    PubMed Central

    Liu, Yuelin; Tabata, Daisuke; Imai, Ryozo

    2016-01-01

    DEAD-box RNA helicases comprise a large family and are involved in a range of RNA processing events. Here, we identified one of the Arabidopsis thaliana DEAD-box RNA helicases, AtRH7, as an interactor of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3), which is an RNA chaperone involved in cold adaptation. Promoter:GUS transgenic plants revealed that AtRH7 is expressed ubiquitously and that its levels of the expression are higher in rapidly growing tissues. Knockout mutant lines displayed several morphological alterations such as disturbed vein pattern, pointed first true leaves, and short roots, which resemble ribosome-related mutants of Arabidopsis. In addition, aberrant floral development was also observed in rh7 mutants. When the mutants were germinated at low temperature (12°C), both radicle and first leaf emergence were severely delayed; after exposure of seedlings to a long period of cold, the mutants developed aberrant, fewer, and smaller leaves. RNA blots and circular RT-PCR revealed that 35S and 18S rRNA precursors accumulated to higher levels in the mutants than in WT under both normal and cold conditions, suggesting the mutants are partially impaired in pre-rRNA processing. Taken together, the results suggest that AtRH7 affects rRNA biogenesis and plays an important role in plant growth under cold. PMID:27116354

  14. Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L.

    PubMed

    Liu, Chenlin; Huang, Xiaohang

    2015-09-01

    DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation. PMID:26174530

  15. Yeast DEAD box protein Mss116p is a transcription elongation factor that modulates the activity of mitochondrial RNA polymerase.

    PubMed

    Markov, Dmitriy A; Wojtas, Ireneusz D; Tessitore, Kassandra; Henderson, Simmone; McAllister, William T

    2014-07-01

    DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions. PMID:24732805

  16. DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition

    PubMed Central

    Samatanga, Brighton; Klostermeier, Dagmar

    2014-01-01

    DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods. Both helicases bind nucleotides predominantly via RecA_N, in agreement with previous studies on Mss116, and with a universal, modular function of RecA_N in nucleotide recognition. In contrast, dsRNA recognition is different: Hera interacts with dsRNA in the absence of nucleotide, involving both RecA domains, whereas for YxiN neither RecA_N nor RecA_C binds dsRNA, and the complete core only interacts with dsRNA after nucleotide has been bound. DEAD-box proteins thus cover a continuum from complete functional independence of their domains, exemplified by Mss116, to various degrees of inter-domain cooperation in dsRNA binding. The different degrees of domain communication and of thermodynamic linkage between dsRNA and nucleotide binding have important implications on the mechanism of dsRNA unwinding, and may help direct RNA helicases to their respective cellular processes. PMID:25123660

  17. The requirement of the DEAD-box protein DDX24 for the packaging of human immunodeficiency virus type 1 RNA

    SciTech Connect

    Ma Jing; Rong Liwei; Zhou Yongdong; Roy, Bibhuti Bushan; Lu, Jennifer; Abrahamyan, Levon; Mouland, Andrew J.; Pan Qinghua; Chen Liang

    2008-05-25

    RNA helicases play important roles in RNA metabolism. Human immunodeficiency virus type 1 (HIV-1) does not carry its own RNA helicase, the virus thus needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. In this study, we report that knockdown of a DEAD-box protein named DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.

  18. Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase

    PubMed Central

    Caruthers, Jonathan M.; Johnson, Eric R.; McKay, David B.

    2000-01-01

    The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 Å resolution; it has a parallel α-β topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 Å resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a “dumbbell” structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI “link” the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding. PMID:11087862

  19. The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly

    PubMed Central

    Tu, Ya-Ting; Barrientos, Antoni

    2015-01-01

    SUMMARY Human mitochondrial ribosomes are specialized in the synthesis of 13 proteins, which are fundamental components of the oxidative phosphorylation system. The pathway of mitoribosome biogenesis, the compartmentalization of the process and factors involved remain largely unknown. Here, we have identified the DEAD box protein DDX28 as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and the mt-LSU. RNAi-mediated DDX28 silencing in HEK293T cells does not affect mitochondrial mRNA stability, 16S rRNA processing or modification. However, it leads to reduced levels of 16S rRNA and mt-LSU proteins, impaired mt-LSU assembly, deeply attenuated mitochondrial protein synthesis and consequent failure to assemble oxidative phosphorylation complexes. Our findings identify DDX28 as essential during the early stages of mitoribosome mt-LSU biogenesis, a process that mainly takes place near the mitochondrial nucleoids, in the compartment defined by the RNA granules. PMID:25683708

  20. Structure of the second domain of the Bacillus subtilis DEAD-box RNA helicase YxiN

    SciTech Connect

    Caruthers, Jonathan M.; Hu, YaoXiong; McKay, David B.

    2006-12-01

    The structure of the second helicase domain of the B. subtilis YxiN protein, a DEAD-box RNA helicase, is presented. The Bacillus subtilis RNA helicase YxiN is a modular three-domain protein. The first two domains form a conserved helicase core that couples an ATPase activity to an RNA duplex-destabilization activity, while the third domain recognizes a stem-loop of 23S ribosomal RNA with high affinity and specificity. The structure of the second domain, amino-acid residues 207–368, has been solved to 1.95 Å resolution, revealing a parallel αβ-fold. The crystallographic asymmetric unit contains two protomers; superposition shows that they differ substantially in two segments of peptide that overlap the conserved helicase sequence motifs V and VI, while the remainder of the domain is isostructural. The conformational variability of these segments suggests that induced fit is intrinsic to the recognition of ligands (ATP and RNA) and the coupling of the ATPase activity to conformational changes.

  1. Coupling between the DEAD-box RNA helicases Ded1p and eIF4A

    PubMed Central

    Gao, Zhaofeng; Putnam, Andrea A; Bowers, Heath A; Guenther, Ulf-Peter; Ye, Xuan; Kindsfather, Audrey; Hilliker, Angela K; Jankowsky, Eckhard

    2016-01-01

    Eukaryotic translation initiation involves two conserved DEAD-box RNA helicases, eIF4A and Ded1p. Here we show that S. cerevisiae eIF4A and Ded1p directly interact with each other and simultaneously with the scaffolding protein eIF4G. We delineate a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as a modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. Our results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F, the complex comprising eIF4G, eIF4A, and eIF4E. DOI: http://dx.doi.org/10.7554/eLife.16408.001 PMID:27494274

  2. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families.

    PubMed

    de la Cruz, J; Kressler, D; Linder, P

    1999-05-01

    Members of the RNA-helicase family are defined by several evolutionary conserved motifs. They are found in all organisms - from bacteria to humans - and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has given new insights into, and confirmed the significance of these proteins for, most cellular RNA metabolic processes. PMID:10322435

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

    PubMed Central

    Yajima, Mamiko; Wessel, Gary M.

    2011-01-01

    Vasa is a broadly conserved ATP-dependent RNA helicase that functions in the germ line of organisms from cnidarians to mammals. Curiously, Vasa is also present in the somatic cells of many animals and functions as a regulator of multipotent cells. Here, we report a mitotic function of Vasa revealed in the sea urchin embryo. We found that Vasa protein is present in all blastomeres of the early embryo and that its abundance oscillates with the cell cycle. Vasa associates with the spindle and the separating sister chromatids at metaphase, and then quickly disappears after telophase. Inhibition of Vasa protein synthesis interferes with proper chromosome segregation, arrests cells at M-phase, and delays overall cell cycle progression. Cdk activity is necessary for the proper localization of Vasa, implying that Vasa is involved in the cyclin-dependent cell cycle network, and Vasa is required for the efficient translation of cyclinB mRNA. Our results suggest an evolutionarily conserved role of Vasa that is independent of its function in germ line determination. PMID:21525076

  4. Mouse D1Pas1, a DEAD-box RNA helicase, is required for the completion of first meiotic prophase in male germ cells.

    PubMed

    Inoue, Hiroki; Ogonuki, Narumi; Hirose, Michiko; Hatanaka, Yuki; Matoba, Shogo; Chuma, Shinichiro; Kobayashi, Kimio; Wakana, Shigeharu; Noguchi, Junko; Inoue, Kimiko; Tanemura, Kentaro; Ogura, Atsuo

    2016-09-16

    D1Pas1 is a mouse autosomal DEAD-box RNA helicase expressed predominantly in the testis. To assess its possible function, we generated D1Pas1-deficient mice using embryonic stem cells with a targeted D1Pas1 allele. Deletion of D1Pas1 did not cause noticeable embryonic defects or death, indicating that D1Pas1 is not essential for embryogenesis. Whereas homozygous knockout female mice showed normal reproductive performance, homozygous knockout male mice were completely sterile. The seminiferous epithelium of D1Pas1-deficient males contained no spermatids or spermatozoa because of spermatogenic arrest at the late pachytene stage. Upregulation of retrotransposons such as LINE-1 was not found in D1Pas1-deficient males, unlike males lacking Mvh, another testicular DEAD-box RNA helicase. Meiotic chromosome behavior in developing spermatocytes of D1Pas1-deficient males was indistinguishable from that in wild-type males, at least until synaptonemal complex formation. Thus, mouse D1Pas1 is the first-identified DEAD-box RNA helicase that plays critical roles in the final step of the first meiotic prophase in male germ cells. PMID:27473657

  5. Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

    PubMed Central

    Yao, Hongjie; Brick, Kevin; Evrard, Yvonne; Xiao, Tiaojiang; Camerini-Otero, R. Daniel; Felsenfeld, Gary

    2010-01-01

    CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function. PMID:20966046

  6. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    DOE PAGESBeta

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; Shuman, Stewart

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to themore » adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.« less

  7. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

    SciTech Connect

    Jacewicz, Agata; Schwer, Beate; Smith, Paul; Shuman, Stewart

    2014-10-10

    Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.

  8. Measuring Helicase Inhibition of the DEAD-box Protein Dbp2 by Yra1

    PubMed Central

    Ma, Wai Kit; Tran, Elizabeth J.

    2016-01-01

    Despite the highly conserved helicase core, individual DEAD-box proteins are specialized in diverse RNA metabolic processes. One mechanism that determines DEAD-box protein specificity is enzymatic regulation by other protein cofactors. In this chapter, we describe a protocol for purifying the Saccharomyces cerevisiae DEAD-box RNA helicase Dbp2 and RNA-binding protein Yra1 and subsequent analysis of helicase regulation. The experiments described here can be adapted to RNA helicase and purified co-factor. PMID:25579587

  9. Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

    SciTech Connect

    Kellner, Julian N.; Meinhart, Anton

    2015-08-25

    The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. 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.

  10. Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin.

    PubMed

    Ma, Wai Kit; Paudel, Bishnu P; Xing, Zheng; Sabath, Ivan G; Rueda, David; Tran, Elizabeth J

    2016-03-27

    RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA-protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)(+)RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2. PMID:26876600

  11. Structure of the RNA binding domain of a DEAD-box helicase bound to its ribosomal RNA target reveals a novel mode of recognition by an RNA recognition motif

    PubMed Central

    Hardin, John W.; Hu, YaoXiong; McKay, David B.

    2010-01-01

    DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif (RRM) domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RRMs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by two orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding. PMID:20673833

  12. A Motif Unique to the Human Dead-Box Protein DDX3 Is Important for Nucleic Acid Binding, ATP Hydrolysis, RNA/DNA Unwinding and HIV-1 Replication

    PubMed Central

    Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-01-01

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication. PMID:21589879

  13. DEAD-box helicase DDX27 regulates 3′ end formation of ribosomal 47S RNA and stably associates with the PeBoW-complex

    SciTech Connect

    Kellner, Markus; Rohrmoser, Michaela; Forné, Ignasi; Voss, Kirsten; Burger, Kaspar; Mühl, Bastian; Gruber-Eber, Anita; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk

    2015-05-15

    PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex. - Highlights: • DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex. • The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1. • Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent. • Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.

  14. Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1

    PubMed Central

    Alessi, Amelia F.; Khivansara, Vishal; Han, Ting; Freeberg, Mallory A.; Moresco, James J.; Tu, Patricia G.; Montoye, Eric; Yates, John R.; Karp, Xantha; Kim, John K.

    2015-01-01

    MicroRNAs (miRNAs) play essential, conserved roles in diverse developmental processes through association with the miRNA-induced silencing complex (miRISC). Whereas fundamental insights into the mechanistic framework of miRNA biogenesis and target gene silencing have been established, posttranslational modifications that affect miRISC function are less well understood. Here we report that the conserved serine/threonine kinase, casein kinase II (CK2), promotes miRISC function in Caenorhabditis elegans. CK2 inactivation results in developmental defects that phenocopy loss of miRISC cofactors and enhances the loss of miRNA function in diverse cellular contexts. Whereas CK2 is dispensable for miRNA biogenesis and the stability of miRISC cofactors, it is required for efficient miRISC target mRNA binding and silencing. Importantly, we identify the conserved DEAD-box RNA helicase, CGH-1/DDX6, as a key CK2 substrate within miRISC and demonstrate phosphorylation of a conserved N-terminal serine is required for CGH-1 function in the miRNA pathway. PMID:26669440

  15. Identification of a putative DEAD-box RNA helicase and a zinc-finger protein in Candida albicans by functional complementation of the S. cerevisiae rok1 mutation.

    PubMed

    Kim, W I; Lee, W B; Song, K; Kim, J

    2000-03-30

    We identified two novel genes, CHR1 and CSR1, of the fungal pathogen Candida albicans, by functional complementation of the Saccharomyces cerevisiae rok1 mutation. The Rok1 protein is a member of the DEAD protein family of ATP-dependent RNA helicases. ROK1 is required for cell cycle progression and also for rRNA processing. The CHR1 gene product of 578 amino acids is highly homologous to the Rok1 protein (54% identity) and is considered to be a putative DEAD-box RNA helicase. We predict that the CSR1 gene encodes a 73 kDa protein of 612 amino acids with five zinc-finger motifs at the C-terminal region. CHR1 or CSR1 on a high-copy number plasmid showed a slow-growth phenotype in a condition where the ROK1 expression is turned on from the GAL1 promoter. This result is consistent with the lethality caused by the ROK1 overexpression. We conclude that CHR1 encodes a functional homologue of Rok1 protein and CSR1 is a heterologous suppressor of the rok1 mutation. PMID:10705369

  16. P(I) Release Limits the Intrinsic and RNA-Stimulated ATPase Cycles of DEAD-Box Protein 5 (Dbp5).

    PubMed

    Wong, Emily V; Cao, Wenxiang; Vörös, Judit; Merchant, Monique; Modis, Yorgo; Hackney, David D; Montpetit, Ben; De La Cruz, Enrique M

    2016-01-29

    mRNA export from the nucleus depends on the ATPase activity of the DEAD-box protein Dbp5/DDX19. Although Dbp5 has measurable ATPase activity alone, several regulatory factors (e.g., RNA, nucleoporin proteins, and the endogenous small molecule InsP6) modulate catalytic activity in vitro and in vivo to facilitate mRNA export. An analysis of the intrinsic and regulator-activated Dbp5 ATPase cycle is necessary to define how these factors control Dbp5 and mRNA export. Here, we report a kinetic and equilibrium analysis of the Saccharomyces cerevisiae Dbp5 ATPase cycle, including the influence of RNA on Dbp5 activity. These data show that ATP binds Dbp5 weakly in rapid equilibrium with a binding affinity (KT~4 mM) comparable to the KM for steady-state cycling, while ADP binds an order of magnitude more tightly (KD~0.4 mM). The overall intrinsic steady-state cycling rate constant (kcat) is limited by slow, near-irreversible ATP hydrolysis and even slower subsequent phosphate release. RNA increases kcat and rate-limiting Pi release 20-fold, although Pi release continues to limit steady-state cycling in the presence of RNA, in conjunction with RNA binding. Together, this work identifies RNA binding and Pi release as important biochemical transitions within the Dbp5 ATPase cycle and provides a framework for investigating the means by which Dbp5 and mRNA export is modulated by regulatory factors. PMID:26730886

  17. The DEAD-box RNA helicase 51 controls non-small cell lung cancer proliferation by regulating cell cycle progression via multiple pathways.

    PubMed

    Wang, Xiaojing; Liu, Hongli; Zhao, Chengling; Li, Wei; Xu, Huanbai; Chen, Yuqing

    2016-01-01

    The genetic regulation of cell cycle progression and cell proliferation plays a role in the growth of non-small cell lung cancer (NSCLC), one of the most common causes of cancer-related mortality. Although DEAD-box RNA helicases are known to play a role in cancer development, including lung cancer, the potential involvement of the novel family member DDX51 has not yet been investigated. In the current study we assessed the role of DDX51 in NSCLC using a siRNA-based approach. DDX51 siRNA-expressing cells exhibited a slower cell proliferation rate and underwent arrest in S-phase of the cell cycle compared with control cells. Microarray analyses revealed that DDX51siRNA expression resulted in the dysregulation of a number of cell signalling pathways. Moreover, injection of DDX51 siRNA into an animal model resulted in the formation of smaller tumours compared with the control group. We also assessed the expression of DDX51 in patients with NSCLC, and the data revealed that the expression was correlated with patient age but no other risk factors. Overall, our data suggest for the first time that DDX51 aids cell cancer proliferation by regulating multiple signalling pathways, and that this protein might be a therapeutic target for NSCLC. PMID:27198888

  18. The DEAD-box RNA helicase 51 controls non-small cell lung cancer proliferation by regulating cell cycle progression via multiple pathways

    PubMed Central

    Wang, Xiaojing; Liu, Hongli; Zhao, Chengling; Li, Wei; Xu, Huanbai; Chen, Yuqing

    2016-01-01

    The genetic regulation of cell cycle progression and cell proliferation plays a role in the growth of non-small cell lung cancer (NSCLC), one of the most common causes of cancer-related mortality. Although DEAD-box RNA helicases are known to play a role in cancer development, including lung cancer, the potential involvement of the novel family member DDX51 has not yet been investigated. In the current study we assessed the role of DDX51 in NSCLC using a siRNA-based approach. DDX51 siRNA-expressing cells exhibited a slower cell proliferation rate and underwent arrest in S-phase of the cell cycle compared with control cells. Microarray analyses revealed that DDX51siRNA expression resulted in the dysregulation of a number of cell signalling pathways. Moreover, injection of DDX51 siRNA into an animal model resulted in the formation of smaller tumours compared with the control group. We also assessed the expression of DDX51 in patients with NSCLC, and the data revealed that the expression was correlated with patient age but no other risk factors. Overall, our data suggest for the first time that DDX51 aids cell cancer proliferation by regulating multiple signalling pathways, and that this protein might be a therapeutic target for NSCLC. PMID:27198888

  19. Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2009-09-02

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/{Delta}598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions ({le}1 mg ml{sup -1}), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50% glycerol to achieve concentrations of {approx}10 mg ml{sup -1}. Initial crystals were obtained by the microbatch method in the presence of a U{sub 10} RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/{Delta}598-664 in complex with AMP-PNP and U{sub 10} belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 {angstrom}, and diffracted X-rays to beyond 1.9 {angstrom} resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.

  20. Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP

    PubMed Central

    Del Campo, Mark; Lambowitz, Alan M.

    2009-01-01

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/Δ598–664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions (≤1 mg ml−1), but its solubility could be increased by adding 50 mM l-arginine plus 50 mM l-glutamate and 50% glycerol to achieve concentrations of ∼10 mg ml−1. Initial crystals were obtained by the microbatch method in the presence of a U10 RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/Δ598–664 in complex with AMP-PNP and U10 belonged to space group P21212, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 Å, and diffracted X-rays to beyond 1.9 Å resolution using synchrotron radiation from sector 21 at the Advanced Photon Source. PMID:19652352

  1. DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

    SciTech Connect

    Kanno, Yuichiro; Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer DP97 interacts with nuclear receptor CAR. Black-Right-Pointing-Pointer DP97 enhances CAR-mediated transcriptional activation. Black-Right-Pointing-Pointer DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1{alpha}. Black-Right-Pointing-Pointer DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1{alpha}, therefore it might act as mediator between hCAR and appropriate co-activators.

  2. An Arabidopsis ATP-Dependent, DEAD-Box RNA Helicase Loses Activity upon IsoAsp Formation but Is Restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE[C][W

    PubMed Central

    Nayak, Nihar R.; Putnam, Andrea A.; Addepalli, Balasubrahmanyam; Lowenson, Jonathan D.; Chen, Tingsu; Jankowsky, Eckhard; Perry, Sharyn E.; Dinkins, Randy D.; Limbach, Patrick A.; Clarke, Steven G.; Downie, A. Bruce

    2013-01-01

    Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75’s complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination. PMID:23903319

  3. PRD-1, a Component of the Circadian System of Neurospora crassa, Is a Member of the DEAD-box RNA Helicase Family.

    PubMed

    Adhvaryu, Keyur; Firoozi, Ghazaleh; Motavaze, Kamyar; Lakin-Thomas, Patricia

    2016-06-01

    The circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (TTFLs). However, TTFL-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. The fungus Neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock. Although a circadian TTFL involving the proteins FRQ, WC-1, and WC-2 is well-characterized in N. crassa, rhythms can still be observed in the absence of this feedback loop. These rhythms are said to be driven by 1 or more FRQ-less oscillator(s) (FLOs). The prd-1 mutation lengthens the period in frq wild type and was previously shown to severely disrupt FRQ-less rhythms in frq null mutants under several different conditions; therefore, the prd-1 gene product is a candidate for a component of a FLO. We report here that prd-1 also disrupts free-running rhythms in wc-1 null mutants, confirming its effects on FRQ-less rhythms. We have now mapped and identified the prd-1 gene as NCU07839, a DEAD-box RNA helicase dbp-2 Complementation with the wild-type gene corrects the rhythm defects of the prd-1 mutant in the complete circadian system (when the FRQ-based TTFL is intact) and also the free-running FRQ-less rhythm on low choline. A PRD-1-GFP fusion protein localizes to the nucleus. The prd-1 mutant has a single base pair change in the first base of an intron that results in abnormally spliced transcripts. FRQ-less rhythms on low choline, or entrained to heat pulses, were only marginally affected in strains carrying deletions of 2 other RNA helicases (prd-6 and msp-8). We conclude that PRD-1 is a member of an RNA helicase family that may be specifically involved in regulating rhythmicity in N. crassa in both the complete circadian system and FLO(s). PMID:27029286

  4. Ezrin Binds to DEAD-Box RNA Helicase DDX3 and Regulates Its Function and Protein Level

    PubMed Central

    Çelik, Haydar; Sajwan, Kamal P.; Selvanathan, Saravana P.; Marsh, Benjamin J.; Pai, Amrita V.; Kont, Yasemin Saygideger; Han, Jenny; Minas, Tsion Z.; Rahim, Said; Erkizan, Hayriye Verda; Toretsky, Jeffrey A.

    2015-01-01

    Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC–MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5′ untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane. PMID:26149384

  5. Embryonic expression of p68, a DEAD-box RNA helicase, in the oligochaete annelid Tubifex tubifex.

    PubMed

    Oyama, Atsuko; Yoshida, Hiroshi; Shimizu, Takashi

    2008-07-01

    We have cloned and characterized the expression of a p68 homologue (designated Ttu-p68) from the oligochaete annelid Tubifex tubifex. Ttu-p68 mRNA is distributed broadly throughout the early stages. Ttu-p68 is expressed in all of the early blastomeres, in which Ttu-p68 RNA associates with pole plasms. Ttu-p68 transcripts are concentrated to 4d cell but not to 2d cell. During gastrulation, expression of Ttu-p68 is restricted to elongating germ bands (GBs) and an anteriormost crescent of micromere descendants on both sides of the embryo. During body elongation that follows gastrulation, expression of Ttu-p68 is further restricted to the stomodaeum (derived from the micromere crescent), ventral ganglia, lateral dots (corresponding to dorsal and ventral setal sacs), ventral large cells (that resemble presumptive primordial germ cells) in segments VIII-XII, and a bilateral pair of cell clusters at the caudal end. At the end of embryogenesis, Ttu-p68 expression persists exclusively in the tail and the lining epithelium of the pharynx. PMID:18381252

  6. Analog sensitive chemical inhibition of the DEAD-box protein DDX3.

    PubMed

    Floor, Stephen N; Barkovich, Krister J; Condon, Kendall J; Shokat, Kevan M; Doudna, Jennifer A

    2016-03-01

    Proper maintenance of RNA structure and dynamics is essential to maintain cellular health. Multiple families of RNA chaperones exist in cells to modulate RNA structure, RNA-protein complexes, and RNA granules. The largest of these families is the DEAD-box proteins, named after their catalytic Asp-Glu-Ala-Asp motif. The human DEAD-box protein DDX3 is implicated in diverse biological processes including translation initiation and is mutated in numerous cancers. Like many DEAD-box proteins, DDX3 is essential to cellular health and exhibits dosage sensitivity, such that both decreases and increases in protein levels can be lethal. Therefore, chemical inhibition would be an ideal tool to probe the function of DDX3. However, most DEAD-box protein active sites are extremely similar, complicating the design of specific inhibitors. Here, we show that a chemical genetic approach best characterized in protein kinases, known as analog-sensitive chemical inhibition, is viable for DDX3 and possibly other DEAD-box proteins. We present an expanded active-site mutant that is tolerated in vitro and in vivo, and is sensitive to chemical inhibition by a novel bulky inhibitor. Our results highlight a course towards analog sensitive chemical inhibition of DDX3 and potentially the entire DEAD-box protein family. PMID:26650549

  7. DEAD-box RNA helicase DDX3 connects CRM1-dependent nuclear export and translation of the HIV-1 unspliced mRNA through its N-terminal domain.

    PubMed

    Fröhlich, Alvaro; Rojas-Araya, Bárbara; Pereira-Montecinos, Camila; Dellarossa, Alessandra; Toro-Ascuy, Daniela; Prades-Pérez, Yara; García-de-Gracia, Francisco; Garcés-Alday, Andrea; Rubilar, Paulina S; Valiente-Echeverría, Fernando; Ohlmann, Théophile; Soto-Rifo, Ricardo

    2016-05-01

    DEAD-box RNA helicase DDX3 is a host factor essential for HIV-1 replication and thus, a potential target for novel therapies aimed to overcome viral resistance. Previous studies have shown that DDX3 promotes nuclear export and translation of the HIV-1 unspliced mRNA. Although the function of DDX3 during both processes requires its catalytic activity, it is unknown whether other domains surrounding the helicase core are involved. Here, we show the involvement of the N- and C-terminal domains of DDX3 in the regulation of HIV-1 unspliced mRNA translation. Our results suggest that the intrinsically disordered N-terminal domain of DDX3 regulates its functions in translation by acting prior to the recruitment of the 43S pre-initiation complex onto the viral 5'-UTR. Interestingly, this regulation was conserved in HIV-2 and was dependent on the CRM1-dependent nuclear export pathway suggesting a role of the RNA helicase in interconnecting nuclear export with ribosome recruitment of the viral unspliced mRNA. This specific function of DDX3 during HIV gene expression could be exploited as an alternative target for pharmaceutical intervention. PMID:27012366

  8. The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA.

    PubMed

    Soto-Rifo, Ricardo; Rubilar, Paulina S; Ohlmann, Théophile

    2013-07-01

    Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5' m(7)GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m(7)GTP 5' cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled. PMID:23630313

  9. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    PubMed

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  10. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

  11. Roles of Four Putative DEAD-Box RNA Helicase Genes in Growth of Listeria monocytogenes EGD-e under Heat, pH, Osmotic, Ethanol, and Oxidative Stress Conditions

    PubMed Central

    Lindström, Miia; Johansson, Per; Björkroth, Johanna; Korkeala, Hannu

    2012-01-01

    To examine the role of the four putative DEAD-box RNA helicase genes of Listeria monocytogenes EGD-e in stress tolerance, the growth of the Δlmo0866, Δlmo1246, Δlmo1450, and Δlmo1722 deletion mutant strains at 42.5°C, at pH 5.6 or pH 9.4, in 6% NaCl, in 3.5% ethanol, and in 5 mM H2O2 was studied. Restricted growth of the Δlmo0866 deletion mutant strain in 3.5% ethanol suggests that Lmo0866 contributes to ethanol stress tolerance of L. monocytogenes EGD-e. The Δlmo1450 mutant strain showed negligible growth at 42.5°C, at pH 9.4, and in 5 mM H2O2 and a lower maximum growth temperature than the wild-type EGD-e, suggesting that Lmo1450 is involved in the tolerance of L. monocytogenes EGD-e to heat, alkali, and oxidative stresses. The altered stress tolerance of the Δlmo0866 and Δlmo1450 deletion mutant strains did not correlate with changes in relative expression levels of lmo0866 and lmo1450 genes under corresponding stresses, suggesting that Lmo0866- and Lmo1450-dependent tolerance to heat, alkali, ethanol, or oxidative stress is not regulated at the transcriptional level. Growth of the Δlmo1246 and Δlmo1722 deletion mutant strains did not differ from that of the wild-type EGD-e under any of the conditions tested, suggesting that Lmo1246 and Lmo1722 have no roles in the growth of L. monocytogenes EGD-e under heat, pH, osmotic, ethanol, or oxidative stress. This study shows that the putative DEAD-box RNA helicase genes lmo0866 and lmo1450 play important roles in tolerance of L. monocytogenes EGD-e to ethanol, heat, alkali, and oxidative stresses. PMID:22820328

  12. Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii

    PubMed Central

    Story, Randall M.; Li, Hong; Abelson, John N.

    2001-01-01

    We have determined the structure of a DEAD box putative RNA helicase from the hyperthermophile Methanococcus jannaschii. Like other helicases, the protein contains two α/β domains, each with a recA-like topology. Unlike other helicases, the protein exists as a dimer in the crystal. Through an interaction that resembles the dimer interface of insulin, the amino-terminal domain's 7-strand β-sheet is extended to 14 strands across the two molecules. Motifs conserved in the DEAD box family cluster in the cleft between domains, and many of their functions can be deduced by mutational data and by comparison with other helicase structures. Several lines of evidence suggest that motif III Ser-Ala-Thr may be involved in binding RNA. PMID:11171974

  13. Potential Regulatory Interactions of Escherichia coli RraA Protein with DEAD-box Helicases*

    PubMed Central

    Pietras, Zbigniew; Hardwick, Steven W.; Swiezewski, Szymon; Luisi, Ben F.

    2013-01-01

    Members of the DEAD-box family of RNA helicases contribute to virtually every aspect of RNA metabolism, in organisms from all domains of life. Many of these helicases are constituents of multicomponent assemblies, and their interactions with partner proteins within the complexes underpin their activities and biological function. In Escherichia coli the DEAD-box helicase RhlB is a component of the multienzyme RNA degradosome assembly, and its interaction with the core ribonuclease RNase E boosts the ATP-dependent activity of the helicase. Earlier studies have identified the regulator of ribonuclease activity A (RraA) as a potential interaction partner of both RNase E and RhlB. We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB. Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å. The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases. PMID:24045937

  14. Unraveling the 'DEAD-box' helicases of Plasmodium falciparum.

    PubMed

    Tuteja, Renu; Pradhan, Arun

    2006-07-01

    The causative agent for the most fatal form of malaria, Plasmodium falciparum, has developed insecticide and drug resistance with time. Therefore combating this disease is becoming increasingly difficult and this calls for finding alternate ways to control malaria. One of the feasible ways could be to find out inhibitors/drugs specific for the indispensable enzymes of malaria parasite such as helicases. These helicases, which contain intrinsic nucleic acid-dependent ATPase activity, are capable of enzymatically unwinding energetically stable duplex nucleic acids into single-stranded templates and are required for all the nucleic acid transactions. Most of the helicases contain a set of nine extremely conserved amino acid sequences, which are called 'helicase motifs'. Due to the presence of the DEAD (Asp-Glu-Ala-Asp) in one of the conserved motifs, this family is also known as the 'DEAD-box' family. In this review, using bioinformatic approach, we describe the 'DEAD-box' helicases of malaria parasite P. falciparum. An in depth analysis shows that the parasite contains 22 full-length genes, some of which are homologues of well-characterized helicases of this family from other organisms. Recently we have cloned and characterized the first member of this family, which is a homologue of p68 and is expressed during the schizont stage of the development of the parasite [Pradhan, A., Chauhan, V.S., Tuteja, R., 2005a. A novel 'DEAD-box' DNA helicase from Plasmodium falciparum is homologous to p68. Mol. Biochem. Parasitol. 140, 55-60.; Pradhan A., Chauhan V.S., Tuteja R., 2005b. Plasmodium falciparum DNA helicase 60 is a schizont stage specific, bipolar and dual helicase stimulated by PKC phosphorylation. Mol. Biochem. Parasitol. 144, 133-141.]. It will be really interesting to clone and characterize other members of the 'DEAD-box' family and understand their role in the replication and transmission of the parasite. These detailed studies may help to identify a parasite

  15. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

    PubMed Central

    Grandori, C; Mac, J; Siëbelt, F; Ayer, D E; Eisenman, R N

    1996-01-01

    The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism. Images PMID:8861962

  16. In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

    PubMed

    Dehghani, Mehrnoush; Lasko, Paul

    2015-01-01

    The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas) is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells), posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk) mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles. PMID:25795910

  17. In vivo mapping of the functional regions of the DEAD-box helicase Vasa

    PubMed Central

    Dehghani, Mehrnoush; Lasko, Paul

    2015-01-01

    The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas) is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells), posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk) mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles. PMID:25795910

  18. Mechanism of Mss116 ATPase reveals functional diversity of DEAD-box proteins

    PubMed Central

    Cao, Wenxiang; Coman, Maria Magdalena; Ding, Steve; Henn, Arnon; Middleton, Elizabeth R.; Bradley, Michael J.; Rhoades, Elizabeth; Hackney, David D.; Pyle, Anna Marie; De La Cruz, Enrique M.

    2011-01-01

    Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for efficient in vivo splicing of all group I and II introns and activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ~7-fold by promoting rate-limiting ATP hydrolysis, such that Pi release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-Pi binds RNA more strongly than with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA binding, Mss116-ADP-Pi state. Strong RNA binding allows Mss116 to fulfill its biological role in stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA binding, ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with Pi release dissociates RNA-Mss116 complexes, regenerating free Mss116. PMID:21501623

  19. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-box Protein Mss116p

    PubMed Central

    Mohr, Georg; Campo, Mark Del; Turner, Kathryn G.; Gilman, Benjamin; Wolf, Rachel Z.; Lambowitz, Alan M.

    2011-01-01

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins, but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends upon ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the C-terminal extension results primarily from a steric block. Finally, we identified two conserved regions, one the previously noted post-II region in the helicase core and the other in the C-terminal extension, which may help displace or sequester the opposite RNA strand during RNA unwinding. PMID:21945532

  20. High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

    SciTech Connect

    Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.; Gilman, Benjamin; Wolf, Rachel Z.; Lambowitz, Alan M.

    2012-03-15

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.

  1. Investigation of the conserved glutamate immediately following the DEAD box in eukaryotic translation initiation factor 4AI.

    PubMed

    Patel, Krishnaben; Shah, Grishma K; Kommaraju, Sai Shilpa; Low, Woon-Kai

    2014-02-01

    The DExD-box family (DEAD-box) of proteins was surveyed for eukaryotic translation initiation factor 4A-specific sequences surrounding the DEAD box. An eIF4A-unique glutamate residue (E186 in eIF4AI) was identified immediately following the D-E-A-D sequence in eIF4AI, II, and III that was found to be conserved from yeast to Man. Mutation to a selection of alternative amino acids was performed within recombinant eIF4AI expressed in Escherichia coli and mutant proteins were surveyed for RNA-dependent ATPase activity. The mutants were also investigated for changes in activity in the presence of the two eIF4AI-binding domains of eIF4GI as well as for co-purification ability to these two domains. The E186 residue was found to be of significance for RNA-dependent ATPase activity for eIF4AI alone and in the presence of eIF4AI-binding domains of eIF4GI through point-mutation analysis. Furthermore, binding interactions between eIF4AI and eIF4GI domains were also significantly influenced by mutation of E186, as observed through co-purification assays. Thus, this residue appears to be of functional significance for eIF4A. PMID:24471916

  2. A role for the cytoplasmic DEAD box helicase Dbp21E2 in rhodopsin maturation and photoreceptor viability

    PubMed Central

    Hibbard, Karen L.; O’Tousa, Joseph. E.

    2013-01-01

    The Dbp21E2 (Dead box protein 21E2) is a member of a family of DEAD box helicases active in RNA processing and stability. We used genetic mosaics to identify mutants in Dbp21E2 that affect rhodopsin biogenesis and the maintenance of photoreceptor structure. Analysis of a GFP-tagged Rh1 rhodopsin construct placed under control of a heat shock promoter showed that Dbp21E21 fails to efficiently transport Rh1 from the photoreceptor cell body to the rhabdomere. Retinal degeneration is not dependent on the Rh1 transport defects. We also show that GFP- and RFP-tagged Dbp21E2 proteins are localized to discrete cytoplasmic structures that are not associated with organelles known to be active in rhodopsin transport. The molecular genetic analysis described here reveals an unexpected role for the Dbp21E2 helicase and provides an experimental system to further characterize its function. PMID:22794106

  3. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    SciTech Connect

    Möhlmann, Sina; Mathew, Rebecca; Neumann, Piotr; Schmitt, Andreas; Lührmann, Reinhard; Ficner, Ralf

    2014-06-01

    The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.

  4. Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

    PubMed Central

    Möhlmann, Sina; Mathew, Rebecca; Neumann, Piotr; Schmitt, Andreas; Lührmann, Reinhard; Ficner, Ralf

    2014-01-01

    The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP. PMID:24914973

  5. DEAD-box proteins, like Leishmania eIF4A, modulate interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha production by human monocytes.

    PubMed

    Barhoumi, M; Meddeb-Garnaoui, A; Tanner, N K; Banroques, J; Kaabi, B; Guizani, I

    2013-01-01

    Previously we showed that His-tagged, recombinant, Leishmania infantum eukaryotic initiation factor (LeIF) was both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described for other members of the DEAD-box helicase family. In addition, we showed that LeIF induces the production of IL-12, IL-10, and TNF-α by human monocytes. This study aims to characterize the cytokine-inducing activity in human monocytes of several proteins belonging to the DEAD-box family from mammals and yeast. All tested proteins contained the 11 conserved motifs (Q, I, Ia, GG Ib, II, III, IV, QxxR, V and VI) characteristic of DEAD-box proteins, but they have different biological functions and different percentages of identities with LeIF. We show that these mammalian or yeast recombinant proteins also are able to induce IL-12, IL-10 and TNF-α secretion by monocytes of healthy human subjects. This cytokine-inducing activity is proteinase K sensitive and polymyxin B resistant. Our results show that the induction of cytokines in human monocytes is not unique to the protein LeIF of Leishmania, and it suggests that the activity of certain DEAD-box proteins can be exploited as adjuvant and/or to direct immune responses towards a Th1 profile in vaccination or immunotherapy protocols. PMID:23363368

  6. Are RNAi and miRNA therapeutics truly dead?

    PubMed

    Conde, João; Artzi, Natalie

    2015-03-01

    Only a few years ago pharmaceutical companies were excited about the potential of RNA interference (RNAi). Now, financial volatility and subsequent dissolutions of in-house facilities by pharmaceutical companies have had media channels pronouncing that RNAi therapeutics are dead. However, advances in nanomedicine may now help the vast potential of RNAi therapeutics to be fulfilled. PMID:25595555

  7. Structural and Functional Analysis of the Interaction Between the Nucleoporin Nup214 and the DEAD-box Helicase Ddx19

    SciTech Connect

    Napetschnig, J.; Kassube, S; Debler, E; Wong, R; Blobel, G; Hoelz, A

    2009-01-01

    Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 {angstrom} resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.

  8. Cloning, characterization, and expression analysis of the DEAD-box family genes, Fc-vasa and Fc-PL10a, in Chinese shrimp ( Fenneropenaeus chinensis)

    NASA Astrophysics Data System (ADS)

    Zhou, Qianru; Shao, Mingyu; Qin, Zhenkui; Kyoung, Ho Kang; Zhang, Zhifeng

    2010-01-01

    RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-vasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.

  9. Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor.

    PubMed

    Iwasaki, Shintaro; Floor, Stephen N; Ingolia, Nicholas T

    2016-06-23

    Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions. PMID:27309803

  10. LIGAND-INDUCED CHANGES IN T BOX ANTITERMINATOR RNA STABILITY

    PubMed Central

    Zhou, S.; Acquaah-Harrison, G.; Jack, K.D.; Bergmeier, S.C.; Hines, J.V.

    2012-01-01

    The T box antiterminator RNA element is an important component of the T box riboswitch that controls the transcription of vital genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened in a fluorescence-monitored thermal denaturation assay to identify ligands that altered the stability of antiterminator model RNA. Several ligands were identified that significantly increased or decreased the melting temperature (Tm) of the RNA. The results indicate that this series of triazole ligands can alter the stability of antiterminator model RNA in a structure-dependent manner. PMID:22117759

  11. The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

    PubMed Central

    Intile, Peter J.; Balzer, Grant J.; Wolfgang, Matthew C.

    2015-01-01

    ABSTRACT The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCE Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box

  12. DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

    SciTech Connect

    Kim, Ki Hyung; Kang, Yun-Jeong; Jo, Jin-Ok; Ock, Mee Sun; Moon, Soo Hyun; Suh, Dong Soo; Yoon, Man Soo; Park, Eun-Sil; Jeong, Namkung; Eo, Wan-Kyu; Kim, Heung Yeol; Cha, Hee-Jae

    2014-05-02

    Highlights: • Germ cell marker DDX4 was significantly increased in ovarian cancer. • Ovarian cancer stem cell marker CD133 was significantly increased in ovarian cancer. • DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. • CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4. • Germ cell marker DDX4 has the potential of ovarian cancer stem cell marker. - Abstract: DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.

  13. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs.

    PubMed

    Soto-Rifo, Ricardo; Rubilar, Paulina S; Limousin, Taran; de Breyne, Sylvain; Décimo, Didier; Ohlmann, Théophile

    2012-09-12

    Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m(7)GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5' of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5' free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation. PMID:22872150

  14. DExD/H-box RNA helicases in ribosome biogenesis

    PubMed Central

    Martin, Roman; Straub, Annika U.; Doebele, Carmen; Bohnsack, Markus T.

    2013-01-01

    Ribosome synthesis requires a multitude of cofactors, among them DExD/H-box RNA helicases. Bacterial RNA helicases involved in ribosome assembly are not essential, while eukaryotes strictly require multiple DExD/H-box proteins that are involved in the much more complex ribosome biogenesis pathway. Here, RNA helicases are thought to act in structural remodeling of the RNPs including the modulation of protein binding, and they are required for allowing access or the release of specific snoRNPs from pre-ribosomes. Interestingly, helicase action is modulated by specific cofactors that can regulate recruitment and enzymatic activity. This review summarizes the current knowledge and focuses on recent findings and open questions on RNA helicase function and regulation in ribosome synthesis. PMID:22922795

  15. Fluorescence anisotropy: analysis of tRNA binding to the T box riboswitch antiterminator RNA.

    PubMed

    Zhou, S; Anupam, R; Hines, J V

    2015-01-01

    Fluorescence anisotropy can be utilized in drug discovery screening assays to identify compounds that disrupt medicinally important RNA-macromolecular complexes. Here we describe the application of this technique to monitor tRNA binding to T box riboswitch antiterminator RNA. PMID:25352143

  16. Pea p68, a DEAD-Box Helicase, Provides Salinity Stress Tolerance in Transgenic Tobacco by Reducing Oxidative Stress and Improving Photosynthesis Machinery

    PubMed Central

    Tuteja, Narendra; Banu, Mst. Sufara Akhter; Huda, Kazi Md. Kamrul; Gill, Sarvajeet Singh; Jain, Parul; Pham, Xuan Hoi; Tuteja, Renu

    2014-01-01

    Background The DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant. Results The pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities. Conclusions To the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance. PMID:24879307

  17. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

    PubMed Central

    Khoshnevis, Sohail; Askenasy, Isabel; Dattolo, Maria D.; Young-Erdos, Crystal L.; Stroupe, M. Elizabeth; Karbstein, Katrin

    2016-01-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  18. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

    PubMed

    Khoshnevis, Sohail; Askenasy, Isabel; Johnson, Matthew C; Dattolo, Maria D; Young-Erdos, Crystal L; Stroupe, M Elizabeth; Karbstein, Katrin

    2016-06-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  19. The T box riboswitch: a novel regulatory RNA that utilizes tRNA as its ligand

    PubMed Central

    Henkin, Tina M.

    2016-01-01

    The T box riboswitch is a cis-acting regulatory RNA that controls expression of amino acid-related genes in response to the aminoacylation state of a specific tRNA. Multiple genes in the same organism can utilize this mechanism, with each gene responding independently to its cognate tRNA. The uncharged tRNA interacts directly with the regulatory RNA element, and this interaction promotes readthrough of an intrinsic transcriptional termination site upstream of the regulated coding sequence. A second class of T box elements uses a similar tRNA-dependent response to regulate translation initiation. This review will describe the current state of our knowledge about this regulatory system. PMID:24816551

  20. Dbp73D, a Drosophila gene expressed in ovary, encodes a novel D-E-A-D box protein.

    PubMed Central

    Patterson, L F; Harvey, M; Lasko, P F

    1992-01-01

    Proteins of the D-E-A-D family of putative ATP-dependent RNA helicases have been implicated in translation initiation and RNA splicing in a variety of organisms from E. coli to man. The Drosophila vasa protein, a member of this family, is required in the female germ line for fertility and for specification of germ line and posterior positional information in progeny embryos. We report the isolation of another D-E-A-D gene from Drosophila, which, like vasa, is expressed in germ line tissue. The predicted amino acid sequence of this new gene, Dbp73D, contains all of the highly conserved helicase motifs, but is otherwise the farthest-diverged member of the family so far identified. Images PMID:1620603

  1. Electrophoretic mobility shift assays: analysis of tRNA binding to the T box riboswitch antiterminator RNA.

    PubMed

    Anupam, R; Zhou, S; Hines, J V

    2015-01-01

    Changes in electrophoretic mobility upon complex formation with RNA can be used to probe structure-function relationships that are critical for complex formation. Here, we describe the application of this technique to monitor tRNA binding to the T box riboswitch antiterminator RNA. PMID:25352142

  2. Structural organization of box C/D RNA-guided RNA methyltransferase

    PubMed Central

    Ye, Keqiong; Jia, Ru; Lin, Jinzhong; Ju, Minghua; Peng, Jin; Xu, Anbi; Zhang, Liman

    2009-01-01

    Box C/D guide RNAs are abundant noncoding RNAs that primarily function to direct the 2′-O-methylation of specific nucleotides by base-pairing with substrate RNAs. In archaea, a bipartite C/D RNA assembles with L7Ae, Nop5, and the methyltransferase fibrillarin into a modification enzyme with unique substrate specificity. Here, we determined the crystal structure of an archaeal C/D RNA–protein complex (RNP) composed of all 3 core proteins and an engineered half-guide RNA at 4 Å resolution, as well as 2 protein substructures at higher resolution. The RNP structure reveals that the C-terminal domains of Nop5 in the dimeric complex provide symmetric anchoring sites for 2 L7Ae-associated kink-turn motifs of the C/D RNA. A prominent protrusion in Nop5 seems to be important for guide RNA organization and function and for discriminating the structurally related U4 snRNA. Multiple conformations of the N-terminal domain of Nop5 and its associated fibrillarin in different structures indicate the inherent flexibility of the catalytic module, suggesting that a swinging motion of the catalytic module is part of the enzyme mechanism. We also built a model of a native C/D RNP with substrate and fibrillarin in an active conformation. Our results provide insight into the overall organization and mechanism of action of C/D RNA–guided RNA methyltransferases. PMID:19666563

  3. Immunosuppressive Yersinia Effector YopM Binds DEAD Box Helicase DDX3 to Control Ribosomal S6 Kinase in the Nucleus of Host Cells

    PubMed Central

    Rumm, Andreas; Trasak, Claudia; Ruckdeschel, Klaus; Alawi, Malik; Grundhoff, Adam; Kikhney, Alexey G.; Koch-Nolte, Friedrich; Buck, Friedrich; Perbandt, Markus; Betzel, Christian; Svergun, Dmitri I.; Hentschke, Moritz; Aepfelbacher, Martin

    2016-01-01

    Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines. PMID:27300509

  4. APOBEC3 inhibits DEAD-END function to regulate microRNA activity

    PubMed Central

    2013-01-01

    The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3′-untranslated region (3′-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3′-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3′-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3−/− mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function. PMID:23890083

  5. APOBEC3 inhibits DEAD-END function to regulate microRNA activity.

    PubMed

    Ali, Sara; Karki, Namrata; Bhattacharya, Chitralekha; Zhu, Rui; MacDuff, Donna A; Stenglein, Mark D; Schumacher, April J; Demorest, Zachary L; Harris, Reuben S; Matin, Angabin; Aggarwal, Sita

    2013-01-01

    The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3'-untranslated region (3'-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3'-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3'-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3-/- mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function. PMID:23890083

  6. The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

    PubMed Central

    Granneman, Sander; Zhu, Jieyi; Gill, Michael; Papoulas, Ophelia; Marcotte, Edward M.; Tollervey, David; Correll, Carl C.; Johnson, Arlen W.

    2015-01-01

    In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins. PMID:25710520

  7. The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.

    PubMed

    Sardana, Richa; Liu, Xin; Granneman, Sander; Zhu, Jieyi; Gill, Michael; Papoulas, Ophelia; Marcotte, Edward M; Tollervey, David; Correll, Carl C; Johnson, Arlen W

    2015-02-01

    In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins. PMID:25710520

  8. Single-molecule studies reveal that DEAD box protein DDX1 promotes oligomerization of HIV-1 Rev on the Rev response element.

    PubMed

    Robertson-Anderson, Rae M; Wang, Jun; Edgcomb, Stephen P; Carmel, Andrew B; Williamson, James R; Millar, David P

    2011-07-29

    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. PMID:21763499

  9. Factors that influence T box riboswitch efficacy and tRNA affinity.

    PubMed

    Zeng, C; Zhou, S; Bergmeier, S C; Hines, J V

    2015-09-01

    The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors. PMID:26220520

  10. Importance and Determinants of Induction of Cold-Induced DEAD RNA Helicase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

    PubMed Central

    Nagaoka, Eriko; Hidese, Ryota; Imanaka, Tadayuki

    2013-01-01

    Thermococcus kodakarensis, which grows optimally at 85°C, expresses cold stress-inducible DEAD box RNA helicase (Tk-deaD) when shifted to 60°C. A DDA1 deletion (ΔTk-deaD) mutant exhibited decreased cell growth, and cells underwent lysis at 60°C in nutrient broth in the absence of elemental sulfur. In contrast, cells in medium containing elemental sulfur at 60°C did not undergo lysis, suggesting that Tk-deaD is necessary for cell growth in sulfur-free medium. To identify the element responsible for the cold response, a pTKR expression probe plasmid was constructed using thermostable catalase from Pyrobaculum calidifontis as a reporter. The plasmid pTKRD, which contained the transcription factor B recognition element, TATA region, and Shine-Dalgarno (SD) region, including the initiation codon of the Tk-deaD gene, exhibited cold inducibility. We also constructed a series of deletion and chimeric constructs with the glutamate dehydrogenase (gdh) promoter, whose expression is constitutive independent of culture temperatures and catalase expression. Reporter assay experiments indicated that the regulatory element is located in the region between the SD region and the initiation codon (ATG). Nucleotide sequences in the upstream regions of Tk-deaD and gdh were compared and revealed a five-adenosine (AAAAA) sequence between SD and ATG of Tk-deaD that was not present in gdh. Replacement of the repeated adenosine sequence with other sequences revealed that the AAAAA sequence is important for cold induction. This sequence-specific mechanism is unique and is one that has not been identified in other known cold-inducible genes. PMID:23729644

  11. Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues.

    PubMed

    Filippova, J A; Stepanov, G A; Semenov, D V; Koval, O A; Kuligina, E V; Rabinov, I V; Richter, V A

    2015-01-01

    Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans. PMID:26085946

  12. Acquired Dependence Of Acute Myeloid Leukemia On The DEAD-BOX RNA Helicase DDX5

    PubMed Central

    Mazurek, Anthony; Park, Youngkyu; Miething, Cornelius; Wilkinson, John E.; Gillis, Jesse; Lowe, Scott W.; Vakoc, Christopher R.; Stillman, Bruce

    2014-01-01

    SUMMARY Acute myeloid leukemia (AML) therapy involves compounds that are cytotoxic to both normal and cancer cells and relapsed AML is resistant to subsequent chemotherapy. Thus agents are needed that selectively kill AML cells with minimal toxicity. Here we report that AML is dependent on DDX5 and that inhibiting DDX5 expression slows AML cell proliferation in vitro and AML progression in vivo, but is not toxic to cells from normal bone marrow. Inhibition of DDX5 expression in AML cells induces apoptosis via induction of reactive oxygen species (ROS). This apoptotic response can be blocked either by BCL2 overexpression or treatment with the ROS scavenger N-Acetyl-L-cysteine (NAC). Combining DDX5 knockdown with a BCL2 family inhibitor cooperate to induce cell death in AML cells. By inhibiting DDX5 expression in vivo we show that DDX5 is dispensable for normal hematopoiesis and tissue homeostasis. These results validate DDX5 as a potential target for blocking AML. PMID:24910429

  13. Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites.

    PubMed

    Semlow, Daniel R; Blanco, Mario R; Walter, Nils G; Staley, Jonathan P

    2016-02-25

    During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3' splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3' to 5' translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome. PMID:26919433

  14. The T box mechanism: tRNA as a regulatory molecule

    PubMed Central

    Green, Nicholas J.; Grundy, Frank J.; Henkin, Tina M.

    2009-01-01

    The T box mechanism is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase genes and genes involved in amino acid biosynthesis and uptake. Binding of a specific uncharged tRNA to a riboswitch element in the nascent transcript causes a structural change in the transcript that promotes expression of the downstream coding sequence. In most cases, this occurs by stabilization of an antiterminator element that competes with formation of a terminator helix. Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA. PMID:19932103

  15. Identification of neomycin B-binding site in T box antiterminator model RNA.

    PubMed

    Anupam, Rajaneesh; Denapoli, Leyna; Muchenditsi, Abigael; Hines, Jennifer V

    2008-04-15

    The T box transcription antitermination mechanism regulates the expression of unique genes in many Gram-positive bacteria by responding, in a magnesium-dependent manner, to uncharged cognate tRNA base pairing with an antiterminator RNA element and other regions of the 5'-untranslated region. Model T box antiterminator RNA is known to bind aminoglycosides, ligands that typically bind RNA in divalent metal ion-binding sites. In this study, enzymatic footprinting and spectroscopic assays were used to identify and characterize the binding site of neomycin B to an antiterminator model RNA. Neomycin B binds the antiterminator bulge nucleotides in an electrostatic-dependent manner and displaces 3-4 monovalent cations, indicating that the antiterminator likely contains a divalent metal ion-binding site. Neomycin B facilitates rather than inhibits tRNA binding indicating that bulge-targeted inhibitors that bind the antiterminator via non-electrostatic interactions may be the more optimal candidates for antiterminator-targeted ligand design. PMID:18329274

  16. RGG boxes within the TET/FET family of RNA-binding proteins are functionally distinct.

    PubMed

    Chau, Bess Ling; Ng, King Pan; Li, Kim K C; Lee, Kevin A W

    2016-08-01

    The multi-functional TET (TAF15/EWS/TLS) or FET (FUS/EWS/TLS) protein family of higher organisms harbor a transcriptional-activation domain (EAD) and an RNA-binding domain (RBD). The transcriptional activation function is, however, only revealed in oncogenic TET-fusion proteins because in native TET proteins it is auto-repressed by RGG-boxes within the TET RBD. Auto-repression is suggested to involve direct cation-pi interactions between multiple Arg residues within RGG boxes and EAD aromatics. Via analysis of TET transcriptional activity in different organisms, we report herein that repression is not autonomous but instead requires additional trans-acting factors. This finding is not supportive of a proposed model whereby repression occurs via a simple intramolecular EAD/RGG-box interaction. We also show that RGG-boxes present within reiterated YGGDRGG repeats that are unique to TAF15, are defective for repression due to the conserved Asp residue. Thus, RGG boxes within TET proteins can be functionally distinguished. While our results show that YGGDRGG repeats are not involved in TAF15 auto-repression, their remarkable number and conservation strongly suggest that they may confer specialized properties to TAF15 and thus contribute to functional differentiation within the TET/FET protein family. PMID:27159574

  17. CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases.

    PubMed Central

    Yoshinari, S; Nagy, P D; Simon, A E; Dreher, T W

    2000-01-01

    It has previously been observed that the only specific requirement for transcriptional initiation on viral RNA in vitro by the RNA-dependent RNA polymerase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdRp, and Qbeta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multiple CCA boxes but lacking specific viral sequences. Each enzyme is able to initiate transcription from several CCA boxes within these RNAs, and no special reaction conditions are required for these activities. The transcriptional yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redundant sequences presents a challenge to the specificity of viral transcription and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit initiation among potential CCA initiation sites. These two specificity mechanisms are used to different degrees by the three RdRps. The finding that three viral RdRps representing two of the three supergroups within the positive-strand RNA viral RdRp phylogeny support substantial transcription in the absence of unique promoters suggests that this phenomenon may be common among positive-strand viruses. A framework is presented arguing that replication of viral RNA in the absence of unique promoter elements is feasible. PMID:10836791

  18. A Cajal body-specific pseudouridylation guide RNA is composed of two box H/ACA snoRNA-like domains

    PubMed Central

    Kiss, Arnold M.; Jády, Beáta E.; Darzacq, Xavier; Verheggen, Céline; Bertrand, Edouard; Kiss, Tamás

    2002-01-01

    Site-specific post-transcriptional conversion of uridines to pseudouridine in ribosomal RNAs and small nuclear RNAs (snRNAs) is directed by guide RNAs which possess the conserved box H and ACA sequence elements and fold into the consensus ‘hairpin–hinge–hairpin–tail’ secondary structure. Here, we describe an unusual mammalian pseudouridylation guide RNA, called U93, that is composed of two tandemly arranged box H/ACA RNA domains. The U93 RNA therefore carries two H and two ACA box motifs, all of which are essential for accumulation of the full-length RNA. The human U93 RNA accumulates in Cajal (coiled) bodies and it is predicted to function in pseudouridylation of the U2 spliceosomal snRNA. Our results lend further support to the notion that modification of the RNA polymerase II-transcribed spliceosomal snRNAs takes place in Cajal bodies. PMID:12409454

  19. The structure of the box C/D enzyme reveals regulation of RNA methylation.

    PubMed

    Lapinaite, Audrone; Simon, Bernd; Skjaerven, Lars; Rakwalska-Bange, Magdalena; Gabel, Frank; Carlomagno, Teresa

    2013-10-24

    Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2'-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding. PMID:24121435

  20. Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing

    PubMed Central

    van Nues, Robert Willem; Granneman, Sander; Kudla, Grzegorz; Sloan, Katherine Elizabeth; Chicken, Matthew; Tollervey, David; Watkins, Nicholas James

    2011-01-01

    2′-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C′/D′ motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This ‘extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA–protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C′/D′ motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA–rRNA interactions required for efficient methylation and the structural organization of the snoRNPs. PMID:21556049

  1. A Unique HMG-Box Domain of Mouse Maelstrom Binds Structured RNA but Not Double Stranded DNA

    PubMed Central

    Genzor, Pavol; Bortvin, Alex

    2015-01-01

    Piwi-interacting piRNAs are a major and essential class of small RNAs in the animal germ cells with a prominent role in transposon control. Efficient piRNA biogenesis and function require a cohort of proteins conserved throughout the animal kingdom. Here we studied Maelstrom (MAEL), which is essential for piRNA biogenesis and germ cell differentiation in flies and mice. MAEL contains a high mobility group (HMG)-box domain and a Maelstrom-specific domain with a presumptive RNase H-fold. We employed a combination of sequence analyses, structural and biochemical approaches to evaluate and compare nucleic acid binding of mouse MAEL HMG-box to that of canonical HMG-box domain proteins (SRY and HMGB1a). MAEL HMG-box failed to bind double-stranded (ds)DNA but bound to structured RNA. We also identified important roles of a novel cluster of arginine residues in MAEL HMG-box in these interactions. Cumulatively, our results suggest that the MAEL HMG-box domain may contribute to MAEL function in selective processing of retrotransposon RNA into piRNAs. In this regard, a cellular role of MAEL HMG-box domain is reminiscent of that of HMGB1 as a sentinel of immunogenic nucleic acids in the innate immune response. PMID:25807393

  2. Structural features of the guide:target RNA duplex required for archaeal box C/D sRNA-guided nucleotide 2′-O-methylation

    PubMed Central

    Appel, C. Denise; Maxwell, E. Stuart

    2007-01-01

    Archaeal box C/D sRNAs guide the 2′-O-methylation of target nucleotides using both terminal box C/D and internal C′/D′ RNP complexes. In vitro assembly of a catalytically active Methanocaldococcus jannaschii sR8 box C/D RNP provides a model complex to determine those structural features of the guide:target RNA duplex important for sRNA-guided nucleotide methylation. Watson–Crick pairing of guide and target nucleotides was found to be essential for methylation, and mismatched bases within the guide:target RNA duplex also disrupted nucleotide modification. However, dependence upon Watson–Crick base-paired guide:target nucleotides for methylation was compromised in elevated Mg2+ concentrations where mismatched target nucleotides were modified. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA duplex as a target ribonucleotide within a guide RNA:target DNA duplex that was not methylated. Interestingly, D and D′ target RNAs exhibited different levels of methylation when deoxynucleotides were inserted into the target RNA or when target methylation was carried out in elevated Mg2+ concentrations. These observations suggested that unique structural features of the box C/D and C′/D′ RNPs differentially affect their respective methylation capabilities. The ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested that the sRNP can facilitate unwinding of double-stranded target RNAs. Finally, increasing target RNA length to extend beyond those nucleotides that base pair with the sRNA guide sequence significantly increased sRNP turnover and thus nucleotide methylation. This suggests that target RNA interaction with the sRNP core proteins is also important for box C/D sRNP-guided nucleotide methylation. PMID:17438123

  3. Structural features of the guide:target RNA duplex required for archaeal box C/D sRNA-guided nucleotide 2'-O-methylation.

    PubMed

    Appel, C Denise; Maxwell, E Stuart

    2007-06-01

    Archaeal box C/D sRNAs guide the 2'-O-methylation of target nucleotides using both terminal box C/D and internal C'/D' RNP complexes. In vitro assembly of a catalytically active Methanocaldococcus jannaschii sR8 box C/D RNP provides a model complex to determine those structural features of the guide:target RNA duplex important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was found to be essential for methylation, and mismatched bases within the guide:target RNA duplex also disrupted nucleotide modification. However, dependence upon Watson-Crick base-paired guide:target nucleotides for methylation was compromised in elevated Mg(2+) concentrations where mismatched target nucleotides were modified. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA duplex as a target ribonucleotide within a guide RNA:target DNA duplex that was not methylated. Interestingly, D and D' target RNAs exhibited different levels of methylation when deoxynucleotides were inserted into the target RNA or when target methylation was carried out in elevated Mg(2+) concentrations. These observations suggested that unique structural features of the box C/D and C'/D' RNPs differentially affect their respective methylation capabilities. The ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested that the sRNP can facilitate unwinding of double-stranded target RNAs. Finally, increasing target RNA length to extend beyond those nucleotides that base pair with the sRNA guide sequence significantly increased sRNP turnover and thus nucleotide methylation. This suggests that target RNA interaction with the sRNP core proteins is also important for box C/D sRNP-guided nucleotide methylation. PMID:17438123

  4. Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing ▿

    PubMed Central

    Nayak, Bina S.; Badgley, Brian; Harwood, Valerie J.

    2011-01-01

    Environmental Enterococcus spp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups. PMID:21622792

  5. Overexpression of an Apocynum venetum DEAD-Box Helicase Gene (AvDH1) in Cotton Confers Salinity Tolerance and Increases Yield in a Saline Field

    PubMed Central

    Chen, Jie; Wan, Sibao; Liu, Huaihua; Fan, Shuli; Zhang, Yujuan; Wang, Wei; Xia, Minxuan; Yuan, Rui; Deng, Fenni; Shen, Fafu

    2016-01-01

    Soil salinity is a major environmental stress limiting plant growth and productivity. We have reported previously the isolation of an Apocynum venetum DEAD-box helicase 1 (AvDH1) that is expressed in response to salt exposure. Here, we report that the overexpression of AvDH1 driven by a constitutive cauliflower mosaic virus-35S promoter in cotton plants confers salinity tolerance. Southern and Northern blotting analyses showed that the AvDH1 gene was integrated into the cotton genome and expressed. In this study, the growth of transgenic cotton expressing AvDH1 was evaluated under saline conditions in a growth chamber and in a saline field trial. Transgenic cotton overexpressing AvDH1 was much more resistant to salt than the wild-type plants when grown in a growth chamber. The lower membrane ion leakage, along with increased activity of superoxide dismutase, in AvDH1 transgenic lines suggested that these characteristics may prevent membrane damage, which increases plant survival rates. In a saline field, the transgenic cotton lines expressing AvDH1 showed increased boll numbers, boll weights and seed cotton yields compared with wild-type plants, especially at high soil salinity levels. This study indicates that transgenic cotton expressing AvDH1 is a promising option for increasing crop productivity in saline fields. PMID:26779246

  6. Alternative Forms of Y-Box Binding Protein 1 and YB-1 mRNA

    PubMed Central

    Lyabin, Dmitry N.; Doronin, Alexander N.; Eliseeva, Irina A.; Guens, Gelena P.; Kulakovskiy, Ivan V.; Ovchinnikov, Lev P.

    2014-01-01

    The multifunctional eukaryotic protein YB-1 (Y-box binding protein 1) plays a role in DNA reparation, transcription regulation, splicing, and mRNA translation, thereby participating in many crucial events in cells. Its effect is dependent mostly on its amount, and hence, on regulation of its synthesis. Published data on regulation of synthesis of YB-1 mediated by its mRNA 5′ UTR, and specifically on the 5′ UTR length and the presence of TOP-like motifs in this region, are contradictory. Here we report that 5′ UTRs of major forms of human, rabbit, and mouse YB-1 mRNAs are about 140 nucleotides long and contain no TOP-like motifs mentioned in the literature. Also, we have found that YB-1 specifically interacts with the 5′ UTR of its own mRNA within a region of about 100 nucleotides upstream from the start codon. Apart from YB-1, translation of YB-1 mRNA in a cell free system gives an additional product with an extended N-terminus and lower electrophoretic mobility. The start codon for synthesis of the additional product is AUC at position –(60–58) of the same open reading frame as that for the major product. Also, in the cell there is an alternative YB-1 mRNA with exon 1 replaced by a part of intron 1; YB-1 synthesized in vitro from this mRNA contains, instead of its N-terminal A/P domain, 10–11 amino acids encoded by intron 1. PMID:25116735

  7. Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers

    PubMed Central

    Zhang, Jinwei; Ferré-DAmaré, Adrian R.

    2016-01-01

    Length determination is a fundamental problem in biology and chemistry. Numerous proteins measure distances on linear biopolymers to exert effects with remarkable spatial precision. Recently, ruler-like devices made of noncoding RNAs have been structurally and biochemically characterized. Two prominent examples are the RNase P ribozyme and the T-box riboswitch. Both act as molecular calipers. The two RNAs clamp onto the elbow of tRNA (or pre-tRNA) and make distance measurements orthogonal to each other. Here, we compare and contrast the molecular ruler characteristics of these RNAs. RNase P appears pre-configured to measure a fixed distance on pre-tRNA to ensure the fidelity of its maturation. RNase P is a multiple-turnover ribozyme, and its rigid structure efficiently selects pre-tRNAs, cleaves, and releases them. In contrast, the T-box is flexible and segmented, an architecture that adapts to the intrinsically flexible tRNA. The tripartite T-box inspects the overall shape, anticodon sequence, and aminoacylation status of an incoming tRNA while it folds co-transcriptionally, leading to a singular, conditional genetic switching event. The elucidation of the structures and mechanisms of action of these two RNA molecular rulers may augur the discovery of new RNA measuring devices in noncoding and viral transcriptomes, and inform the design of artificial RNA rulers. PMID:27043647

  8. Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers.

    PubMed

    Zhang, Jinwei; Ferré-DAmaré, Adrian R

    2016-01-01

    Length determination is a fundamental problem in biology and chemistry. Numerous proteins measure distances on linear biopolymers to exert effects with remarkable spatial precision. Recently, ruler-like devices made of noncoding RNAs have been structurally and biochemically characterized. Two prominent examples are the RNase P ribozyme and the T-box riboswitch. Both act as molecular calipers. The two RNAs clamp onto the elbow of tRNA (or pre-tRNA) and make distance measurements orthogonal to each other. Here, we compare and contrast the molecular ruler characteristics of these RNAs. RNase P appears pre-configured to measure a fixed distance on pre-tRNA to ensure the fidelity of its maturation. RNase P is a multiple-turnover ribozyme, and its rigid structure efficiently selects pre-tRNAs, cleaves, and releases them. In contrast, the T-box is flexible and segmented, an architecture that adapts to the intrinsically flexible tRNA. The tripartite T-box inspects the overall shape, anticodon sequence, and aminoacylation status of an incoming tRNA while it folds co-transcriptionally, leading to a singular, conditional genetic switching event. The elucidation of the structures and mechanisms of action of these two RNA molecular rulers may augur the discovery of new RNA measuring devices in noncoding and viral transcriptomes, and inform the design of artificial RNA rulers. PMID:27043647

  9. Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing.

    PubMed

    Falaleeva, Marina; Pages, Amadis; Matuszek, Zaneta; Hidmi, Sana; Agranat-Tamir, Lily; Korotkov, Konstantin; Nevo, Yuval; Eyras, Eduardo; Sperling, Ruth; Stamm, Stefan

    2016-03-22

    C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2'-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex,SNORD27 regulates the alternative splicing of the transcription factor E2F7p re-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes. PMID:26957605

  10. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    PubMed

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product. PMID:26925607

  11. Conservation and divergence of transcriptional coregulations between box C/D snoRNA and ribosomal protein genes in Ascomycota

    PubMed Central

    Diao, Li-Ting; Xiao, Zhen-Dong; Leng, Xiao-Min; Li, Bin; Li, Jun-Hao; Luo, Yu-Ping; Li, Si-Guang; Yu, Chuan-He; Zhou, Hui

    2014-01-01

    Coordinated assembly of the ribosome is essential for proper translational activity in eukaryotic cells. It is therefore critical to coordinate the expression of components of ribosomal programs with the cell's nutritional status. However, coordinating expression of these components is poorly understood. Here, by combining experimental and computational approaches, we systematically identified box C/D snoRNAs in four fission yeasts and found that the expression of box C/D snoRNA and ribosomal protein (RP) genes were orchestrated by a common Homol-D box, thereby ensuring a constant balance of these two genetic components. Interestingly, such transcriptional coregulations could be observed in most Ascomycota species and were mediated by different cis-regulatory elements. Via the reservation of cis elements, changes in spatial configuration, the substitution of cis elements, and gain or loss of cis elements, the regulatory networks of box C/D snoRNAs evolved to correspond with those of the RP genes, maintaining transcriptional coregulation between box C/D snoRNAs and RP genes. Our results indicate that coregulation via common cis elements is an important mechanism to coordinate expression of the RP and snoRNA genes, which ensures a constant balance of these two components. PMID:25002674

  12. S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA

    PubMed Central

    Fuchs, Ryan T.; Grundy, Frank J.; Henkin, Tina M.

    2007-01-01

    The SMK box is a conserved riboswitch motif found in the 5′ untranslated region of metK genes [encoding S-adenosylmethionine (SAM) synthetase] in lactic acid bacteria, including Enterococcus, Streptococcus, and Lactococcus sp. Previous studies showed that this RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine–Dalgarno (SD) sequence by pairing with an anti-SD (ASD) element. A model was proposed in which SAM binding inhibits metK translation by preventing binding of the ribosome to the SD region of the mRNA. In the current work, the addition of SAM was shown to inhibit binding of 30S ribosomal subunits to SMK box RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA, which has a disrupted SD-ASD pairing, was defective in SAM binding and showed no reduction of ribosome binding in the presence of SAM, whereas a compensatory mutation that restored SD-ASD pairing restored the response to SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that SMK box translational repression operates through occlusion of the ribosome binding site and that SAM binding requires the SD-ASD pairing. PMID:17360376

  13. T box transcription antitermination riboswitch: Influence of nucleotide sequence and orientation on tRNA binding by the antiterminator element

    PubMed Central

    Fauzi, Hamid; Agyeman, Akwasi; Hines, Jennifer V.

    2008-01-01

    Many bacteria utilize riboswitch transcription regulation to monitor and appropriately respond to cellular levels of important metabolites or effector molecules. The T box transcription antitermination riboswitch responds to cognate uncharged tRNA by specifically stabilizing an antiterminator element in the 5′-untranslated mRNA leader region and precluding formation of a thermodynamically more stable terminator element. Stabilization occurs when the tRNA acceptor end base pairs with the first four nucleotides in the seven nucleotide bulge of the highly conserved antiterminator element. The significance of the conservation of the antiterminator bulge nucleotides that do not base pair with the tRNA is unknown, but they are required for optimal function. In vitro selection was used to determine if the isolated antiterminator bulge context alone dictates the mode in which the tRNA acceptor end binds the bulge nucleotides. No sequence conservation beyond complementarity was observed and the location was not constrained to the first four bases of the bulge. The results indicate that formation of a structure that recognizes the tRNA acceptor end in isolation is not the determinant driving force for the high phylogenetic sequence conservation observed within the antiterminator bulge. Additional factors or T box leader features more likely influenced the phylogenetic sequence conservation. PMID:19152843

  14. Transcripts that associate with the RNA binding protein, DEAD-END (DND1), in embryonic stem (ES) cells

    PubMed Central

    2011-01-01

    Background The RNA binding protein, DEAD END (DND1), is essential for maintaining viable germ cells in vertebrates. It is also a testicular germ cell tumor susceptibility factor in mice. DND1 has been shown to interact with the 3'-untranslated region (3'-UTR) of mRNAs such as P27 and LATS2. Binding of DND1 to the 3'-UTRs of these transcripts blocks the inhibitory function of microRNAs (miRNA) from these transcripts and in this way DND1 helps maintain P27 and LATS2 protein expression. We found that DND1 is also expressed in embryonic stem (ES) cells. Because ES cells share similar gene expression patterns as germ cells, we utilized ES cells to identify additional candidate mRNAs that associate with DND1. Results ES cells are readily amenable to genetic modification and easier to culture in vitro compared to germ cells. Therefore, for the purpose of our study, we made a genetically modified, stable, human embryonic stem (hES) cell line that expresses hemagluttinin (HA)-tagged DND1 in a doxycycline (dox) regulatable manner. This line expresses modest levels of HA-DND1 and serves as a good system to study DND1 function in vitro. We used this stable cell line to identify the transcripts that physically interact with DND1. By performing ribonucleoprotein immunoprecipitation (RIP) followed by RT-PCR, we identified that transcripts encoding pluripotency factors (OCT4, SOX2, NANOG, LIN28), cell cycle regulators (TP53, LATS2) and apoptotic factors (BCLX, BAX) are specifically associated with the HA-DND1 ribonucleoprotein complex. Surprisingly, in many cases, bioinformatics analysis of the pulled-down transcripts did not reveal the presence of known DND1 interacting motifs. Conclusions Our results indicate that the inducible ES cell line system serves as a suitable in vitro system to identify the mRNA targets of DND1. The RIP-RT results hint at the broad spectrum of mRNA targets that interact with DND1 in ES cells. Based on what is known about DND1 function, our results

  15. The BAT1 gene in the MHC encodes an evolutionarily conserved putative nuclear RNA helicase of the DEAD family

    SciTech Connect

    Peelman, L.J.; Van Zeveren, A.; Coppeiters, W.

    1995-03-20

    The BAT1 gene has previously been identified about 30 kb upstream from the tumor necrosis factor (TNF) locus and close to a NF{sub kb}-related gene of the nuclear factor family in the major histocompatibility complex (MHC) of human, mouse, and pig. We now show that the BAT1 translation product is the homolog of the rat p47 nuclear protein, the WM6 Drosophila gene product, and probably also Ce08102 of Caenorhabditis elegans, all members of the DEAD protein family of ATP-dependent RNA helicases. This family has more than 40 members, including the eukaryotic translation initiation factor-4A (eIF-4A), the human nuclear protein p68, and the Drosophila oocyte polar granule component vasa. BAT1 spans about 10 kb, is split into 10 exons of varying length, and encodes a protein of 428 amino acids ({approximately}48 kDa). Human and pig BAT1 cDNAs display 95.6% identity in the coding region and 80% identity in the 5{prime} and 3{prime} noncoding regions. Several repeat sequences of different types were identified in introns of the porcine BAT1 gene. Three different mRNAs, 4.1,1.7, and 0.9 kb, respectively, were detected in all tissues analyzed upon hybridization with porcine BAT1 cDNA. Transfection and expression of human BAT1 cDNA after tagging with a heterologous antibody recognition epitope revealed a nuclear localization of the hybrid protein. An MspI RFLP was detected in an SLA class I typed family, confirming the localization of the BAT1 gene in the porcine MHC. BAT1 thus encodes a putative nuclear ATP-dependent RNA helicase and is likely to have an indispensable function. 35 refs., 6 figs., 1 tab.

  16. DExD-box RNA-helicases in Listeria monocytogenes are important for growth, ribosomal maturation, rRNA processing and virulence factor expression

    PubMed Central

    Bäreclev, Caroline; Vaitkevicius, Karolis; Netterling, Sakura; Johansson, Jörgen

    2014-01-01

    RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37°C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26°C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20°C, whereas the effect was marginal at 37°C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells. PMID:25590644

  17. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  18. Efficient RNA 2′-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C′/D′ RNPs

    PubMed Central

    Tran, Elizabeth J.; Zhang, Xinxin; Maxwell, E.Stuart

    2003-01-01

    Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2′-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C′/D′ RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C′/D′ motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2′-O-methylation requires that both the box C/D and C′/D′ RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C′/D′ motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles. PMID:12881427

  19. Efficient RNA 2'-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C'/D' RNPs.

    PubMed

    Tran, Elizabeth J; Zhang, Xinxin; Maxwell, E Stuart

    2003-08-01

    Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2'-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C'/D' RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C'/D' motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2'-O-methylation requires that both the box C/D and C'/D' RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C'/D' motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles. PMID:12881427

  20. Assembly into snoRNP controls 5'-end maturation of a box C/D snoRNA in Saccharomyces cerevisiae

    SciTech Connect

    Preti, Milena; Guffanti, Elisa; Valitutto, Eleonora; Dieci, Giorgio . E-mail: giorgio.dieci@unipr.it

    2006-12-15

    The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymerase (Pol) III in Saccharomyces cerevisiae. Pol III transcription generates a precisely terminated primary transcript that undergoes extensive 5'-end processing. Here, we show that mutations of the box C/D core motif required for snoRNP assembly compromise 5'-end maturation of the SNR52 snoRNA. Upstream processing was also impaired by specific depletion of either Nop1p or Nop58p snoRNP proteins. We further show that the nuclear exosome is required for 3'-end maturation of SNR52 snoRNA, at variance with all the other known Pol III transcripts. Our data suggest a functional coupling between snoRNP assembly and 5'-end maturation of independently transcribed box C/D snoRNAs.

  1. Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal.

    PubMed

    Jády, Beáta E; Bertrand, Edouard; Kiss, Tamás

    2004-03-01

    Telomerase is a ribonucleoprotein reverse transcriptase that uses its RNA component as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes. Here, fluorescence in situ hybridization demonstrates that in HeLa cancer cells, human telomerase RNA (hTR) accumulates in the nucleoplasmic Cajal bodies (CBs). Localization of transiently expressed hTR to CBs is supported by a short sequence motif (411-UGAG-414) that is located in the 3'-terminal box H/ACA RNA-like domain of hTR and that is structurally and functionally indistinguishable from the CB-specific localization signal of box H/ACA small CB-specific RNAs. In synchronized HeLa cells, hTR shows the most efficient accumulation in CBs during S phase, when telomeres are most likely synthesized. CBs may function in post-transcriptional maturation (e.g., cap hypermethylation of hTR), but they may also play a role in the assembly and/or function of telomerase holoenzyme. PMID:14981093

  2. Transcription of the Xenopus laevis selenocysteine tRNA(Ser)Sec gene: a system that combines an internal B box and upstream elements also found in U6 snRNA genes.

    PubMed Central

    Carbon, P; Krol, A

    1991-01-01

    The transcription mode of the Xenopus tRNA(Ser)Sec gene by RNA polymerase III was deciphered by injection of mutant templates into Xenopus oocyte nuclei. tRNA(Ser)Sec represents the paradigm of a new class of RNA polymerase III genes combining tRNA and U snRNA gene regulatory elements. Its promoter is tripartite, constituted by two upstream elements, a PSE and a TATA motif that are interchangeable with those of U6 snRNA genes and an internal box B as in other tRNAs. The B box enables the transcription level dependent on the upstream promoter to be increased. Data obtained indicate that U1 snRNA (Pol II) and tRNA(Ser)Sec (Pol III) genes share at least one transcription factor, implying that the border between transcription systems is less tight than expected. Images PMID:2001675

  3. Dissecting the role of conserved box C/D sRNA sequences in di-sRNP assembly and function

    PubMed Central

    Bleichert, Franziska; Baserga, Susan J.

    2010-01-01

    In all three kingdoms of life, nucleotides in ribosomal RNA (rRNA) are post-transcriptionally modified. One type of chemical modification is 2′-O-ribose methylation, which is, in eukaryotes and archaea, performed by box C/D small ribonucleoproteins (box C/D sRNPs in archaea) and box C/D small nucleolar ribonucleoproteins (box C/D snoRNPs in eukaryotes), respectively. Recently, the first structure of any catalytically active box C/D s(no)RNP determined by electron microscopy and single particle analysis surprisingly demonstrated that they are dimeric RNPs. Mutational analyses of the Nop5 protein interface suggested that di-sRNP formation is also required for the in vitro catalytic activity. We have now analyzed the functional relevance of the second interface, the sRNA interface, within the box C/D di-sRNP. Mutations in conserved sequence elements of the sRNA, which allow sRNP assembly but which severely interfere with the catalytic activity of box C/D sRNPs, prevent formation of the di-sRNP. In addition, we can observe the dimeric box C/D sRNP architecture with a different box C/D sRNP, suggesting that this architecture is conserved. Together, these results provide further support for the functional relevance of the di-sRNP architecture and also provide a structural explanation for the observed defects in catalysis of 2′-O-ribose methylation. PMID:20693534

  4. The DEAH-box helicase DHX36 mediates dendritic localization of the neuronal precursor-microRNA-134

    PubMed Central

    Bicker, Silvia; Khudayberdiev, Sharof; Weiß, Kerstin; Zocher, Kathleen; Baumeister, Stefan; Schratt, Gerhard

    2013-01-01

    Specific microRNAs (miRNAs), including miR-134, localize to neuronal dendrites, where they control synaptic protein synthesis and plasticity. However, the mechanism of miRNA transport is unknown. We found that the neuronal precursor-miRNA-134 (pre-miR-134) accumulates in dendrites of hippocampal neurons and at synapses in vivo. Dendritic localization of pre-miR-134 is mediated by the DEAH-box helicase DHX36, which directly associates with the pre-miR-134 terminal loop. DHX36 function is required for miR-134-dependent inhibition of target gene expression and the control of dendritic spine size. Dendritically localized pre-miR-134 could provide a local source of miR-134 that can be mobilized in an activity-dependent manner during plasticity. PMID:23651854

  5. Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling.

    PubMed

    Gagnon, Keith T; Zhang, Xinxin; Agris, Paul F; Maxwell, E Stuart

    2006-10-01

    Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active

  6. Polymorphisms in MicroRNA Target Sites of Forkhead Box O Genes Are Associated with Hepatocellular Carcinoma

    PubMed Central

    Zeng, Xiaoyun; Yu, Hongping; Li, Anhua; Bei, Chunhua; Qiu, Xiaoqiang

    2015-01-01

    The forkhead box O (FOXO) transcription factors play important roles in various cancer development including Hepatocellular Carcinoma (HCC). In this study we conducted a hospital-based case control study including 1049 cases (HCC patients) and 1052 controls (non-tumor patients) to examine whether single nucleotide polymorphisms (SNPs) within microRNA (miRNA) target sites of FOXO genes confer HCC susceptibility. A total of three miRNA target site SNPs in the 3’ untranslated regions (UTR) of FOXO1 (rs17592236), FOXO3 (rs4946936) and FOXO4 (rs4503258) were analyzed. No statistically significant differences were found in genotype distribution for rs17592236, rs4946936, and rs4503258 between the HCC patient group and the tumor-free control group using single factor chi-square analysis (P>0.05). However, multivariate logistic regression analysis showed that the CT/TT genotype in rs17592236 was significantly associated with decreased risk of HCC development (P = 0.010, OR = 0.699, 95% CI: 0.526–0.927) as compared to the CC genotype in rs17592236. Additionally, a genetic interaction was found between rs17592236 and rs4503258 (P = 0.003, OR = 0.755, 95% CI: 0.628–0.908). Functional dual luciferase reporter assays verified that the rs17592236 SNP was a target site of human miRNA miR-137. Together, these results indicate that the rs17592236 polymorphism is associated with decreasing of HCC hereditary susceptibility likely through modulating the binding affinity of miR-137 to the 3’UTR in FOXO1 messenger RNA (mRNA). Further knowledge obtained from this study may provide important evidence for the prevention and targeted therapy of HCC. PMID:25739100

  7. Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription.

    PubMed Central

    Newman, D R; Kuhn, J F; Shanab, G M; Maxwell, E S

    2000-01-01

    The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) essential for ribosome biogenesis. The box C/D snoRNA family possesses conserved nucleotide boxes C and D that are multifunctional elements required for snoRNA processing, snoRNA transport to the nucleolus, and 2'-O-methylation of ribosomal RNA. We have previously demonstrated that the assembly of an snoRNP complex is essential for processing the intronic box C/D snoRNAs and that specific nuclear proteins associate with the box C/D core motif in vitro. Using a box C/D motif derived from mouse U14 snoRNA, we have now affinity purified and defined four mouse proteins that associate with this minimal RNA substrate. These four proteins consist of two protein pairs: members of each pair are highly related in sequence. One protein pair corresponds to the essential yeast nucleolar proteins Nop56p and Nop58p. Affinity purification of mouse Nop58 confirms observations made in yeast that Nop58 is a core protein of the box C/D snoRNP complex. Isolation of Nop56 using this RNA motif defines an additional snoRNP core protein. The second pair of mouse proteins, designated p50 and p55, are also highly conserved among eukaryotes. Antibody probing of nuclear fractions revealed a predominance of p55 and p50 in the nucleoplasm, suggesting a possible role for the p50/p55 pair in snoRNA production and/or nucleolar transport. The reported interaction of p55 with TATA-binding protein (TBP) and replication A protein as well as the DNA helicase activity of p55 and p50 may suggest the coordination of snoRNA processing and snoRNP assembly with replication and/or transcriptional events in the nucleus. Homologs for both snoRNA-associated protein pairs occur in Archaea, strengthening the hypothesis that the box C/D RNA elements and their interacting proteins are of ancient evolutionary origin. PMID:10864044

  8. A role for the TATA-box-binding protein component of the transcription factor IID complex as a general RNA polymerase III transcription factor.

    PubMed Central

    White, R J; Jackson, S P; Rigby, P W

    1992-01-01

    The major class of vertebrate genes transcribed by RNA polymerase (EC 2.7.7.6) III, which includes 5S rRNA genes, tRNA genes, and the adenovirus VA genes, is characterized by split internal promoters and no absolute dependence upon specific upstream sequences. Fractionation experiments have shown that transcription of such genes requires two general RNA polymerase III-specific factors, TFIIIB and TFIIIC. We now demonstrate that a third general factor is also employed by these genes. This is the TATA-box-binding protein originally identified as being a component of the general RNA polymerase II transcription factor TFIID. This protein is involved in the transcription by RNA polymerase III of every template tested, even though the promoters of VA and most vertebrate tRNA and 5S rRNA genes do not contain recognizable TATA elements. Images PMID:1542692

  9. Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

    PubMed

    Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed; Santiago, Ace George; Chen, Tai-Yen; Jung, Won; Helmann, John D; Chen, Peng

    2015-11-01

    Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription. PMID:26483469

  10. Physical and Functional Interaction between the Methyltransferase Bud23 and the Essential DEAH-Box RNA Helicase Ecm16

    PubMed Central

    Sardana, Richa; Zhu, Jieyi; Gill, Michael

    2014-01-01

    The small ribosomal subunit assembles cotranscriptionally on the nascent primary transcript. Cleavage at site A2 liberates the pre-40S subunit. We previously identified Bud23 as a conserved eukaryotic methyltransferase that is required for efficient cleavage at A2. Here, we report that Bud23 physically and functionally interacts with the DEAH-box RNA helicase Ecm16 (also known as Dhr1). Ecm16 is also required for cleavage at A2. We identified mutations in ECM16 that suppressed the growth and A2 cleavage defects of a bud23Δ mutant. RNA helicases often require protein cofactors to provide substrate specificity. We used yeast (Saccharomyces cerevisiae) two-hybrid analysis to map the binding site of Bud23 on Ecm16. Despite the physical and functional interaction between these factors, mutations that disrupted the interaction, as assayed by two-hybrid analysis, did not display a growth defect. We previously identified mutations in UTP2 and UTP14 that suppressed bud23Δ. We suggest that a network of protein interactions may mask the loss of interaction that we have defined by two-hybrid analysis. A mutation in motif I of Ecm16 that is predicted to impair its ability to hydrolyze ATP led to accumulation of Bud23 in an ∼45S particle containing Ecm16. Thus, Bud23 enters the pre-40S pathway at the time of Ecm16 function. PMID:24710271

  11. Physical and functional interaction between the methyltransferase Bud23 and the essential DEAH-box RNA helicase Ecm16.

    PubMed

    Sardana, Richa; Zhu, Jieyi; Gill, Michael; Johnson, Arlen W

    2014-06-01

    The small ribosomal subunit assembles cotranscriptionally on the nascent primary transcript. Cleavage at site A2 liberates the pre-40S subunit. We previously identified Bud23 as a conserved eukaryotic methyltransferase that is required for efficient cleavage at A2. Here, we report that Bud23 physically and functionally interacts with the DEAH-box RNA helicase Ecm16 (also known as Dhr1). Ecm16 is also required for cleavage at A2. We identified mutations in ECM16 that suppressed the growth and A2 cleavage defects of a bud23Δ mutant. RNA helicases often require protein cofactors to provide substrate specificity. We used yeast (Saccharomyces cerevisiae) two-hybrid analysis to map the binding site of Bud23 on Ecm16. Despite the physical and functional interaction between these factors, mutations that disrupted the interaction, as assayed by two-hybrid analysis, did not display a growth defect. We previously identified mutations in UTP2 and UTP14 that suppressed bud23Δ. We suggest that a network of protein interactions may mask the loss of interaction that we have defined by two-hybrid analysis. A mutation in motif I of Ecm16 that is predicted to impair its ability to hydrolyze ATP led to accumulation of Bud23 in an ∼45S particle containing Ecm16. Thus, Bud23 enters the pre-40S pathway at the time of Ecm16 function. PMID:24710271

  12. Antiapoptotic Effect of Recombinant HMGB1 A-box Protein via Regulation of microRNA-21 in Myocardial Ischemia-Reperfusion Injury Model in Rats.

    PubMed

    Han, Qiang; Zhang, Hua-Yong; Zhong, Bei-Long; Zhang, Bing; Chen, Hua

    2016-04-01

    The ~80 amino acid A box DNA-binding domain of high mobility group box 1 (HMGB1) protein antagonizes proinflammatory responses during myocardial ischemia reperfusion (I/R) injury. The exact role of microRNA-21 (miR-21) is unknown, but its altered levels are evident in I/R injury. This study examined the roles of HMGB1 A-box and miR-21 in rat myocardial I/R injury model. Sixty Sprague-Dawley rats were randomly divided into six equal groups: (1) Sham; (2) I/R; (3) Ischemic postconditioning (IPost); (4) AntagomiR-21 post-treatment; (5) Recombinant HMGB1 A-box pretreatment; and (6) Recombinant HMGB1 A-box + antagomiR-21 post-treatment. Hemodynamic indexes, arrhythmia scores, ischemic area and infarct size, myocardial injury, and related parameters were studied. Expression of miR-21 was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to quantify apoptosis. Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximal rate of pressure rise (+dp/dtmax), and decline (-dp/dtmax) showed clear reduction upon treatment with recombinant HMGB1 A-box. Arrhythmia was relieved and infarct area decreased in the group pretreated with recombinant HMGB1 A-box, compared with other groups. Circulating lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels increased in response to irreversible cellular injury, while creatine kinase MB isoenzymes (CK-MB) and superoxide dismutase (SOD) activities were reduced in the I/R group, which was reversed following recombinant HMGB1 A-box treatment. Interestingly, pretreatment with recombinant HMGB1 A-box showed the most dramatic reductions in miR-21 levels, compared with other groups. Significantly reduced apoptotic index (AI) was seen in recombinant HMGB1 A-box pretreatment group and recombinant HMGB1 A-box + antagomiR-21 post-treatment group, with the former showing a more

  13. Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

    PubMed Central

    Schmid, S R; Linder, P

    1991-01-01

    The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products. Images PMID:2046664

  14. Control of Dead end localization and activity--implications for the function of the protein in antagonizing miRNA function.

    PubMed

    Slanchev, Krasimir; Stebler, Juerg; Goudarzi, Mehdi; Cojocaru, Vlad; Weidinger, Gilbert; Raz, Erez

    2009-01-01

    Dead end (dnd) is a vertebrate-specific component of the germ plasm and germ-cell granules that is crucial for germ-cell development in zebrafish and mouse. Dnd counteracts the inhibitory function of miRNAs, thereby facilitating the expression of proteins such as Nanos and Tdrd7 in the germ cells. Here, we show that cis-acting elements within dnd mRNA and the RNA recognition motive (RRM) of the protein are essential for targeting protein expression to the germ cells and to the perinuclear granules, respectively. We demonstrate that as it executes its function, Dnd translocates between the germ-cell nucleus and germ-cell granules. This phenomenon is not observed in proteins mutated in the RRM motif, correlating with loss of function of Dnd. Based on molecular modeling, we identify the putative RNA binding domain of Dnd as a canonical RRM and propose that this domain is important for protein subcellular localization and function. PMID:19013519

  15. Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences.

    PubMed

    Arahal, D R; Dewhirst, F E; Paster, B J; Volcani, B E; Ventosa, A

    1996-10-01

    Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study. Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions. The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864. Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera--Haloferax, Halobacterium, and Haloarcula. Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum. However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively. Strains E2 and E11 could represent two new species of the genus Haloarcula. However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized. PMID:8837434

  16. Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters*

    PubMed Central

    Verma, Neha; Hung, Ko-Hsuan; Kang, Jin Joo; Barakat, Nermeen H.; Stumph, William E.

    2013-01-01

    In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459–469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription. PMID:23955442

  17. Non-Conserved Residues in Clostridium acetobutylicum tRNAAla Contribute to tRNA Tuning for Efficient Antitermination of the alaS T Box Riboswitch

    PubMed Central

    Liu, Liang-Chun; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination. PMID:26426057

  18. Magical Boxes

    ERIC Educational Resources Information Center

    Costello, Judith

    2005-01-01

    Students get excited when they realize that they can transform a flat sheet of paper into a box. By using different sizes of paper, they can make different sizes of boxes and put a box inside a box, inside a box. These magical boxes within boxes can contain unwanted emotions or special treasures. The project described in this article incorporates…

  19. The bipartite architecture of the sRNA in an archaeal box C/D complex is a primary determinant of specificity

    PubMed Central

    Hardin, John W.; Batey, Robert T.

    2006-01-01

    The archaeal box C/D sRNP, the enzyme responsible for 2′-O-methylation of rRNA and tRNA, possesses a nearly perfect axis of symmetry and bipartite structure. This RNP contains two platforms for the assembly of protein factors, the C/D and C′/D′ motifs, acting in conjunction with two guide sequences to direct methylation of a specific 2′-hydroxyl group in a target RNA. While this suggests that a functional asymmetric single-site complex complete with guide sequence and a single box C/D motif should be possible, previous work has demonstrated such constructs are not viable. To understand the basis for a bipartite RNP, we have designed and assayed the activity and specificity of a series of synthetic RNPs that represent a systematic reduction of the wild-type RNP to a fully single-site enzyme. This reduced RNP is active and exhibits all of the characteristics of wild-type box C/D RNPs except it is nonspecific with respect to the site of 2′-O-methylation. Our results demonstrate that protein–protein crosstalk through Nop5p dimerization is not required, but that architecture plays a crucial role in directing methylation activity with both C/D and C′/D′ motifs being required for specificity. PMID:16984968

  20. Binding of the Bacteriophage P22 N-Peptide to the boxB RNA Motif Studied by Molecular Dynamics Simulations

    PubMed Central

    Bahadur, Ranjit P.; Kannan, Srinivasaraghavan; Zacharias, Martin

    2009-01-01

    Abstract Protein-RNA interactions are important for many cellular processes. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics simulations were used to investigate the dynamics of the P22 N-peptide-boxB complex and to elucidate the energetic contributions to binding. In addition, the free-energy changes of RNA and peptide conformational adaptation to the bound forms, as well as the role of strongly bound water molecules at the peptide-RNA interface, were studied. The influence of peptide amino acid substitutions and the salt dependence of interaction were investigated and showed good agreement with available experimental results. Several tightly bound water molecules were found at the RNA-binding interface in both the presence and absence of N-peptide. Explicit consideration of the waters resulted in shifts of calculated contributions during the energetic analysis, but overall similar binding energy contributions were found. Of interest, it was found that the electrostatic field of the RNA has a favorable influence on the coil-to-α-helix transition of the N-peptide already outside of the peptide-binding site. This result may have important implications for understanding peptide-RNA complex formation, which often involves coupled folding and association processes. It indicates that electrostatic interactions near RNA molecules can lead to a shift in the equilibrium toward the bound form of an interacting partner before it enters the binding pocket. PMID:20006951

  1. deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2.

    PubMed Central

    Toone, W M; Rudd, K E; Friesen, J D

    1991-01-01

    We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli. Images PMID:2045359

  2. Jeweled Boxes

    ERIC Educational Resources Information Center

    Coy, Mary

    2009-01-01

    While an empty cardboard box from a ream of copy paper may be the most coveted box among teachers in the author's school, for other people, brass boxes from India, Khokhlova lacquer boxes from Russia, and puzzle boxes from Japan are more the type that are collected and admired. Whether it is used for storage or decoration, a box can evoke a sense…

  3. TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine

    PubMed Central

    Yuan, Zhi-Yi; Lu, Xi; Lei, Fan; Chai, Yu-Shuang; Wang, Yu-Gang; Jiang, Jing-Fei; Feng, Tian-Shi; Wang, Xin-Pei; Yu, Xuan; Yan, Xiao-Jin; Xing, Dong-Ming; Du, Li-Jun

    2015-01-01

    Berberine (BBR) is a natural compound with variable pharmacological effects and a broad panel of target genes. We investigated berberine’s pharmacological activities from the perspective of its nucleotide-binding ability and discovered that BBR directly regulates gene expression by targeting TATA boxes in transcriptional regulatory regions as well as the poly adenine (poly (A)) tail at the mRNA terminus. BBR inhibits gene transcription by binding the TATA boxes in the transcriptional regulatory region, but it promotes higher levels of expression by targeting the poly (A) tails of mRNAs. The present study demonstrates that TATA boxes and poly (A) tails are the first and second primary targets by which BBR regulates gene expression. The final outcome of gene regulation by BBR depends on the structure of the individual gene. This is the first study to reveal that TATA boxes and poly (A) tails are direct targets for BBR in its regulation of gene expression. Our findings provide a novel explanation for the complex activities of a small molecule compound in a biological system and a novel horizon for small molecule-compound pharmacological studies. PMID:26671652

  4. Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry1[OPEN

    PubMed Central

    Xia, Rui; Ye, Songqing; Liu, Zongrang; Meyers, Blake C.; Liu, Zhongchi

    2015-01-01

    The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants. PMID:26143249

  5. Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry.

    PubMed

    Xia, Rui; Ye, Songqing; Liu, Zongrang; Meyers, Blake C; Liu, Zhongchi

    2015-09-01

    The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants. PMID:26143249

  6. A Weak C′ Box Renders U3 snoRNA Levels Dependent on hU3-55K Binding ▿

    PubMed Central

    Knox, Andrew Alexander; McKeegan, Kenneth Scott; Debieux, Charles Maurice; Traynor, Adele; Richardson, Hannah; Watkins, Nicholas James

    2011-01-01

    The rate of ribosome biogenesis, which is downregulated in terminally differentiated cells and upregulated in most cancers, regulates the growth rate and is linked to the cell's proliferative potential. The U3 box C/D small nucleolar RNP (snoRNP) is an integral component of the small subunit (SSU) processome and is essential for 18S rRNA processing. We show that U3 snoRNP assembly, and therefore U3 snoRNA accumulation, is regulated through the U3-specific protein hU3-55K. Furthermore, we report that the levels of several SSU processome components, including the U3 snoRNA but not other box C/D snoRNAs, are specifically downregulated during human lung (CaCo-2) and colon (CaLu-3) epithelial cell differentiation. c-Myc is reported to play an integral role in regulating ribosome production by controlling the expression of many ribosome biogenesis factors. Our data, however, indicate that this regulation is not dependent on c-Myc since the level of this protein does not change during epithelial cell differentiation. In addition, depletion of c-Myc had only a mild affect on the levels of SSU processome proteins. CaCo-2 cells are colon adenocarcinoma epithelial cells that are believed to revert to their precancerous state during differentiation. This suggests a significant increase in the levels of specific SSU processome components during tumorogenesis. PMID:21505065

  7. The Saccharomyces cerevisiae RNA polymerase III recruitment factor subunits Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box.

    PubMed

    Tsihlis, Nick D; Grove, Anne

    2006-01-01

    Association of the TATA-binding protein (TBP) with its cognate site within eukaryotic promoters is key to accurate and efficient transcriptional initiation. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB. Previous data have suggested that the structure or dynamics of the TBP-DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. We show here, using the altered specificity TBP mutant TBPm3 and an iterative in vitro selection assay, that entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene. We suggest that the selected T*A base pair step at the downstream end of the 8 bp TBP site may provide a DNA flexure that promotes TFIIIB-DNA complex formation. PMID:17028095

  8. A truncated hnRNP A1 isoform, lacking the RGG-box RNA binding domain, can efficiently regulate HIV-1 splicing and replication.

    PubMed

    Jean-Philippe, Jacques; Paz, Sean; Lu, Michael L; Caputi, Massimo

    2014-01-01

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is one of the most abundant RNA binding proteins. hnRNP A1 is localized prevalently in the nucleus but it can relocate to the cytoplasm in response to specific stimuli shuttling between nuclear and cytoplasmic compartments. The cellular localization of this protein is regulated by a short C-terminus motif (M9) and other less defined sequences. The RNA binding specificity of this protein is dependent on multiple RNA binding domains (RBDs), which regulate its role in RNA processing and expression. hnRNP A1 plays multiple roles in gene expression by regulating the biogenesis and translation of messengers RNAs, the processing of miRNAs, affecting transcription and controlling telomere maintenance. The multiple functions of this protein correlate with diverse roles in genetic disease, cancer and the replication of viral pathogens. Utilizing a tagged hnRNP A1 deletion library we have shown that the three hnRNP A1 RBDs contribute to the prevalent nuclear distribution of the protein. Our data also indicate that a truncated form of the protein, lacking one of the RBDs, the RGG-box, can regulate splicing of a splicing reporter minigene and down-regulate replication of the HIV-1 virus with efficiency comparable to the wild-type protein. This functional hnRNP A1 deletion mutant is similar to a predicted hnRNP A1 isoform, which had not been previously experimentally characterized. PMID:24530421

  9. Forkhead box C1 promoter upstream transcript, a novel long non-coding RNA, regulates proliferation and migration in basal-like breast cancer.

    PubMed

    Liu, Juntao; Shen, Lei; Yao, Jie; Li, Yi; Wang, Yongcheng; Chen, Hong; Geng, Peiliang

    2015-04-01

    Recent studies have shown that long non‑coding RNAs (lncRNAs) have crucial regulating roles in carcinogenesis. Forkhead box C1 (FOXC1) is an important cancer‑associated gene in basal‑like breast cancer (BLBC). In the present study, a novel lncRNA, FOXC1 promoter upstream transcript (FOXCUT) was investigated in BLBC patients using polymerase chain reaction analysis. The results showed that the expression of FOXCUT and FOXC1 were positively correlated. When the expression of FOXCUT was downregulated by small interfering RNA, the expression of FOXC1 was similarly reduced. Furthermore, in MDA‑MB‑231 and MDA‑MB‑468 breast cancer cells, knockdown of FOXCUT markedly inhibited cell proliferation and migration in vitro. In conclusion, FOXCUT lncRNA may be functionally involved in the tumor progression of BLBCs through the regulation of its paired mRNA, FOXC1, demonstrating that FOXCUT may serve as a novel biomarker and therapeutic target in BLBCs. PMID:25516208

  10. The DEAH-box ATPases Prp16 and Prp43 cooperate to proofread 5′ splice site cleavage during pre-mRNA splicing

    PubMed Central

    Koodathingal, Prakash; Novak, Thaddeus; Piccirilli, Joseph A.; Staley, Jonathan P.

    2010-01-01

    SUMMARY To investigate the mechanism of fidelity in pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5′ splice site cleavage. We inactivated spliceosomes by disrupting a critical metal-ligand interaction at the catalytic center and discovered that when the DEAH-box ATPase Prp16 was disabled these spliceosomes successfully catalyzed 5′ splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5′ splice site cleavage, we found that Prp16 can associate with spliceosomes before 5′ splice site cleavage, consistent with a role for Prp16 in proofreading 5′ splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH-box ATPase Prp43, a spliceosome disassembly factor. These data provide evidence that splicing fidelity mechanisms discriminate against slow substrates and that the mechanisms for establishing the fidelity of 5′ splice site cleavage and exon ligation share a common ATP-dependent framework. PMID:20705241

  11. Microdiversity of Deep-Sea Bacillales Isolated from Tyrrhenian Sea Sediments as Revealed by ARISA, 16S rRNA Gene Sequencing and BOX-PCR Fingerprinting

    PubMed Central

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  12. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  13. MiRNA-101 inhibits oral squamous-cell carcinoma growth and metastasis by targeting zinc finger E-box binding homeobox 1

    PubMed Central

    Wu, Baolei; Lei, Delin; Wang, Lei; Yang, Xinjie; Jia, Sen; Yang, Zihui; Shan, Chun; Yang, Xi; Zhang, Chenping; Lu, Bin

    2016-01-01

    MicroRNAs (miRNAs) are implicated in the pathogenesis of oral squamous-cell carcinoma (OSCC). miR-101 is involved in the development and progression of OSCC, but the biological functions and underlying molecular mechanisms of this miRNA remain largely unknown. In this study, we showed that miR-101 was underexpressed in OSCC tissues and cell lines. miR-101 downregulation was inversely correlated with zinc finger E-box binding homeobox 1 (ZEB1) expression, lymph-node metastasis, and poor prognosis in OSCC patients. Enhanced expression of miR-101 significantly inhibited OSCC cell proliferation, apoptosis resistance, migration and invasion in vitro, and suppressed tumor growth and lung metastasis in vivo. Bioinformatics analyses showed that miR-101 directly targeted ZEB1, as confirmed by a dual-luciferase reporter assay. The inhibitory effects of miR-101 on OSCC growth and metastasis were attenuated and phenocopied by ZEB1 overexpression and knockdown, respectively. Overall, our findings indicated that miRNA-101 reduced OSCC growth and metastasis by targeting ZEB1 and provided new evidence of miR-101 as a potential therapeutic target for OSCC patients. PMID:27429852

  14. Formation of C-terminally truncated version of the Taz1 protein employs cleavage-box structure in mRNA

    SciTech Connect

    Gunisova, Stanislava; Bartosova, Zdenka; Kramara, Juraj; Nosek, Jozef; Tomaska, Lubomir

    2010-02-12

    When expressed in various hosts the taz1{sup +} gene encoding the fission yeast telomere-binding protein produces two forms of polypeptides: full-length (Taz1p) and truncated (Taz1p{Delta}C) version lacking almost entire Myb-domain. Whereas Taz1p binds telomeric DNA in vitro, Taz1p{Delta}C forms long filaments unable of DNA binding. The formation of Taz1p{Delta}C is a result of neither site-specific proteolysis, nor premature termination of transcription. In silico analysis of the taz1{sup +} RNA transcript revealed a stem-loop structure at the site of cleavage (cleavage box; CB). In order to explore whether it possesses inherent destabilizing effects, we cloned CB sequence into the open reading frame (ORF) of glutathione-S-transferase (GST) and observed that when expressed in Escherichia coli the engineered gene produced two forms of the reporter protein. The formation of the truncated version of GST was abolished, when CB was replaced with recoded sequence containing synonymous codons thus indicating that the truncation is based on structural properties of taz1{sup +} mRNA.

  15. Genome-Wide Comparative In Silico Analysis of the RNA Helicase Gene Family in Zea mays and Glycine max: A Comparison with Arabidopsis and Oryza sativa

    PubMed Central

    Huang, Jinguang; Zheng, Chengchao

    2013-01-01

    RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of

  16. Controlled Delivery of T-box21 Small Interfering RNA Ameliorates Autoimmune Alopecia (Alopecia Areata) in a C3H/HeJ Mouse Model

    PubMed Central

    Nakamura, Motonobu; Jo, Jun-ichiro; Tabata, Yasuhiko; Ishikawa, Osamu

    2008-01-01

    Autoimmune alopecia (alopecia areata) is considered to be triggered by a collapse of immune privilege in hair follicles. Here we confirmed that infiltrating CD4 T lymphocytes around hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells, suggesting a dominant role of Th1 cells in the alopecic lesion. Given this finding, we sought to elucidate the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant interleukin-4 and neutralizing anti-interferon-γ antibody. We found that local injections of both interleukin-4 and neutralizing anti-interferon-γ antibody effectively treated alopecia in C3H/HeJ mice. Results from immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction demonstrated that intralesional injection of interleukin-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon-γ mRNA expression in the affected alopecic skin. Furthermore, Th1 transcription factor T-box21 small interfering RNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. Taken together, the use of gelatin–small interfering RNA conjugates promises to be a novel, efficient, and safe tool as an alternative gene therapy for the treatment of various human diseases. To our knowledge, this is the first report of effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease. PMID:18245811

  17. Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

    PubMed Central

    Gagnon, Keith T.; Zhang, Xinxin; Qu, Guosheng; Biswas, Shyamasri; Suryadi, Jimmy; Brown, Bernard A.; Maxwell, E. Stuart

    2010-01-01

    The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs. PMID:19926724

  18. Controlled delivery of T-box21 small interfering RNA ameliorates autoimmune alopecia (Alopecia Areata) in a C3H/HeJ mouse model.

    PubMed

    Nakamura, Motonobu; Jo, Jun-ichiro; Tabata, Yasuhiko; Ishikawa, Osamu

    2008-03-01

    Autoimmune alopecia (alopecia areata) is considered to be triggered by a collapse of immune privilege in hair follicles. Here we confirmed that infiltrating CD4 T lymphocytes around hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells, suggesting a dominant role of Th1 cells in the alopecic lesion. Given this finding, we sought to elucidate the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant interleukin-4 and neutralizing anti-interferon-gamma antibody. We found that local injections of both interleukin-4 and neutralizing anti-interferon-gamma antibody effectively treated alopecia in C3H/HeJ mice. Results from immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction demonstrated that intralesional injection of interleukin-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon-gamma mRNA expression in the affected alopecic skin. Furthermore, Th1 transcription factor T-box21 small interfering RNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. Taken together, the use of gelatin-small interfering RNA conjugates promises to be a novel, efficient, and safe tool as an alternative gene therapy for the treatment of various human diseases. To our knowledge, this is the first report of effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease. PMID:18245811

  19. Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp

    PubMed Central

    d’Orval, Béatrice Clouet; Bortolin, Marie-Line; Gaspin, Christine; Bachellerie, Jean-Pierre

    2001-01-01

    Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2′-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5′ exon but also onto position 39 (universal tRNA numbering) in the 3′ exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon–intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs. PMID:11713301

  20. Bento Boxes

    ERIC Educational Resources Information Center

    Hasio, Cindy

    2010-01-01

    Bento boxes are common objects in Japanese culture, designed to hold enough lunch for one person. They have individual compartments and sometimes multiple tiers for rice, vegetables, and other side dishes. They are made of materials ranging from wood, cloth, aluminum, or plastic. In general, the greater the number of foods, the better the box is…

  1. Film Boxes.

    ERIC Educational Resources Information Center

    Osterer, Irv

    2002-01-01

    Presents an art lesson in which students created three-dimensional designs for 35mm film packages to improve graphic arts learning. Describes how the students examined and created film boxes using QuarkXPress software. (CMK)

  2. Day of the Dead

    ERIC Educational Resources Information Center

    Dann, Tammy; Murphy, Amy

    2012-01-01

    Foreign Language in Elementary School (FLES) teachers in the West Des Moines schools incorporate the Day of the Dead into the fourth grade curriculum each year. The teachers discuss the Day of the Dead celebration at the Art Center, and many ask for volunteers from fourth grade to participate in the event. Student presentations include a wide…

  3. Investigating Aquatic Dead Zones

    ERIC Educational Resources Information Center

    Testa, Jeremy; Gurbisz, Cassie; Murray, Laura; Gray, William; Bosch, Jennifer; Burrell, Chris; Kemp, Michael

    2010-01-01

    This article features two engaging high school activities that include current scientific information, data, and authentic case studies. The activities address the physical, biological, and chemical processes that are associated with oxygen-depleted areas, or "dead zones," in aquatic systems. Students can explore these dead zones through both…

  4. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  5. In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

    PubMed Central

    Zamrod, Z; Tyree, C M; Song, Y; Stumph, W E

    1993-01-01

    Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter. Images PMID:8355718

  6. [The dead mother].

    PubMed

    Green, A

    1993-03-01

    This study is not concerned, as the title might suggest, with the actual death of the mother but with the child's experience of a mother who is physically present but internally absent due to depression. The child simultaneously introjects and splits off the mother imago, making mourning and "burial" equally impossible. The consequence of this cathectic deprivation is what the author calls "psychic holes" or "white depression". Green attributes to the dead mother a similar structuring function for the psychic apparatus to that attributed to the dead father in Freud's Totem and Taboo, and places the dead mother complex side by side with the Oedipus complex. PMID:8465007

  7. Boxing clever.

    PubMed

    Toon, P D

    1988-06-01

    This is the first contribution to a new JME column, "At the coalface," to which readers are invited to relate ethical problems they have encountered in their work. An adolescent patient requested that the author, a general practitioner, certify that he was medically fit to box. Toon attempted to dissuade him from boxing by explaining its dangers. When the boy persisted, the physician rapidly considered the ethical principles involved in the encounter and decided to "wash his hands" by telling his patient that if "he insisted on damaging his, or someone else's brain, then he must find another medical accomplice." PMID:3392720

  8. Dead Sea Scrolls

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A consortium of researchers from Jet Propulsion Laboratory and three other organizations used charged coupled devices (CCDs) and other imaging enhancement technology to decipher previously unreadable portions of the Dead Sea Scrolls. The technique has potentially important implications for archeology.

  9. Exploding Boxes

    ERIC Educational Resources Information Center

    Kinney; Jan

    2011-01-01

    How do you teach the "same old, same old" in an interesting and inexpensive way? Art teachers are forever looking for new angles on the good-old elements and principles. And, as budgets tighten, they are trying to be as frugal as possible while still holding their students' attention. Enter exploding boxes! In conceptualizing the three types of…

  10. Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells.

    PubMed

    Di Gioia, Sante; Sardo, Carla; Belgiovine, Giuliana; Triolo, Daniela; d'Apolito, Maria; Castellani, Stefano; Carbone, Annalucia; Giardino, Ida; Giammona, Gaetano; Cavallaro, Gennara; Conese, Massimo

    2015-08-01

    High-mobility group box 1 (HMGB1) is a nonhistone protein secreted by airway epithelial cells in hyperinflammatory diseases such as asthma. In order to down-regulate HMGB1 expression in airway epithelial cells, siRNA directed against HMGB1 was delivered through nanocomplexes based on a cationic copolymer of poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) by using H441 cells. Two copolymers were used in these experiments bearing respectively spermine side chains (PHEA-Spm) and both spermine and PEG2000 chains (PHEA-PEG-Spm). PHEA-Spm and PHEA-PEG-Spm derivatives complexed dsDNA oligonucleotides with a w/w ratio of 1 and higher as shown by a gel retardation assay. PHEA-Spm and PHEA-PEG-Spm siRNA polyplexes were sized 350-650 nm and 100-400 nm respectively and ranged from negativity/neutrality (at 0.5 ratio) to positivity (at 5 ratio) as ζ potential. Polyplexes formed either at a ratio of 0.5 (partially complexing) or at the ratio of 5 (fully complexing) were tested in subsequent experiments. Epifluorescence revealed that nanocomplexes favored siRNA entry into H441 cells in comparison with naked siRNA. As determined by flow cytometry and a trypan blue assay, PHEA-Spm and PHEA-PEG-Spm allowed siRNA uptake in 42-47% and 30% of cells respectively, however only with PHEA-Spm at w/w ratio of 5 these percentages were significantly higher than those obtained with naked siRNA (20%). Naked siRNA or complexed scrambled siRNA did not exert any effect on HMGB1mRNA levels, whereas PHEA-Spm/siRNA at the w/w ratio of 5 down-regulated HMGB1 mRNA up to 58% of control levels (untransfected cells). PEGylated PHEA-Spm/siRNA nanocomplexes were able to down-regulate HMGB1 mRNA levels up to 61% of control cells. MTT assay revealed excellent biocompatibility of copolymer/siRNA polyplexes with cells. In conclusion, we have found optimal conditions for down-regulation of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers in airway epithelial cells in the absence of cytotoxicity

  11. ON HYDRODYNAMIC MOTIONS IN DEAD ZONES

    SciTech Connect

    Oishi, Jeffrey S.; Mac Low, Mordecai-Mark E-mail: mordecai@amnh.or

    2009-10-20

    We investigate fluid motions near the midplane of vertically stratified accretion disks with highly resistive midplanes. In such disks, the magnetorotational instability drives turbulence in thin layers surrounding a resistive, stable dead zone. The turbulent layers in turn drive motions in the dead zone. We examine the properties of these motions using three-dimensional, stratified, local, shearing-box, non-ideal, magnetohydrodynamical simulations. Although the turbulence in the active zones provides a source of vorticity to the midplane, no evidence for coherent vortices is found in our simulations. It appears that this is because of strong vertical oscillations in the dead zone. By analyzing time series of azimuthally averaged flow quantities, we identify an axisymmetric wave mode particular to models with dead zones. This mode is reduced in amplitude, but not suppressed entirely, by changing the equation of state from isothermal to ideal. These waves are too low frequency to affect sedimentation of dust to the midplane, but may have significance for the gravitational stability of the resulting midplane dust layers.

  12. "Living versus Dead":

    PubMed Central

    Chakrabarti, Pratik

    2010-01-01

    Summary The Semple antirabies vaccine was developed by David Semple in India in 1911. Semple introduced a peculiarly British approach within the Pasteurian tradition by using carbolized dead virus. This article studies this unique phase of vaccine research between 1910 and 1935 to show that in the debates and laboratory experiments around the potency and safety of vaccines, categories like "living" and "dead" were often used as ideological and moral denominations. These abstract and ideological debates were crucial in defining the final configuration of the Semple vaccine, the most popular antirabies vaccine used globally, and also in shaping international vaccination policies. PMID:21037397

  13. The Bacillus subtilis tyrZ Gene Encodes a Highly Selective Tyrosyl-tRNA Synthetase and Is Regulated by a MarR Regulator and T Box Riboswitch

    PubMed Central

    Williams-Wagner, Rebecca N.; Grundy, Frank J.; Raina, Medha; Ibba, Michael

    2015-01-01

    ABSTRACT Misincorporation of d-tyrosine (d-Tyr) into cellular proteins due to mischarging of tRNATyr with d-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation of Bacillus subtilis. Furthermore, many B. subtilis strains lack a functional gene encoding d-aminoacyl-tRNA deacylase, which prevents misincorporation of d-Tyr in most organisms. B. subtilis has two genes that encode tyrosyl-tRNA synthetase: tyrS is expressed under normal growth conditions, and tyrZ is known to be expressed only when tyrS is inactivated by mutation. We hypothesized that tyrZ encodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow when d-Tyr is present. We show that TyrZ is more selective for l-Tyr over d-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression of tyrZ is required for growth and biofilm formation in the presence of d-Tyr. Both tyrS and tyrZ are preceded by a T box riboswitch, but tyrZ is found in an operon with ywaE, which is predicted to encode a MarR family transcriptional regulator. Expression of tyrZ is repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression of tyrZ may allow growth when excess d-Tyr is present. IMPORTANCE Accurate protein synthesis requires correct aminoacylation of each tRNA with the cognate amino acid and discrimination against related compounds. Bacillus subtilis produces d-Tyr, an analog of l-Tyr that is toxic when incorporated into protein, during stationary phase. Most organisms utilize a d-aminoacyl-tRNA deacylase to prevent misincorporation of d-Tyr. This work demonstrates that the increased selectivity of the TyrZ form of tyrosyl-tRNA synthetase may provide a mechanism by which B. subtilis prevents misincorporation of d-Tyr in the absence of a functional d-aminoacyl-tRNA deacylase gene. PMID:25733610

  14. The Dead Sea

    NASA Technical Reports Server (NTRS)

    2006-01-01

    The Dead Sea is the lowest point on Earth at 418 meters below sea level, and also one of the saltiest bodies of water on Earth with a salinity of about 300 parts-per-thousand (nine times greater than ocean salinity). It is located on the border between Jordan and Israel, and is fed by the Jordan River. The Dead Sea is located in the Dead Sea Rift, formed as a result of the Arabian tectonic plate moving northward away from the African Plate. The mineral content of the Dead Sea is significantly different from that of ocean water, consisting of approximately 53% magnesium chloride, 37% potassium chloride and 8% sodium chloride. In the early part of the 20th century, the Dead Sea began to attract interest from chemists who deduced that the Sea was a natural deposit of potash and bromine. From the Dead Sea brine, Israel and Jordan produce 3.8 million tons potash, 200,000 tons elemental bromine, 45,000 tons caustic soda, 25, 000 tons magnesium metal, and sodium chloride. Both countries use extensive salt evaporation pans that have essentially diked the entire southern end of the Dead Sea.

    With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER images Earth to map and monitor the changing surface of our planet.

    ASTER is one of five Earth-observing instruments launched December 18, 1999, on NASA's Terra satellite. The instrument was built by Japan's Ministry of Economy, Trade and Industry. A joint U.S./Japan science team is responsible for validation and calibration of the instrument and the data products.

    The broad spectral coverage and high spectral resolution of ASTER provides scientists in numerous disciplines with critical information for surface mapping, and monitoring of dynamic conditions and temporal change. Example applications are: monitoring glacial advances and retreats; monitoring potentially active volcanoes; identifying crop

  15. The Analysis of the Inflorescence miRNome of the Orchid Orchis italica Reveals a DEF-Like MADS-Box Gene as a New miRNA Target

    PubMed Central

    Aceto, Serena; Sica, Maria; De Paolo, Sofia; D'Argenio, Valeria; Cantiello, Piergiuseppe; Salvatore, Francesco; Gaudio, Luciano

    2014-01-01

    Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the “orchid code” theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs. PMID:24832004

  16. In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNATrp

    PubMed Central

    Bortolin, Marie-Line; Bachellerie, Jean-Pierre; Clouet-d'Orval, Béatrice

    2003-01-01

    Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre-tRNATrp represents an outstanding specimen able to guide in cis, instead of in trans, two 2′-O-methylations in the pre-tRNA exons. Remarkably, both sites of methylation involve nucleotides within the bulge–helix–bulge (BHB) splicing motif, while the RNA-guided methylation and pre-tRNA splicing events depend on mutually exclusive RNA folding patterns. Using the three recombinant core proteins of archaeal C/D RNPs, we have analyzed in vitro RNP assembly of the pre-tRNA and tested its site-specific methylation activity. Recognition by L7Ae of hallmark K-turns at the C/D and C′/D′ motifs appears as a crucial assembly step required for subsequent binding of a Nop5p–aFib heterodimer at each site. Unexpectedly, however, even without L7Ae but at a higher concentration of Nop5p–aFib, a substantially active RNP complex can still form, possibly reflecting the higher propensity of the cis-acting system to form guide RNA duplex(es) relative to classical trans- acting C/D RNA guides. Moreover, footprinting data of RNPs, consistent with Nop5p interacting with the non-canonical stem of the K-turn, suggest that binding of Nop5p–aFib to the pre-tRNA–L7Ae complex might direct transition from a splicing-competent structure to an RNA conformer displaying the guide RNA duplexes required for site-specific methylation. PMID:14602911

  17. The RNA Helicase eIF4A Is Required for Sapovirus Translation

    PubMed Central

    Hosmillo, Myra; Sweeney, Trevor R.; Chaudhry, Yasmin; Leen, Eoin; Curry, Stephen

    2016-01-01

    The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5′ untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5′ UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation. PMID:26937032

  18. Dead Sea rhodopsins revisited.

    PubMed

    Bodaker, Idan; Suzuki, Marcelino T; Oren, Aharon; Béjà, Oded

    2012-12-01

    The Dead Sea is a unique hypersaline ecosystem with near toxic magnesium levels (∼2 M), dominance of divalent cations and a slightly acidic pH. Previously, we reported a haloarchaeon related to Halobacterium salinarum to dominate in a microbial bloom that developed in 1992 in the upper water layers of the lake following massive freshwater runoff. Whether this clade also dominated an earlier bloom in 1980-1982 cannot be ascertained as no samples for cultivation-independent analysis were preserved. The presence of the light-driven proton pump bacteriorhodopsin was reported in the 1980-1982 bloom of prokaryotes that had developed in the Dead Sea. To test the hypothesis that bacteriorhodopsin proton pumping may play a major role in determining what type of haloarchaea may dominate in specific bloom conditions, we compared rhodopsin genes recovered from Dead Sea biomass collected in different periods with genes coding for retinal proteins in isolated haloarchaea. Novel bacteriorhodopsin and sensory rhodopsin genes were found in samples collected in 2007 and 2010. The fact that no rhodopsin genes were recovered from samples collected during the 1992 bloom, which was dominated by a single species, suggests that different clades were present in the 1980-1982 and 1992 blooms, and that bacteriorhodopsin proton pumping did not necessarily play a determinative role in the dominance of specific halophiles in the blooms. PMID:23760932

  19. Thinking beside the box: Should we care about the non-coding strand of the 16S rRNA gene?

    PubMed

    Garcia-Mazcorro, Jose F; Barcenas-Walls, Jose R

    2016-08-01

    The 16S rRNA gene (16S rDNA) codes for RNA that plays a fundamental role during translation in the ribosome and is used extensively as a marker gene to establish relationships among bacteria. However, the complementary non-coding 16S rDNA (nc16S rDNA) has been ignored. An idea emerged in the course of analyzing bacterial 16S rDNA sequences in search for nucleotide composition and substitution patterns: Does the nc16S rDNA code? If so, what does it code for? More importantly: Does 16S rDNA evolution reflect its own evolution or the evolution of its counterpart nc16S rDNA? The objective of this minireview is to discuss these thoughts. nc strands often encode small RNAs (sRNAs), ancient components of gene regulation. nc16S rDNA sequences from different bacterial groups were used to search for possible matches in the Bacterial Small Regulatory RNA Database. Intriguingly, the sequence of one published sRNA obtained from Legionella pneumophila (GenBank: AE0173541) showed high non-random similarity with nc16S rDNA corresponding in part to the V5 region especially from Legionella and relatives. While the target(s) of this sRNA is unclear at the moment, its mere existence might open up a new chapter in the use of the 16S rDNA to study relationships among bacteria. PMID:27412167

  20. Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C/D box snoRNA cluster in Prader–Willi syndrome

    PubMed Central

    Duker, Angela L; Ballif, Blake C; Bawle, Erawati V; Person, Richard E; Mahadevan, Sangeetha; Alliman, Sarah; Thompson, Regina; Traylor, Ryan; Bejjani, Bassem A; Shaffer, Lisa G; Rosenfeld, Jill A; Lamb, Allen N; Sahoo, Trilochan

    2010-01-01

    Prader–Willi syndrome (PWS) is a neurobehavioral disorder manifested by infantile hypotonia and feeding difficulties in infancy, followed by morbid obesity secondary to hyperphagia. It is caused by deficiency of paternally expressed transcript(s) within the human chromosome region 15q11.2. PWS patients harboring balanced chromosomal translocations with breakpoints within small nuclear ribonucleoprotein polypeptide N (SNRPN) have provided indirect evidence for a role for the imprinted C/D box containing small nucleolar RNA (snoRNA) genes encoded downstream of SNRPN. In addition, recently published data provide strong evidence in support of a role for the snoRNA SNORD116 cluster (HBII-85) in PWS etiology. In this study, we performed detailed phenotypic, cytogenetic, and molecular analyses including chromosome analysis, array comparative genomic hybridization (array CGH), expression studies, and single-nucleotide polymorphism (SNP) genotyping for parent-of-origin determination of the 15q11.2 microdeletion on an 11-year-old child expressing the major components of the PWS phenotype. This child had an ∼236.29 kb microdeletion at 15q11.2 within the larger Prader–Willi/Angelman syndrome critical region that included the SNORD116 cluster of snoRNAs. Analysis of SNP genotypes in proband and mother provided evidence in support of the deletion being on the paternal chromosome 15. This child also met most of the major PWS diagnostic criteria including infantile hypotonia, early-onset morbid obesity, and hypogonadism. Identification and characterization of this case provide unequivocal evidence for a critical role for the SNORD116 snoRNA molecules in PWS pathogenesis. Array CGH testing for genomic copy-number changes in cases with complex phenotypes is proving to be invaluable in detecting novel alterations and enabling better genotype–phenotype correlations. PMID:20588305

  1. Comparative Study of Two Box H/ACA Ribonucleoprotein Pseudouridine-Synthases: Relation between Conformational Dynamics of the Guide RNA, Enzyme Assembly and Activity

    PubMed Central

    Leclerc, Fabrice; Branlant, Christiane; Charpentier, Bruno

    2013-01-01

    Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10–L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21. PMID:23922977

  2. An RNA degradosome assembly in Caulobacter crescentus

    PubMed Central

    Hardwick, Steven W.; Chan, Vivian S. Y.; Broadhurst, R. William; Luisi, Ben F.

    2011-01-01

    In many bacterial species, the multi-enzyme RNA degradosome assembly makes key contributions to RNA metabolism. Powering the turnover of RNA and the processing of structural precursors, the RNA degradosome has differential activities on a spectrum of transcripts and contributes to gene regulation at a global level. Here, we report the isolation and characterization of an RNA degradosome assembly from the α-proteobacterium Caulobacter crescentus, which is a model organism for studying morphological development and cell-cycle progression. The principal components of the C. crescentus degradosome are the endoribonuclease RNase E, the exoribonuclease polynucleotide phosphorylase (PNPase), a DEAD-box RNA helicase and the Krebs cycle enzyme aconitase. PNPase and aconitase associate with specific segments in the C-terminal domain of RNase E that are predicted to have structural propensity. These recognition ‘microdomains’ punctuate structurally an extensive region that is otherwise predicted to be natively disordered. Finally, we observe that the abundance of RNase E varies through the cell cycle, with maxima at morphological differentiation and cell division. This variation may contribute to the program of gene expression during cell division. PMID:20952404

  3. Dead of night.

    PubMed

    Balter, Leon

    2010-07-01

    Dead of Night, the first psychoanalytic horror film, was produced in England in 1945, immediately after the end of World War II--that is, after the English population had suffered systematic Nazi terror from imminent invasion, incessant aerial bombing, and rocket-bombs. This film continued the prewar format of horror films based on themes of the supernatural and the hubris and excesses of science. However, it introduced psychoanalysis as the science in question. The film is structured on two levels: a genteel English country weekend to which witty and urbane guests have been invited; and five horror stories told by the guests. Psychoanalytic insights into this film structure are used here to explain how the film induces horror in the audience. PMID:20726184

  4. New Life From Dead Trees

    ERIC Educational Resources Information Center

    DeGraaf, Richard M.

    1978-01-01

    There are numerous bird species that will nest only in dead or dying trees. Current forestry practices include clearing forests of these snags, or dead trees. This practice is driving many species out of the forests. An illustrated example of bird succession in and on a tree is given. (MA)

  5. Black Box Chimera Check (B2C2): a Windows-Based Software for Batch Depletion of Chimeras from Bacterial 16S rRNA Gene Datasets.

    PubMed

    Gontcharova, Viktoria; Youn, Eunseog; Wolcott, Randall D; Hollister, Emily B; Gentry, Terry J; Dowd, Scot E

    2010-01-01

    The existing chimera detection programs are not specifically designed for "next generation" sequence data. Technologies like Roche 454 FLX and Titanium have been adapted over the past years especially with the introduction of bacterial tag-encoded FLX/Titanium amplicon pyrosequencing methodologies to produce over one million 250-600 bp 16S rRNA gene reads that need to be depleted of chimeras prior to downstream analysis. Meeting the needs of basic scientists who are venturing into high-throughput microbial diversity studies such as those based upon pyrosequencing and specifically providing a solution for Windows users, the B2C2 software is designed to be able to accept files containing large multi-FASTA formatted sequences and screen for possible chimeras in a high throughput fashion. The graphical user interface (GUI) is also able to batch process multiple files. When compared to popular chimera screening software the B2C2 performed as well or better while dramatically decreasing the amount of time required generating and screening results. Even average computer users are able to interact with the Windows .Net GUI-based application and define the stringency to which the analysis should be done. B2C2 may be downloaded from http://www.researchandtesting.com/B2C2. PMID:21339894

  6. Metagenomic insights into important microbes from the Dead Zone

    NASA Astrophysics Data System (ADS)

    Thrash, C.; Baker, B.; Seitz, K.; Temperton, B.; Gillies, L.; Rabalais, N. N.; Mason, O. U.

    2015-12-01

    Coastal regions of eutrophication-driven oxygen depletion are widespread and increasing in number. Also known as dead zones, these regions take their name from the deleterious effects of hypoxia (dissolved oxygen less than 2 mg/L) on shrimp, demersal fish, and other animal life. Dead zones result from nutrient enrichment of primary production, concomitant consumption by chemoorganotrophic aerobic microorganisms, and strong stratification that prevents ventilation of bottom water. One of the largest dead zones in the world occurs seasonally in the northern Gulf of Mexico (nGOM), where hypoxia can reach up to 22,000 square kilometers. While this dead zone shares many features with more well-known marine oxygen minimum zones, it is nevertheless understudied with regards to the microbial assemblages involved in biogeochemical cycling. We performed metagenomic and metatranscriptomic sequencing on six samples from the 2013 nGOM dead zone from both hypoxic and oxic bottom waters. Assembly and binning led to the recovery of over fifty partial to nearly complete metagenomes from key microbial taxa previously determined to be numerically abundant from 16S rRNA data, such as Thaumarcheaota, Marine Group II Euryarchaeota, SAR406, SAR324, Synechococcus spp., and Planctomycetes. These results provide information about the roles of these taxa in the nGOM dead zone, and opportunities for comparing this region of low oxygen to others around the globe.

  7. Is Piaget's Epistemic Subject Dead?

    ERIC Educational Resources Information Center

    Lawson, Anton E.

    1991-01-01

    Argues that the Piaget's epistemic subject is not supported by evidence and contains weaknesses. Concludes that the epistemic subject is dead and that continued acceptance of this aspect of Piagetian theory would be counterproductive. (PR)

  8. Climate change and dead zones.

    PubMed

    Altieri, Andrew H; Gedan, Keryn B

    2015-04-01

    Estuaries and coastal seas provide valuable ecosystem services but are particularly vulnerable to the co-occurring threats of climate change and oxygen-depleted dead zones. We analyzed the severity of climate change predicted for existing dead zones, and found that 94% of dead zones are in regions that will experience at least a 2 °C temperature increase by the end of the century. We then reviewed how climate change will exacerbate hypoxic conditions through oceanographic, ecological, and physiological processes. We found evidence that suggests numerous climate variables including temperature, ocean acidification, sea-level rise, precipitation, wind, and storm patterns will affect dead zones, and that each of those factors has the potential to act through multiple pathways on both oxygen availability and ecological responses to hypoxia. Given the variety and strength of the mechanisms by which climate change exacerbates hypoxia, and the rates at which climate is changing, we posit that climate change variables are contributing to the dead zone epidemic by acting synergistically with one another and with recognized anthropogenic triggers of hypoxia including eutrophication. This suggests that a multidisciplinary, integrated approach that considers the full range of climate variables is needed to track and potentially reverse the spread of dead zones. PMID:25385668

  9. DESERVE - Dead Sea Research Venue

    NASA Astrophysics Data System (ADS)

    Mohsen, A.; Weber, M. H.; Kottmeier, C.

    2013-12-01

    DESERVE 'Dead Sea Research Venue' focuses on the Dead Sea region as it is a unique environment and may be considered as one of the most inspiring natural laboratories on Earth. The Dead Sea Region is an exceptional ecosystem whose seismic activity has influenced all facets of the development, from ground water availability to human evolution. DESERVE addresses three grand challenges: Environmental Risks, Water Availability, Climate Change and comprises long term monitoring of geophysical parameters, studies of coupled processes in the atmosphere, hydrosphere and lithosphere as well as modeling of prediction and remediation strategies of geogenic risks. The Dead Sea has been selected for this integrated approach because it constitutes an outstanding 'natural laboratory' to study these phenomena, as - all 3 challenges are critical in this region. - the region is especially sensitive to climate change and human influences such as ground and surface water over-exploitation for agriculture and industrial purposes. - environmental processes are subject to boundary conditions that cannot be found elsewhere on Earth - understanding their interactions and the future evolution of the whole Dead Sea region are of key importance for economic development in peaceful cooperation. Results obtained in the Dead Sea region are also of prototype relevance for other (semi)-arid terminal basins of the world.

  10. Dead Star Rumbles

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site] Composite of Supernova Remnant Cassiopeia A This Spitzer Space Telescope composite shows the supernova remnant Cassiopeia A (white ball) and surrounding clouds of dust (gray, orange and blue). It consists of two processed images taken one year apart. Dust features that have not changed over time appear gray, while those that have changed are colored blue or orange. Blue represents an earlier time and orange, a later time.

    These observations illustrate that a blast of light from Cassiopeia A is waltzing outward through the dusty skies. This dance, called an 'infrared echo,' began when the remnant erupted about 50 years ago.

    Cassiopeia A is the remnant of a once massive star that died in a violent supernova explosion 325 years ago. It consists of a dead star, called a neutron star, and a surrounding shell of material that was blasted off as the star died. This remnant is located 10,000 light-years away in the northern constellation Cassiopeia.

    An infrared echo is created when a star explodes or erupts, flashing light into surrounding clumps of dust. As the light zips through the dust clumps, it heats them up, causing them to glow successively in infrared, like a chain of Christmas bulbs lighting up one by one. The result is an optical illusion, in which the dust appears to be flying outward at the speed of light. This apparent motion can be seen here by the shift in colored dust clumps.

    Echoes are distinct from supernova shockwaves, which are made up material that is swept up and hurled outward by exploding stars.

    This infrared echo is the largest ever seen, stretching more than 50 light-years away from Cassiopeia A. If viewed from Earth, the entire movie frame would take up the same amount of space as two full moons.

    Hints of an older infrared echo from Cassiopeia A's supernova explosion hundreds of years ago can also be seen.

    The earlier Spitzer image was taken on November 30

  11. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans.

    PubMed

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V

    2015-08-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans. PMID:26195740

  12. RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans

    PubMed Central

    Seo, Mihwa; Seo, Keunhee; Hwang, Wooseon; Koo, Hee Jung; Hahm, Jeong-Hoon; Yang, Jae-Seong; Han, Seong Kyu; Hwang, Daehee; Kim, Sanguk; Jang, Sung Key; Lee, Yoontae; Nam, Hong Gil; Lee, Seung-Jae V.

    2015-01-01

    The homeostatic maintenance of the genomic DNA is crucial for regulating aging processes. However, the role of RNA homeostasis in aging processes remains unknown. RNA helicases are a large family of enzymes that regulate the biogenesis and homeostasis of RNA. However, the functional significance of RNA helicases in aging has not been explored. Here, we report that a large fraction of RNA helicases regulate the lifespan of Caenorhabditis elegans. In particular, we show that a DEAD-box RNA helicase, helicase 1 (HEL-1), promotes longevity by specifically activating the DAF-16/forkhead box O (FOXO) transcription factor signaling pathway. We find that HEL-1 is required for the longevity conferred by reduced insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) and is sufficient for extending lifespan. We further show that the expression of HEL-1 in the intestine and neurons contributes to longevity. HEL-1 enhances the induction of a large fraction of DAF-16 target genes. Thus, the RNA helicase HEL-1 appears to promote longevity in response to decreased IIS as a transcription coregulator of DAF-16. Because HEL-1 and IIS are evolutionarily well conserved, a similar mechanism for longevity regulation via an RNA helicase-dependent regulation of FOXO signaling may operate in mammals, including humans. PMID:26195740

  13. RNA clamping by Vasa assembles a piRNA amplifier complex on transposon transcripts.

    PubMed

    Xiol, Jordi; Spinelli, Pietro; Laussmann, Maike A; Homolka, David; Yang, Zhaolin; Cora, Elisa; Couté, Yohann; Conn, Simon; Kadlec, Jan; Sachidanandam, Ravi; Kaksonen, Marko; Cusack, Stephen; Ephrussi, Anne; Pillai, Ramesh S

    2014-06-19

    Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5' sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons. PMID:24910301

  14. 46 CFR 171.117 - Dead covers.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Dead covers. 171.117 Section 171.117 Shipping COAST... Dead covers. (a) Except as provided in paragraph (b) of this section, each port light with the sill located below the margin line must have a hinged, inside dead cover. (b) The dead cover on a port...

  15. Autogenous Translational Regulation of the Borna Disease Virus Negative Control Factor X from Polycistronic mRNA Using Host RNA Helicases

    PubMed Central

    Watanabe, Yohei; Ohtaki, Naohiro; Hayashi, Yohei; Ikuta, Kazuyoshi; Tomonaga, Keizo

    2009-01-01

    Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs): upstream ORF (uORF), X, and P in the 5′ to 3′ direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5′ untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases. PMID:19893625

  16. Determination of the role of DDX3 a factor involved in mammalian RNAi pathway using an shRNA-expression library.

    PubMed

    Kasim, Vivi; Wu, Shourong; Taira, Kazunari; Miyagishi, Makoto

    2013-01-01

    RNA interference (RNAi) is an endogenous RNA-destruction phenomenon induced by certain double-stranded RNAs (dsRNAs). In RNAi, dsRNAs are processed into small interfering RNAs (siRNAs) which in turn trigger the cleavage of the target mRNA. Here, using a short hairpin RNA-expression library, we identified a DEAD-box helicase 3, DDX3, as an essential factor involved in RNAi pathway and revealed that DDX3 is colocalized with Ago2, an essential factor in RNAi pathway that cleaves target mRNA. Results of experiments with a dominant negative mutant of DDX3 further confirmed that this factor affects the RNAi activity. Together, DDX3 functions to assure mammalian RNAi pathway. Together, our results indicate that DDX3 is a new key molecule to understand the molecular mechanism underlying RNAi pathway in mammals. PMID:23527197

  17. An Improved Box Theater

    NASA Astrophysics Data System (ADS)

    Huster, Michael E.

    2011-09-01

    While designing an optics lab for a conceptual physics course, I came across a "box theater" activity. The box theater is a pinhole camera obscura made from a box that students put over their heads and shoulders. I use the activity as a capstone experience to explain optical systems. (Classroom demonstrations of the camera obscura have been described by others.2) First, the students build and experiment with a camera obscura made from a plastic cup and a convex lens with a focal length of 7.5 cm, and then "wear" the box theater. The difficulty with the box theater is the dimness of the image. A cloth drape has to be hung from the bottom of the box around the shoulders of the students to prevent light leakage, and the students have to wait a few minutes for their eyes to adjust to the darkness.

  18. GLOVE BOX ATTACHMENT

    DOEpatents

    Butts, H.L.

    1962-02-13

    This invention comprises a housing unit to be fitted between a glove box port and a glove so that a slidable plate within the housing seals off the glove box port for evacuation of the glove box without damage to the glove. The housing and the glove may be evacuated without damage to the glove since movement of the glove is restricted during evacuation by the slidable plate. (AEC)

  19. HER4 D-box sequences regulate mitotic progression and degradation of the nuclear HER4 cleavage product, s80HER4

    PubMed Central

    Strunk, Karen E.; Husted, Carty; Miraglia, Leah C.; Sandahl, Melissa; Rearick, William A.; Hunter, Debra M.; Earp, H. Shelton; Muraoka-Cook, Rebecca S.

    2010-01-01

    Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80HER4, containing nuclear localization sequences) and results in G2/M delay by unknown signaling mechanisms. We report herein that s80HER4 contains a functional cyclin B-like sequence known as a D-box, which targets proteins for degradation by APC/C, a multisubunit ubiquitin ligase. s80HER4 ubiquitination and ptoteosomal degradation occurred during mitosis but not during S-phase. Inhibition of an APC subunit (APC2) using siRNA knock-down impaired s80HER4 degradation. Mutation of the s80HER4 D-box sequence stabilized s80HER4 during mitosis, and s80HER4-dependent growth inhibition via G2/M delay was significantly greater with the D-box mutant. Polyomvirus middle-T antigen-transformed HC11 cells expressing s80HER4 resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80HER4 or the empty vector. Cells expressing s80HER4 with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle-dependent degradation of s80HER4 limits its growth inhibitory action, and stabilization of s80HER4 enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control. PMID:17638867

  20. [Parasitic dead-end: update].

    PubMed

    Magnaval, J F

    2006-08-01

    Parasitic dead-ends occur when a parasite is unable to establish a permanent interaction in an unnatural host. Although the likelihood of successful reproduction by the pathogenic agent is nul, parasitic dead-end heralds capture of new parasites and therefore expansion of the host range. Angiostrongyliasis due to A. cantonensis or A. costaricensis, anisakiasis, Ancylostoma caninum infection, gnathostomiasis and sparganosis are undoubtedly emerging zoonoses of particular medical interest. Prevention of these diseases relies on abstinence from eating raw meat from invertebrates or cold-blooded (poikilotherm) vertebrates (e.g. used in exotic dishes). These guidelines must be included in recommendations to travelers. PMID:16999036

  1. RNA chaperones buffer deleterious mutations in E. coli

    PubMed Central

    Rudan, Marina; Schneider, Dominique; Warnecke, Tobias; Krisko, Anita

    2015-01-01

    Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability. DOI: http://dx.doi.org/10.7554/eLife.04745.001 PMID:25806682

  2. Who are the Unclaimed Dead?

    PubMed

    Quinet, Kenna; Nunn, Samuel; Ballew, Alfarena

    2016-01-01

    Unclaimed dead are deceased persons with no known next of kin (NoK) or NoK was located but did not claim the deceased. Unclaimed dead in Marion County, Indiana, 2004-2011, are examined. Comparisons are provided of the unclaimed to the claimed dead population and county death patterns. Race, gender, marital status, age, location, manner and cause of death, NoK, and days to disposition are analyzed. The unclaimed dead were disproportionately male, slightly more likely to be Black, younger at death, died from natural causes, had unknown marital status, were equally likely as not to have NoK, did not die in a hospital, and were subject to autopsy. Nearly half the unclaimed had NoK who did not claim the body; the other half had no identifiable NoK. Unclaimed were more likely to have an autopsy and to die from external causes. Most unclaimed were identified by means outside fingerprints or DNA. PMID:26524620

  3. Boxing with neutrino oscillations

    NASA Astrophysics Data System (ADS)

    Wagner, D. J.; Weiler, Thomas J.

    1999-06-01

    We develop a characterization of neutrino oscillations based on the coefficients of the oscillating terms. These coefficients are individually observable; although they are quartic in the elements of the unitary mixing matrix, they are independent of the conventions chosen for the angle and phase parametrization of the mixing matrix. We call these reparametrization-invariant observables ``boxes'' because of their geometric relation to the mixing matrix, and because of their association with the Feynman box diagram that describes oscillations in field theory. The real parts of the boxes are the coefficients for the CP- or T-even oscillation modes, while the imaginary parts are the coefficients for the CP- or T-odd oscillation modes. Oscillation probabilities are linear in the boxes, so measurements can straightforwardly determine values for the boxes (which can then be manipulated to yield magnitudes of mixing matrix elements). We examine the effects of unitarity on the boxes and discuss the reduction of the number of boxes to a minimum basis set. For the three-generation case, we explicitly construct the basis. Using the box algebra, we show that CP violation may be inferred from measurements of neutrino flavor mixing even when the oscillatory factors have averaged. The framework presented here will facilitate general analyses of neutrino oscillations among n>=3 flavors.

  4. Boxing with neutrino oscillations

    SciTech Connect

    Wagner, D.J.; Weiler, T.J.

    1999-06-01

    We develop a characterization of neutrino oscillations based on the coefficients of the oscillating terms. These coefficients are individually observable; although they are quartic in the elements of the unitary mixing matrix, they are independent of the conventions chosen for the angle and phase parametrization of the mixing matrix. We call these reparametrization-invariant observables {open_quotes}boxes{close_quotes} because of their geometric relation to the mixing matrix, and because of their association with the Feynman box diagram that describes oscillations in field theory. The real parts of the boxes are the coefficients for the {ital CP}- or {ital T}-even oscillation modes, while the imaginary parts are the coefficients for the {ital CP}- or {ital T}-odd oscillation modes. Oscillation probabilities are linear in the boxes, so measurements can straightforwardly determine values for the boxes (which can then be manipulated to yield magnitudes of mixing matrix elements). We examine the effects of unitarity on the boxes and discuss the reduction of the number of boxes to a minimum basis set. For the three-generation case, we explicitly construct the basis. Using the box algebra, we show that {ital CP} violation may be inferred from measurements of neutrino flavor mixing even when the oscillatory factors have averaged. The framework presented here will facilitate general analyses of neutrino oscillations among n{ge}3 flavors. {copyright} {ital 1999} {ital The American Physical Society}

  5. Thinking outside the Box

    ERIC Educational Resources Information Center

    Fanshawe, Simon; Sriskandarajah, Dhananjayan

    2010-01-01

    Britain is not only more diverse than ever before, but that diversity itself is growing more diverse. Britain's simplistic "tick-box" approach to identity is in danger of inhibiting the very equality it seeks to promote. To question the tick-box is not to accuse local authorities of "political correctness gone mad". The notion of political…

  6. The C-terminal region of the RNA helicase CshA is required for the interaction with the degradosome and turnover of bulk RNA in the opportunistic pathogen Staphylococcus aureus

    PubMed Central

    Giraud, Caroline; Hausmann, Stéphane; Lemeille, Sylvain; Prados, Julien; Redder, Peter; Linder, Patrick

    2015-01-01

    Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes. PMID:25997461

  7. FAQ: West Nile Virus and Dead Birds

    MedlinePlus

    ... Education Public Service Videos West Nile Virus & Dead Birds Recommend on Facebook Tweet Share Compartir On this ... dead bird sightings to local authorities. How do birds get infected with West Nile virus? West Nile ...

  8. Structure and mechanism of the T-box riboswitches

    PubMed Central

    Zhang, Jinwei

    2015-01-01

    In most Gram-positive bacteria, including many clinically devastating pathogens from genera such as Bacillus, Clostridium, Listeria and Staphylococcus, T-box riboswitches sense and regulate intracellular availability of amino acids through a multipartite mRNA-tRNA interaction. The T-box mRNA leaders respond to nutrient starvation by specifically binding cognate tRNAs and sensing whether the bound tRNA is aminoacylated, as a proxy for amino acid availability. Based on this readout, T-boxes direct a transcriptional or translational switch to control the expression of downstream genes involved in various aspects of amino acid metabolism: biosynthesis, transport, aminoacylation, transamidation, etc. Two decades after its discovery, the structural and mechanistic underpinnings of the T-box riboswitch were recently elucidated, producing a wealth of insights into how two structured RNAs can recognize each other with robust affinity and exquisite selectivity. The T-box paradigm exemplifies how natural non-coding RNAs can interact not just through sequence complementarity, but can add molecular specificity by precisely juxtaposing RNA structural motifs, exploiting inherently flexible elements and the biophysical properties of post-transcriptional modifications, ultimately achieving a high degree of shape complementarity through mutually induced fit. The T-box also provides a proof-of-principle that compact RNA domains can recognize minute chemical changes (such as tRNA aminoacylation) on another RNA. The unveiling of the structure and mechanism of the T-box system thus expands our appreciation of the range of capabilities and modes of action of structured non-coding RNAs, and hints at the existence of networks of non-coding RNAs that communicate through both, structural and sequence specificity. PMID:25959893

  9. Glove box shield

    DOEpatents

    Brackenbush, Larry W.; Hoenes, Glenn R.

    1981-01-01

    According to the present invention, a shield for a glove box housing radioactive material is comprised of spaced apart clamping members which maintain three overlapping flaps in place therebetween. There is a central flap and two side flaps, the side flaps overlapping at the interior edges thereof and the central flap extending past the intersection of the side flaps in order to insure that the shield is always closed when the user withdraws his hand from the glove box. Lead loaded neoprene rubber is the preferred material for the three flaps, the extent of lead loading depending upon the radiation levels within the glove box.

  10. Glove box shield

    DOEpatents

    Brackenbush, L.W.; Hoenes, G.R.

    A shield for a glove box housing radioactive material is comprised of spaced apart clamping members which maintain three overlapping flaps in place therebetween. There is a central flap and two side flaps, the side flaps overlapping at the interior edges thereof and the central flap extending past the intersection of the side flaps in order to insure that the shield is always closed when the user wthdraws his hand from the glove box. Lead loaded neoprene rubber is the preferred material for the three flaps, the extent of lead loading depending upon the radiation levels within the glove box.

  11. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  12. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 5 2012-01-01 2012-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  13. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 5 2014-01-01 2014-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  14. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 5 2011-01-01 2011-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  15. 7 CFR 322.29 - Dead bees.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 5 2013-01-01 2013-01-01 false Dead bees. 322.29 Section 322.29 Agriculture..., DEPARTMENT OF AGRICULTURE BEES, BEEKEEPING BYPRODUCTS, AND BEEKEEPING EQUIPMENT Importation and Transit of Restricted Articles § 322.29 Dead bees. (a) Dead bees imported into or transiting the United States must...

  16. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 4 2013-07-01 2013-07-01 false Deadly force. 632.4 Section 632.4 National... INVESTIGATIONS USE OF FORCE BY PERSONNEL ENGAGED IN LAW ENFORCEMENT AND SECURITY DUTIES § 632.4 Deadly force. (a) Deadly force is destructive physical force directed against a person or persons (e.g., firing a...

  17. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 4 2014-07-01 2013-07-01 true Deadly force. 632.4 Section 632.4 National... INVESTIGATIONS USE OF FORCE BY PERSONNEL ENGAGED IN LAW ENFORCEMENT AND SECURITY DUTIES § 632.4 Deadly force. (a) Deadly force is destructive physical force directed against a person or persons (e.g., firing a...

  18. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 4 2011-07-01 2011-07-01 false Deadly force. 632.4 Section 632.4 National... INVESTIGATIONS USE OF FORCE BY PERSONNEL ENGAGED IN LAW ENFORCEMENT AND SECURITY DUTIES § 632.4 Deadly force. (a) Deadly force is destructive physical force directed against a person or persons (e.g., firing a...

  19. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 4 2010-07-01 2010-07-01 true Deadly force. 632.4 Section 632.4 National... INVESTIGATIONS USE OF FORCE BY PERSONNEL ENGAGED IN LAW ENFORCEMENT AND SECURITY DUTIES § 632.4 Deadly force. (a) Deadly force is destructive physical force directed against a person or persons (e.g., firing a...

  20. 32 CFR 632.4 - Deadly force.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 4 2012-07-01 2011-07-01 true Deadly force. 632.4 Section 632.4 National... INVESTIGATIONS USE OF FORCE BY PERSONNEL ENGAGED IN LAW ENFORCEMENT AND SECURITY DUTIES § 632.4 Deadly force. (a) Deadly force is destructive physical force directed against a person or persons (e.g., firing a...

  1. DDX19A Senses Viral RNA and Mediates NLRP3-Dependent Inflammasome Activation.

    PubMed

    Li, Jiangnan; Hu, Liang; Liu, Yuanyuan; Huang, Li; Mu, Yang; Cai, Xuehui; Weng, Changjiang

    2015-12-15

    The NLRP3 inflammasome plays a major role in innate immune responses by activating caspase-1, resulting in secretion of IL-1β and inflammatory pathologic responses. Viral RNA can induce NLRP3 inflammasome activation. However, none of the components of NLRP3 inflammasome has the ability to bind viral RNA. Therefore, it had been proposed that there might have been some unidentified cytosolic RNA sensors that could bind viral RNA and NLRP3 to initiate NLRP3 inflammasome activation. In this study, DDX19A, a member of the DEAD/H-box protein family, was identified as a novel component of NLRP3 inflammasome using arterivirus infection as a model. We found that DDX19A interacted with viral RNA and NLRP3. Knockdown of DDX19A expression efficiently inhibited procaspase-1 cleavage and IL-1β secretion in porcine reproductive and respiration syndrome virus (PRRSV)-infected or PRRSV RNA-stimulated primary porcine alveolar macrophages. Overall, DDX19A was identified as a novel cytosolic RNA sensor that bridged PRRSV RNA and NLRP3 to activate NLRP3 inflammasome. PMID:26538395

  2. Climate in a Box

    NASA Video Gallery

    NASA's Climate in a Box Project is exploring the utility of supercomputers in providing a complete, pre-packaged, ready-to-use toolkit of climate research products and on-demand access to a high-pe...

  3. Boxing with Neutrino Oscillations

    NASA Astrophysics Data System (ADS)

    Wagner, Dj; Weiler, Thomas J.

    1998-03-01

    We have developed a model-independent ``box'' parameterization of neutrino oscillations. Oscillation probabilities are linear in these new parameters, so measurements can straighforwardly determine the box parameters which can then be manipulated to yield magnitudes of mixing matrix elements. We will present these new parameters and examine the effects of unitarity which reduce the number of independent parameters to the minimum set. The framework presented here will facilitate general analyses of neutrino oscillations among n >= 3 flavors.

  4. Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation

    PubMed Central

    Weston, Andrew; Sommerville, John

    2006-01-01

    The DEAD-box RNA helicase Xp54 is an integral component of the messenger ribonucleoprotein (mRNP) particles of Xenopus oocytes. In oocytes, several abundant proteins bind pre-mRNA transcripts to modulate nuclear export, RNA stability and translational fate. Of these, Xp54, the mRNA-masking protein FRGY2 and its activating protein kinase CK2α, bind to nascent transcripts on chromosome loops, whereas an Xp54-associated factor, RapA/B, binds to the mRNP complex in the cytoplasm. Over-expression, mutation and knockdown experiments indicate that Xp54 functions to change the conformation of mRNP complexes, displacing one subset of proteins to accommodate another. The sequence of Xp54 is highly conserved in a wide spectrum of organisms. Like Xp54, Drosophila Me31B and Caenorhabditis CGH-1 are required for proper meiotic development, apparently by regulating the translational activation of stored mRNPs and also for sorting certain mRNPs into germplasm-containing structures. Studies on yeast Dhh1 and mammalian rck/p54 have revealed a key role for these helicases in mRNA degradation and in earlier remodelling of mRNP for entry into translation, storage or decay pathways. The versatility of Xp54 and related helicases in modulating the metabolism of mRNAs at all stages of their lifetimes marks them out as key regulators of post-transcriptional gene expression. PMID:16769775

  5. Automatic box loader

    DOEpatents

    Eldridge, Harry H.; Jones, Robert A.; Lindner, Gordon M.; Hight, Paul H.

    1976-01-01

    This invention relates to a system for repetitively forming an assembly consisting of a single layer of tubes and a row of ferromagnetic armatures underlying the same, electromagnetically conveying the resulting assembly to a position overlying a storage box, and depositing the assembly in the box. The system includes means for simultaneously depositing a row of the armatures on the inclined surface of a tube retainer. Tubes then are rolled down the surface to form a single tube layer bridging the armatures. A magnet assembly carrying electromagnets respectively aligned with the armatures is advanced close to the tube layer, and in the course of this advance is angularly displaced to bring the pole pieces of the electromagnets into parallelism with the tube layer. The magnets then are energized to pick up the assembly. The loaded magnet assembly is retracted to a position overlying the box, and during this retraction is again displaced to bring the pole pieces of the electromagnets into a horizontal plane. Means are provided for inserting the loaded electromagnets in the box and then de-energizing the electromagnets to deposit the assembly therein. The system accomplishes the boxing of fragile tubes at relatively high rates. Because the tubes are boxed as separated uniform layers, subsequent unloading operations are facilitated.

  6. Cable Tester Box

    NASA Technical Reports Server (NTRS)

    Lee, Jason H.

    2011-01-01

    Cables are very important electrical devices that carry power and signals across multiple instruments. Any fault in a cable can easily result in a catastrophic outcome. Therefore, verifying that all cables are built to spec is a very important part of Electrical Integration Procedures. Currently, there are two methods used in lab for verifying cable connectivity. (1) Using a Break-Out Box and an ohmmeter this method is time-consuming but effective for custom cables and (2) Commercial Automated Cable Tester Boxes this method is fast, but to test custom cables often requires pre-programmed configuration files, and cables used on spacecraft are often uniquely designed for specific purposes. The idea is to develop a semi-automatic continuity tester that reduces human effort in cable testing, speeds up the electrical integration process, and ensures system safety. The JPL-Cable Tester Box is developed to check every single possible electrical connection in a cable in parallel. This system indicates connectivity through LED (light emitting diode) circuits. Users can choose to test any pin/shell (test node) with a single push of a button, and any other nodes that are shorted to the test node, even if they are in the same connector, will light up with the test node. The JPL-Cable Tester Boxes offers the following advantages: 1. Easy to use: The architecture is simple enough that it only takes 5 minutes for anyone to learn how operate the Cable Tester Box. No pre-programming and calibration are required, since this box only checks continuity. 2. Fast: The cable tester box checks all the possible electrical connections in parallel at a push of a button. If a cable normally takes half an hour to test, using the Cable Tester Box will improve the speed to as little as 60 seconds to complete. 3. Versatile: Multiple cable tester boxes can be used together. As long as all the boxes share the same electrical potential, any number of connectors can be tested together.

  7. Assessing dead space. A meaningful variable?

    PubMed

    Hedenstierna, G; Sandhagen, B

    2006-06-01

    The recording of dead space will give information on how much of total ventilation that reaches both ventilated and perfused alveoli and thus allows gas exchange between alveoli and pulmonary blood. Realising that CO2 retention can be an effect not only of low total ventilation but also of increased dead space is one important information. Moreover, dead space will give insight into the matching of ventilation and perfusion. This is because dead space is affected by a number of factors: 1) tubings and valves that the subject has to rebreath through (apparatus dead space), 2/ Airways (anatomical dead space), 3/ Non-perfused but ventilated alveoli, e.g. pulmonary embolus (alveolar dead space), 4/ Excessive ventilation of alveoli in relation to their perfusion that can be seen in chronic obstructive lung disease (another form of alveolar dead space), and 5/ So called "shunt dead space" that is an erroneous description of right to left lung shunt that brings the higher CO2 concentration in venous blood to the arterial side thereby producing an arterial-to-end-tidal CO2 difference. The dead spaces 2-5 are called physiological dead space. The recording of dead spaces can be done according to the Riley three-compartment model or by analysis of the expired CO2 curve. However, both are subjected to potential errors that have to be considered to make a dead space recording meaningful. A correct measurement and calculation of the dead space will give valuable information on the ventilatory support of the critically ill patient and can also be a valuable diagnostic tool. It should therefore not be forgotten in the intensive care setting. PMID:16682925

  8. Are Brain Dead Individuals Dead? Grounds for Reasonable Doubt.

    PubMed

    Brugger, E Christian

    2016-06-01

    According to the biological definition of death, a human body that has not lost the capacity to holistically organize itself is the body of a living human individual. Reasonable doubt against the conclusion that it has lost the capacity exists when the body appears to express it and no evidence to the contrary is sufficient to rule out reasonable doubt against the conclusion that the apparent expression is a true expression (i.e., when the conclusion that what appears to be holistic organization is in fact holistic organization remains a reasonable explanatory hypothesis in light of the best evidence to the contrary). This essay argues that the evidence and arguments against the conclusion that the signs of complex bodily integration exhibited in ventilated brain dead bodies are true expressions of somatic integration are unpersuasive; that is, they are not adequate to exclude reasonable doubt against the conclusion that BD bodies are dead. Since we should not treat as corpses what for all we know might be living human beings, it follows that we have an obligation to treat BD individuals as if they were living human beings. PMID:27075192

  9. Is Piaget's epistemic subject dead?

    NASA Astrophysics Data System (ADS)

    Lawson, Anton E.

    Niaz (1990) presents arguments in favor of the retention of Piaget's epistemic subject as a theoretical construct to guide research and practice in science education and psychology. The intent of this article is to point out the weaknesses of those arguments and to suggest that the weight of evidence argues against the existence of the logical thinker postulated by Piaget. Therefore, contrary to Niaz's conclusion that the acceptance of Piaget's epistemic subject will facilitate the development of cognitive theories with greater explanatory power, the conclusion is reached that Piaget's epistemic subject is dead and that continued acceptance of this aspect of Piagetian theory would be counterproductive.

  10. The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing

    PubMed Central

    Zhang, Zhao; Wang, Jie; Schultz, Nadine; Zhang, Fan; Parhad, Swapnil S.; Tu, Shikui; Vreven, Thom; Zamore, Phillip D.; Weng, Zhiping; Theurkauf, William E.

    2014-01-01

    Summary piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino co-localizes to germline nuclear foci with Rai1/DXO related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced, and that rhino, cuff and uap56 mutations increase expression of spliced cluster transcripts over 100 fold. LacI∷Rhino fusion protein binding suppresses splicing of a reporter transgene, and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing, and speculate that stalled splicing differentiates piRNA precursors from mRNAs. PMID:24906152

  11. A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

    PubMed

    Lu, Junyan; Jiang, Chenxiao; Li, Xiaojing; Jiang, Lizhi; Li, Zengxia; Schneider-Poetsch, Tilman; Liu, Jianwei; Yu, Kunqian; Liu, Jun O; Jiang, Hualiang; Luo, Cheng; Dang, Yongjun

    2015-12-01

    Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation. However, the mechanism of its coupled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release controlled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA unwinding. Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases. PMID:26464436

  12. Eating the dead in Madagascar.

    PubMed

    Campbell, Gwyn

    2013-12-01

    Cannibalism has been poorly understood and has seldom been studied, since it was often suppressed by missionaries and colonial administrators, and very few societies still practise it. Cannibalistic practices are more complex than was originally thought. They may be supported in societies under stress or in times of famine, to reflect aggression and antisocial behaviour (in cases where the bodies of enemies killed in battle or people who have harmed the family are eaten), or to honour a dead kinsman. It was, for example, noted in Madagascar during the imperial campaigns of Ranavalona I in the period 1829 - 1853. Two types of cannibalism have been described: exocannibalism, where enemies were consumed, and endocannibalism, where dead relatives were eaten to assist their passing to the world of the ancestors, or to prolong contact with beloved and admired family members and absorb their good qualities. This article reviews some of the beliefs and motivations that surrounded the cannibalistic practices of the people of Madagascar in the 19th century.  PMID:24300654

  13. Upstream box/TATA box order is the major determinant of the direction of transcription.

    PubMed Central

    Xu, L C; Thali, M; Schaffner, W

    1991-01-01

    Mammalian gene promoters for transcription by RNA polymerase II are typically organized in the following order: upstream sequence motif(s)/TATA box/initiation site. Here we report studies in which the order, orientation and DNA sequences of these three elements are varied to determine how these affect polarity of transcription. We have constructed promoters with an 'octamer' upstream sequence ATTTGCAT (or its complement ATGCAAAT) in combination with several different TATA boxes and initiation (cap) sites, and tested these promoters in transfection experiments with cultured cells. TATA boxes derived from the adenovirus major late promoter (TATAAAA), immunoglobulin kappa light chain (TTATATA) and heavy chain (TAAATATA) promoter functioned equally well or even better when inverted. Only the beta-globin TATA box (CATAAAA) was poorly active when inverted. In addition, a symmetrical TATA box (TATATATA) derived from a casein gene was very active. Our results suggest that the asymmetry of most TATA boxes (consensus TATAAAA) is not a primary determinant of the polarity of transcription. We also found that the initiation (cap) site, which usually consists of an adenine embedded in a pyrimidine-rich region (PyPyCAPyPyPyPyPy), was permissive towards sequence alterations; even a randomly composed sequence worked well. However, an inverted, hence purine-rich, cap site reduced transcript levels to 1/7th, as did an oligo G sequence. Irrespective of the presence of a cap site, the configuration: 'TATA box/octamer' yielded a strong leftward, rather than rightward transcription. From this, we conclude that the polarity of transcription is primarily determined by the linear order of an upstream sequence relative to a TATA box, rather than by the individual orientations of either of these two elements. Images PMID:1762900

  14. Evaluating dead lodgepole pine for products

    SciTech Connect

    Fahey, T.D.

    1980-12-01

    Dead lodgepole pine is a resource in abundance in the intermountain region of the US. Possible uses for dead pine range from small power poles to fuel and fiber. The potential to use significant volumes depends on how well the resource meets specifications for products and the volume of products that the market will accept. In this report values for products that can be produced from dead trees are evaluated based on ovendry tons of fiber for both logs and products.

  15. Detection and quantification of RNA 2'-O-methylation and pseudouridylation.

    PubMed

    Huang, Chao; Karijolich, John; Yu, Yi-Tao

    2016-07-01

    RNA-guided RNA modification is a naturally occurring process that introduces 2'-O-methylation and pseudouridylation into rRNA, spliceosomal snRNA and several other types of RNA. The Box C/D ribonucleoproteins (RNP) and Box H/ACA RNP, each containing one unique guide RNA (Box C/D RNA or Box H/ACA RNA) and a set of core proteins, are responsible for 2'-O-methylation and pseudouridylation respectively. Box C/D RNA and Box H/ACA RNA provide the modification specificity through base pairing with their RNA substrate. These post-transcriptional modifications could profoundly alter the properties and functions of substrate RNAs. Thus it is desirable to establish reliable and standardized modification methods to study biological functions of modified nucleotides in RNAs. Here, we present several sensitive and efficient methods and protocols for detecting and quantifying post-transcriptional 2'-O-methylation and pseudouridylation. PMID:26853326

  16. Thinking "Inside" the Box

    ERIC Educational Resources Information Center

    Jeffries, Carolyn

    2011-01-01

    The authors conducted a test to determine whether they could incorporate a discovery box into a preschool setting was successful. It stimulated the students' natural inquiry processes while promoting understanding of healthy foods and allowing for practice of fine-motor skills. It was easily incorporated into the curriculum and classroom space.…

  17. Teaching with Box Tops.

    ERIC Educational Resources Information Center

    Raiser, Lynne; D'Zamko, Mary Elizabeth

    1984-01-01

    Using environmental materials (such as the phone book and placemats from fast food restaurants) can be a motivating way to teach learning disabled students skills and concepts, as shown in an approach to reading, math, science and nutrition, and social studies instruction using a JELL-O brand gelatin box. (CL)

  18. PHOTOCHEMICAL BOX MODEL (PBM)

    EPA Science Inventory

    This magnetic tape contains the FORTRAN source code, sample input data, and sample output data for the Photochemical Box Model (PBM). The PBM is a simple stationary single-cell model with a variable height lid designed to provide volume-integrated hour averages of O3 and other ph...

  19. Drawing inside the Box

    ERIC Educational Resources Information Center

    Franklin, Ranella

    2007-01-01

    When working with very young children and/or students with special needs, it is beneficial for teachers to think "outside the box" in order to preserve and enhance a child's natural curiosity. In an effort to teach young children to control their drawing tools, they are often presented with coloring book-type pages and instructed to "stay inside…

  20. Cereal Box Totems.

    ERIC Educational Resources Information Center

    Jones, AnnMarie

    2002-01-01

    Presents a multicultural project used with fourth-grade students in which they created a three-dimensional totem pole using leftover cereal boxes. Discusses in detail how to create the totem pole. Explains that students learned about Northwest American Indians in class. (CMK)

  1. Shoe Box Circuits

    ERIC Educational Resources Information Center

    Sandifer, Cody

    2009-01-01

    Students' eyes grow wide with wonder as they get a motor to work or make a bulb light for the first time. As these daunting feats of electrical engineering remind us, teaching electricity is invariably rewarding and worthwhile. In this inquiry-based science project, elementary students work in pairs to design and wire a shoe box "room" that meets…

  2. "Can" the Black Box

    ERIC Educational Resources Information Center

    Lestingi, Francis S.

    1975-01-01

    Describes the use of the "Arcane (mysterious) Can" which is a "tin" can which is permanently sealed, both air- and water-tight, by means of a home canning device. The canning procedure permits the use of a large variety of materials which can not be utilized in the ordinary mystery box. This Can activity is valuable for illustrating in an…

  3. Hydrophobic, Porous Battery Boxes

    NASA Technical Reports Server (NTRS)

    Bragg, Bobby J.; Casey, John E., Jr.

    1995-01-01

    Boxes made of porous, hydrophobic polymers developed to contain aqueous potassium hydroxide electrolyte solutions of zinc/air batteries while allowing air to diffuse in as needed for operation. Used on other types of batteries for in-cabin use in which electrolytes aqueous and from which gases generated during operation must be vented without allowing electrolytes to leak out.

  4. 6. VIEW OF INTERIOR GLOVE BOX DURING CONSTRUCTION. GLOVE BOXES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. VIEW OF INTERIOR GLOVE BOX DURING CONSTRUCTION. GLOVE BOXES CONTAINED ALL PRODUCTION OPERATIONS AND WERE INTERCONNECTED BY CONVEYORS. (9/21/59) - Rocky Flats Plant, Plutonium Fabrication, Central section of Plant, Golden, Jefferson County, CO

  5. Looking Southwest at Reactor Box Furnaces With Reactor Boxes and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Looking Southwest at Reactor Box Furnaces With Reactor Boxes and Repossessed Uranium in Recycle Recovery Building - Hematite Fuel Fabrication Facility, Recycle Recovery Building, 3300 State Road P, Festus, Jefferson County, MO

  6. Degradation of Dead Microbial Biomass in a Marine Sediment

    PubMed Central

    Novitsky, James A.

    1986-01-01

    The availability of dead microbial biomass in a marine beach sand to degradation and mineralization was examined. Microbial sand populations were labeled with [14C]glutamic acid, [3H]adenine, or [3H]thymidine and killed with chloroform. Live sand or seawater (or both) was added to the sterile labeled sand, and biochemical components of the populations were monitored for 10 days. Labeled RNA was degraded more quickly than labeled DNA, but both nucleic acids were degraded to approximately the same extent (60 to 70%). 3H2O was a major acid-soluble breakdown product. RNA (and possibly DNA) breakdown products were reincorporated into DNA (and possibly RNA) during the incubation period. In addition to metabolite salvage, 32% of the total macromolecular 14C was respired in the 10-day period regardless of whether sand or seawater was used as the inoculum. Respiration was essentially complete in 3 days, whereas nucleic acid degradation continued throughout the 10-day incubation. The results indicate that dead microbial biomass is a labile component of the sediment ecosystem. PMID:16347148

  7. More Dead than Dead: Perceptions of Persons in the Persistent Vegetative State

    ERIC Educational Resources Information Center

    Gray, Kurt; Knickman, T. Anne; Wegner, Daniel M.

    2011-01-01

    Patients in persistent vegetative state (PVS) may be biologically alive, but these experiments indicate that people see PVS as a state curiously more dead than dead. Experiment 1 found that PVS patients were perceived to have less mental capacity than the dead. Experiment 2 explained this effect as an outgrowth of afterlife beliefs, and the…

  8. Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

    PubMed Central

    Kaake, Robyn; Dawson, Anthony R.; Matheos, Dina; Nagashima, Kunio; Sitlani, Parth; Patterson, Kurt; Chang, Ivan; Huang, Lan; Sandmeyer, Suzanne

    2015-01-01

    Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs. PMID:26421679

  9. Molecular recognition of RhlB and RNase D in the Caulobacter crescentus RNA degradosome

    PubMed Central

    Voss, Jarrod E.; Luisi, Ben F.; Hardwick, Steven W.

    2014-01-01

    The endoribonuclease RNase E is a key enzyme in RNA metabolism for many bacterial species. In Escherichia coli, RNase E contributes to the majority of RNA turnover and processing events, and the enzyme has been extensively characterized as the central component of the RNA degradosome assembly. A similar RNA degradosome assembly has been described in the α-proteobacterium Caulobacter crescentus, with the interacting partners of RNase E identified as the Kreb's cycle enzyme aconitase, a DEAD-box RNA helicase RhlB and the exoribonuclease polynucleotide phosphorylase. Here we report that an additional degradosome component is the essential exoribonuclease RNase D, and its recognition site within RNase E is identified. We show that, unlike its E. coli counterpart, C. crescentus RhlB interacts directly with a segment of the N-terminal catalytic domain of RNase E. The crystal structure of a portion of C. crescentus RNase E encompassing the helicase-binding region is reported. This structure reveals that an inserted segment in the S1 domain adopts an α-helical conformation, despite being predicted to be natively unstructured. We discuss the implications of these findings for the organization and mechanisms of the RNA degradosome. PMID:25389270

  10. DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions.

    PubMed

    Huang, Wendy; Thomas, Benjamin; Flynn, Ryan A; Gavzy, Samuel J; Wu, Lin; Kim, Sangwon V; Hall, Jason A; Miraldi, Emily R; Ng, Charles P; Rigo, Frank; Rigo, Frank W; Meadows, Sarah; Montoya, Nina R; Herrera, Natalia G; Domingos, Ana I; Rastinejad, Fraydoon; Myers, Richard M; Fuller-Pace, Frances V; Bonneau, Richard; Chang, Howard Y; Acuto, Oreste; Littman, Dan R

    2015-12-24

    T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases. PMID:26675721