The DEAD-box RNA helicase Ded1 is a translation-elongation factor
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ABSTRACT:
PROVIDER: PRJNA951699 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24055967.fastq.gz | Fastqsanger.gz | |||
| SRR24055968.fastq.gz | Fastqsanger.gz | |||
| SRR24055969.fastq.gz | Fastqsanger.gz | |||
| SRR24055970.fastq.gz | Fastqsanger.gz | |||
| SRR24055971.fastq.gz | Fastqsanger.gz |
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Project description:DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP) to crosslink Ded1 to 4-thiouridine-incorported RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on a relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.
2023-08-01 | GSE228828 | GEO
Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling.The study includes 32 samples, comprised of 16 mRNA-Seq samples and 16 ribosome footprint profiling samples, derived from biological replicates of 3 mutant strains, ded1-cs, ded1-ts and tif1-ts, and the corresponding wild-type strains. The tif1-ts mutant and its wild-type counterpart were analyzed at 30°C and 37°C.
2015-03-02 | E-GEOD-66411 | biostudies-arrayexpress
Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs.
2015-03-02 | GSE66411 | GEO
Project description:DEAD-box ATPases belong to an abundant class of proteins that are involved in virtually all aspects of RNA metabolism and are found in all kingdoms of life. When bound to a DEAD-box ATPase, the RNA substrate is forced into a kinked conformation that is incompatible with helical structures. Distortion of the RNA can result in unwinding of short RNA duplexes (helicase activity) or destabilize RNA-protein interactions, allowing DEAD-box ATPases to remodel mRNPs (RNPase activity). The RNPase activity makes DEAD-box ATPases suitable molecular building blocks for the implementation of checkpoints that confer directionality to the process of RNA biogenesis. Here, we provide data that characterizes the DEAD-box ATPase Dbp2 (SPBP8B7.16c) of the fission yeast Schizosaccharomyces pombe. Using ChIP-seq, we determined the sites where HTP-tagged Dbp2 associates with chromatin. ChIP-seq of Srp2-HTP is included as a reference protein that is known to associate with transcribing RNA polymerase II (RNAPII).
2024-06-30 | E-MTAB-13712 | biostudies-arrayexpress
Project description:Organ-differential Regulation of Vascular Development by DEAD-box Helicase 24
| PRJNA768417 | ENA
Project description:Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade and plays both RING (really interesting new gene)-dependent and RING-independent roles in RIG-I-mediated IFN induction. We report that the PRY-SPRY domain of TRIM25 interacts with the N-terminal extension (NTE) of DEAD-box helicase 3X (DDX3X), a host protein with multiple roles in RLR signalling. Gene reporter assays and knockdown studies reveal DDX3X and TRIM25 cooperate to activate the IFN- promoter following RIG-I activation independent of DDX3X’s catalytic activity. We also show that TRIM25 ubiquitinates DDX3X at several lysine residues in vitro and in cells.
2021-09-08 | PXD028122 | Pride
Project description:How cells regulate gene expression in a precise spatiotemporal manner during organismal development is a fundamental question in biology. Although the role of transcriptional condensates in gene regulation has been established, little is known about the function and regulation of these molecular assemblies in the context of animal development and physiology. Here we show that the evolutionarily conserved DEAD-box helicase DDX-23 controls cell fate in Caenorhabditis elegans by binding to and facilitating the condensation of MAB-10, the Caenorhabditis elegans homolog of mammalian NGFI-A-binding (NAB) protein. MAB-10 is a transcriptional cofactor that functions with the early growth response (EGR) protein LIN-29 to regulate the transcription of genes required for exiting the cell cycle, terminal differentiation, and the larval-to-adult transition. We suggest that DEAD-box helicase proteins function more generally during animal development to control the condensation of NAB proteins important in cell identity and that this mechanism is evolutionarily conserved. In mammals, such a mechanism might underlie terminal cell differentiation and when misregulated might promote cancerous growth.
2023-05-28 | GSE151399 | GEO
Project description:DEAD-box ATPases belong to an abundant class of proteins that are involved in virtually all aspects of RNA metabolism and are found in all kingdoms of life. When bound to a DEAD-box ATPase, the RNA substrate is forced into a kinked conformation that is incompatible with helical structures. Distortion of the RNA can result in unwinding of short RNA duplexes (helicase activity) or destabilize RNA-protein interactions, allowing DEAD-box ATPases to remodel mRNPs (RNPase activity). The RNPase activity makes DEAD-box ATPases suitable molecular building blocks for the implementation of checkpoints that confer directionality to the process of RNA biogenesis. Here, we provide data that characterizes the DEAD-box ATPase Dbp2 (SPBP8B7.16c) of the fission yeast Schizosaccharomyces pombe. Using RNA-seq, we determined RNA expression profiles of a conditional depletion strain of Dbp2 and the corresponding wild type. For this, we placed the endogenous dbp2 gene under the control of the P.nmt1 promoter, which is repressed in the presence of thiamine. Cells were harvested at the beginning (t0) or the end (t5 or t9) of shift to thiamine-containing YES medium. S. cerevisiae spike-in cells were added in a 1:5 OD600 ratio immediately before harvesting.
2024-06-30 | E-MTAB-13717 | biostudies-arrayexpress
Project description:DEAD-box ATPases belong to an abundant class of proteins that are involved in virtually all aspects of RNA metabolism and are found in all kingdoms of life. When bound to a DEAD-box ATPase, the RNA substrate is forced into a kinked conformation that is incompatible with helical structures. Distortion of the RNA can result in unwinding of short RNA duplexes (helicase activity) or destabilize RNA-protein interactions, allowing DEAD-box ATPases to remodel mRNPs (RNPase activity). The RNPase activity makes DEAD-box ATPases suitable molecular building blocks for the implementation of checkpoints that confer directionality to the process of RNA biogenesis. Here, we provide data that characterizes the DEAD-box ATPase Dbp2 (SPBP8B7.16c) of the fission yeast Schizosaccharomyces pombe. Using calibrated RNAPII-ChIP-seq, we determined the transcription profiles of a conditional depletion strain of Dbp2 and the corresponding wild type. For this, we placed the endogenous dbp2 gene under the control of the P.nmt1 promoter, which is repressed in the presence of thiamine. Cells were crosslinked at the beginning (t0) or the end (t9) of a 9h shift to thiamine-containing YES medium. S. cerevisiae spike-in cells were added in a 1:5 OD600 ratio immediately before crosslinking.
2024-06-30 | E-MTAB-13714 | biostudies-arrayexpress
Project description:Ribonucleases (RNases) are central actors in post-transcriptional regulation, a major level of regulation of gene expression in all cells. This control plays an important role in the bacterial pathogen Helicobacter pylori, although only the function of RNase J was characterized so far. Here, we studied the RNase R enzyme from H. pylori, a 3’-5’ exoribonuclease whose ortholog in Escherichia coli was reported to display, in addition, helicase activity and to be able to hydrolyze RNA substrates with double stranded structures. We observed that HpRNase R protein does not carry the domains responsible for helicase activity in E. coli and accordingly that the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The RNase R helicase domain loss is widespread among the Campylobacterota and occurred gradually during their evolution. Furthermore, an in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori, was discovered. Phylogenomics suggests that this interaction might occur in other bacteria of the phylum Campylobacterota. Purified RhpA facilitates the degradation of double stranded RNA substrates by HpRNase R, showing that this complex is functional. HpRNase R has a minor role of in 5S rRNA maturation and, as shown by RNA-Seq, few targets in H. pylori all of them being included in the RhpA regulon. In conclusion, we describe a new type of RNase R that lacks some of the features that were considered as hallmarks of RNase R proteins, but that has co-opted another RNA helicase, which we hypothesize helps it accomplish some of its functions in vivo.
2021-06-19 | E-MTAB-10045 | biostudies-arrayexpress