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At least 19 recordsLinked to original sources

PubMed2000

A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA.

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1-18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1-18 yeast required the RNA2 5' noncoding region (NCR), which also conferred a ded1-18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5' NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1-18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5' NCRs that appears conserved among bromoviruses. The 5' attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Base Sequence↗

PubMed1996

Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35 degrees C, but not at 37 degrees C. A revertant of srm1-1 ded1-21 that became able to grow at 37 degrees C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2.

Cell Cycle Proteins↗

PubMed2001

The Ded1 DEAD box helicase interacts with Chk1 and Cdc2.

Ded1 is a fission yeast DEAD box protein involved in translation. We isolated Ded1 in a screen for multi-copy suppressors of a cold-sensitive, loss-of-function mutant of the cyclin-dependent kinase Cdc2. The checkpoint protein kinase Chk1, required for cell cycle arrest in response to DNA damage, was also isolated in this screen. Ded1 interacts with Chk1 in a two-hybrid screen, and this physical interaction can be recapitulated in Schizosaccharomyces pombe. The Ded1 polypeptide is modified in response to heat shock and depletion of carbon source. These two stressors appear to cause different modifications. Thus, the Ded1 protein appears to respond to particular types of cellular stress and may influence the activity of Cdc2 as a result.

CDC2 Protein Kinase↗

PubMed1997

The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae.

The translation initiation factor eIF4E mediates the binding of the small ribosomal subunit to the cap structure at the 5' end of the mRNA. In Saccharomyces cerevisiae, the cap-binding protein eIF4E is mainly associated with eIF4G, forming the cap-binding complex eIF4F. Other proteins are detected upon purification of the complex on cap-affinity columns. Among them is p20, a protein of unknown function encoded by the CAF20 gene. Here, we show a negative regulatory role for the p20 protein in translation initiation. Deletion of CAF20 partially suppresses mutations in translation initiation factors. Overexpression of the p20 protein results in a synthetic enhancement of translation mutation phenotypes. Similar effects are observed for mutations in the DED1 gene, which we have isolated as a multicopy suppressor of a temperature-sensitive eIF4E mutation. The DED1 gene encodes a putative RNA helicase of the DEAD-box family. The analyses of its suppressor activity, of polysome profiles of ded1 mutant strains, and of synthetic lethal interactions with different translation mutants indicate that the Ded1 protein has a role in translation initiation in S. cerevisiae.

Cloning, Molecular↗

PubMed2006

Discriminatory RNP remodeling by the DEAD-box protein DED1.

DExH/D proteins catalyze NTP-driven rearrangements of RNA and RNA-protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between a large number of RNA and RNP complexes in a physiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which a non-sequence specific DExH/D protein can discriminately remodel similar RNA complexes. We have measured in vitro the disassembly of model RNPs by two distinct DExH/D proteins, DED1 and NPH-II. Both enzymes displace the U1 snRNP from a tightly bound RNA in an active, ATP-dependent fashion. However, DED1 cannot actively displace the protein U1A from its binding site, whereas NPH-II can. The dissociation rate of U1A dictates the rate by which DED1 remodels RNA complexes with U1A bound. We further show that DED1 disassembles RNA complexes with slightly altered U1A binding sites at different rates, but only when U1A is bound to the RNA. These findings suggest that the "inability" to actively displace other proteins from RNA can provide non-sequence specific DExH/D proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. In addition, our study illuminates possible mechanisms for protein displacement by DExH/D proteins.

Adenosine Triphosphate↗

PubMed2005

ATP- and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box protein DED1.

DEAD-box RNA helicases, which are involved in virtually all aspects of RNA metabolism, are generally viewed as enzymes that unwind RNA duplexes or disrupt RNA-protein interactions in an ATP-dependent manner. Here, we show in vitro that the DEAD-box protein DED1 from Saccharomyces cerevisiae promotes not only RNA unwinding but also strand annealing, the latter in such a profound fashion that the physical limit for a bimolecular association rate constant is approached. We further demonstrate that DED1 establishes an ATP-dependent steady state between unwinding and annealing, which enables the enzyme to modulate the balance between the two opposing activities through ATP and ADP concentrations. The ratio between unwinding and annealing and the degree to which both activities are ATP- and ADP-modulated are strongly influenced by structured as well as unstructured regions in the RNA substrate. Collectively, these findings expand the known functional repertoire of DEAD-box proteins and reveal the capacity of DED1 to remodel RNA in response to ADP and ATP concentrations by facilitating not only disruption but also formation of RNA duplexes.

Adenosine Diphosphate↗

PubMed2026

Acetic acid-induced translational repression involves eIF2B body formation and Ded1 sequestration into stress granules in yeast.

Elucidating the physiological impact of acetic acid stress and the corresponding yeast responses is essential for advancing fundamental biology and improving industrial alcoholic fermentation. Despite numerous genome-wide studies, information on the effects of acetic acid stress on yeast translational regulation remains limited. We found that a sublethal concentration of acetic acid (35 mM, 0.2% v/v) causes translational repression, accompanied by the formation of eIF2B bodies and the phosphorylation of eIF2α, both of which are involved in the regulation of translation initiation. Acetic acid also caused the sequestration of Ded1, a DEAD-box RNA helicase crucial for translation initiation, into stress granules. Removal of acetic acid restored translational activity and the proper localization of eIF2B and Ded1, indicating the reversibility of acetic acid-induced translational repression. Furthermore, when yeast cells were pretreated with 0.05% acetic acid, translational repression under subsequent 0.2% acetic acid stress was attenuated in wild-type cells but not in hrk1Δ cells. This indicates that Hrk1, a Pma1 activator, is required to sufficiently enhance tolerance to acetic acid-induced translational repression. These findings provide novel insights into the physiological effects of acetic acid stress on translational activity and translation-related factors in yeast cells.

Saccharomyces cerevisiae↗

PubMed2006

The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases.

Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.

DEAD-box RNA Helicases↗

PubMed2006

A novel Ded1-like RNA helicase interacts with the Y-box protein ctYB-1 in nuclear mRNP particles and in polysomes.

We have characterized a novel mRNA-binding protein, designated hrp84, in the dipteran Chironomus tentans and identified it as a DEAD-box RNA helicase. The protein contains the typical helicase core domain, a glycine-rich C-terminal part and a putative nuclear export signal in the N terminus. The protein belongs to the Ded1 subgroup of DEAD-box helicases, which is highly conserved from yeast (Ded1p) to mammals (DDX3). In tissue culture cells, hrp84 is present both in the nucleus and cytoplasm and, as shown by in vivo UV cross-linking, is bound to mRNA in both compartments. Immunoprecipitation experiments revealed that hpr84 is associated with the C. tentans homologue (ctYB-1) of the vertebrate Y-box protein YB-1 both in the nucleus and cytoplasm, and the two proteins also appear together in polysomes. The interaction is likely to be direct as shown by in vitro binding of purified components. We conclude that the mRNA-bound hrp84.ctYB-1 complex is formed in the nucleus and is translocated with mRNA into the cytoplasm and further into polysomes. As both Ded1 and YB-1 are known to regulate the initiation of translation, we propose that the RNA helicase-Y-box protein complex affects the efficiency of mRNA translation, presumably by modulating the conformation of the mRNP template.

Amino Acid Sequence↗

PubMed1985

Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region.

Genes of the baker's yeast Saccharomyces cerevisiae are densely clustered on 16 linear chromosomes. Here, I characterize a 1.8 kb region of chromosome XV containing the entire structural gene for the histidine biosynthetic enzyme imidazoleglycerolphosphate (IGP) dehydratase (his3) as well as the promoter sequences and 5'-proximal mRNA coding regions for the adjacent genes. The his3 gene encodes several mRNA species averaging 820 bases in length, all of which contain an open reading frame of 219 codons. The location of this open reading frame coincides with the his3 gene as defined by functional criteria, suggesting that the primary translation product of yeast IGP dehydratase has a molecular weight of 23,850. Phenotypic analysis of mutations constructed in vitro indicate that one of the adjacent genes (pet56) is required for mitochondrial function, whereas the other gene (ded1) is essential for cell viability. The pet56 and his3 genes are transcribed divergently from initiation sites that are separated by only 192 bp. Transcription of the ded1 gene is initiated only 130 bp beyond the 3'-end of the his3 mRNA coding region. These results suggest that these unrelated genes are located extremely close together and that the spacer regions between them consist largely of promoter and terminator sequences.

Amino Acid Sequence↗

PubMed1998

DNA unwinding in the CYC1 and DED1 yeast promoters.

The capacity of promoter DNA of two yeast genes to be unwound was studied. Both promoters, those of the CYC1 and DED1 genes, contain long oligopurine.oligopyrimidine (R.Y) tracts. The two promoters were cloned into negatively supercoiled plasmids, and their sensitivity to single-strand specific nuclease P1 was examined. Extensive P1 cleavage was located within the R.Y tracts, and cleavage sites were mapped. The extent of cleavage was only slightly dependent on P1 concentration, indicating a slow conversion of an intermediate form of DNA into the P1 reactive state. The cleavage required negative supercoiling and was suppressed by NaCl, MgCl2 and spermine. Two-dimensional topoisomer analysis showed that six superhelical turns were opened in the plasmids examined. The results indicate that at sufficient torsional stress, the R.Y tracts can intermittently undergo a transition into an unwound, ready-to-separate state. The oligopurine.oligopyrimidine tracts may thus serve as DNA unwinding centers in the gene promoters where they reside.

Base Sequence↗

PubMed1987

Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase.

To distinguish between mechanisms of eukaryotic transcriptional activation, we tested whether yeast upstream promoter elements can stimulate transcription by a heterologous transcription machinery, bacteriophage T7 RNA polymerase. The gal enhancer-like element recognized by GAL4 protein or the ded1 poly(dA-dT) element was placed upstream of the T7 promoter and his3 structural gene, and T7 RNA polymerase was produced in yeast cells. Under conditions where the gal element would normally be either activating or nonactivating, his3 transcription by T7 RNA polymerase was not stimulated above the level observed in the absence of any upstream element. In contrast, the ded1 poly(dA-dT) element stimulated transcription 7-fold, similar to the enhancement observed on the native ded1 promoter. Activation by the ded1 element thus may involve effects on the chromatin template that facilitate entry of the transcription machinery, whereas activation by the gal element may involve specific contacts between GAL4 and the transcriptional machinery.

DNA-Binding Proteins↗

PubMed1990

A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

Base Sequence↗

PubMed2000

A fission yeast general translation factor reveals links between protein synthesis and cell cycle controls.

In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression. These mutations define a single gene which we call ded1. We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species. Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex. When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins. We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G(1) arrest and subsequent mating. Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis.

Cell Cycle↗

PubMed1985

Yeast mRNA initiation sites are determined primarily by specific sequences, not by the distance from the TATA element.

We present evidence suggesting that accurate mRNA initiation in yeast cells, unlike their higher eukaryotic counterparts, is determined primarily by specific sequences downstream from the TATA element. First, changing the distance between the his3 TATA element and the initiation region does not affect the sites of initiation or the level of RNA. Second, reciprocal his3-ded1 and ded1-his3 hybrid promoters containing the upstream and TATA elements of one gene fused to the mRNA coding region of the other gene initiate transcription at sites defined by wild-type mRNA coding sequences, not by the distance from the TATA element. Third, when the his3 or ded1 promoter region is fused to position +2 of the his3 gene, transcripts initiated from a position equivalent to +1 are not observed. The results also suggest that the spacing between the TATA element and initiation site is relatively flexible; distance ranging from 40 to 90 bp appear to be functionally acceptable.

Base Sequence↗

PubMed2006

Unwinding single RNA molecules using helicases involved in eukaryotic translation initiation.

The small (40 S) subunit of the eukaryotic ribosome may have to scan more than 2000 nucleotides (>600 nm) from its 5'cap recruiting point on an mRNA molecule before initiating on a translation start codon. As with many other processes in living cells, including transcription, editing, mRNA splicing, pre-rRNA processing, RNA transport and RNA decay, scanning is facilitated by helicase activity. However, precise quantitative data on the molecular mechanism of scanning, including the roles of helicases, are lacking. Here, we describe a novel atomic force microscopy (AFM)-based procedure to examine the roles of two yeast helicases, eIF4A and Ded1, previously implicated in translation initiation by genetic and biochemical studies. Our results show that eIF4A, especially in the presence of its "cofactor" eIF4B, promotes ATP-dependent unwinding of localised secondary structure in long RNA molecules under tensional loading, albeit only at high protein:RNA ratios. Thus eIF4A can act to separate only a limited number of base-pairs, possibly via a steric unwinding mechanism. In contrast, Ded1 is more effective in reducing (by up to 50 pN at an AFM loading rate of 14 nNs(-1)) the force necessary to disrupt an RNA stem-loop, and thus shows significant kinetic competence to facilitate fast unwinding. These single molecule experiments indicate that Ded1 is likely to act as the more potent unwinding factor on natural mRNA substrates.

Adenosine Triphosphatases↗

PubMed1985

Naturally occurring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast.

pet56, his3, and ded1 are adjacent but unrelated genes located on chromosome XV of the yeast Saccharomyces cerevisiae. his3 and pet56 are transcribed in opposite directions from initiation sites separated by approximately equal to 200 base pairs. Under normal growth conditions, both genes are transcribed at a similar basal level. Deletion analysis of the his3 gene indicates that the upstream promoter element for constitutive expression is defined by a 17-base-pair region that contains 15 thymidine residues in the coding strand. Sequential deletions of the pet56 gene indicate that this same region is required for wild-type transcription levels. Thus, this poly(dA-dT) sequence acts bidirectionally to activate transcription of two unrelated genes. Transcription of the ded1 gene is initiated approximately equal to 300 base pairs downstream from the his3 gene, and it occurs at a 5-fold higher level. This gene contains a 34-base-pair region containing 28 thymidine residues in the coding strand located upstream from the ded1 TATA box. Deletion of this dA-dT stretch significantly reduces transcription below the wild-type level. Thus, for at least three different yeast genes, naturally occurring stretches of poly(dA-dT) serve as upstream promoter elements for constitutive expression. In addition, it appears that longer stretches of poly(dA-dT) are more effective upstream promoter elements. These transcriptional effects may be due to exclusion of nucleosomes from poly(dA-dT) regions.

Base Sequence↗

PubMed1997

Requirement of the DEAD-Box protein ded1p for messenger RNA translation.

The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. It is shown here by genetic and biochemical analysis that translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved.

Animals↗