DDX3 depletion represses translation of mRNAs with complex 5′ UTRs
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA515289 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12595119.fastq.gz | Fastqsanger.gz | |||
| SRR12595120.fastq.gz | Fastqsanger.gz | |||
| SRR8445000.fastq.gz | Fastqsanger.gz | |||
| SRR8445001.fastq.gz | Fastqsanger.gz | |||
| SRR8445002.fastq.gz | Fastqsanger.gz |
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Similar Datasets
Project description:We describe the subset of transcripts that require DDX3 for efficient translation.
2019-03-25 | GSE125114 | GEO
Project description:DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.
| S-EPMC8136831 | biostudies-literature
Project description:Small molecule compounds that sense the nucleic acid sequences, promise the attractive venue for drug development. Such an unusual effect has been observed in the natural product Rocaglamide A (RocA) from Aglaia plant, proving to exhibit anti-tumor effects by clamping eukaryotic initiation factor (eIF) 4A onto mRNA polypurine sequences. Although eIF4A has been speculated the unique target of RocA, the insensitization of eIF4A in human cells only partially rescued the translation repression from RocA, suggesting another alternative target of this compound. Here, we revealed that DDX3 is an alternative target of RocA. Developing a RocA derivative with an O-nitrobenzoxadiazole unit (RocA-O-NBD), which can covalently bind to proximate proteins and provide fluorescence to them, we identified DDX3 bound to RocA. As observed in eIF4A, RocA locked the DDX3 protein onto polypurine sequences of RNA in an ATP-independent manner. De novo assembled Aglaia plant transcriptome uncovered the natural amino acid substitutions in Aglaia DDX3 to protect itself from RocA toxicity. Because of the dominant negative effect of RocA, we also proved the protein abundance of eIF4A and DDX3 in cancer cells determines their sensitivity to RocA. Overall, this study discovered DDX3 as another target of RocA and suggests the probability to predict tumor toxicity of RocA by the target abundance.
2020-12-07 | GSE148636 | GEO
Project description:Small molecule compounds that sense the nucleic acid sequences, promise the attractive venue for drug development. Such an unusual effect has been observed in the natural product Rocaglamide A (RocA) from Aglaia plant, proving to exhibit anti-tumor effects by clamping eukaryotic initiation factor (eIF) 4A onto mRNA polypurine sequences. Although eIF4A has been speculated the unique target of RocA, the insensitization of eIF4A in human cells only partially rescued the translation repression from RocA, suggesting another alternative target of this compound. Here, we revealed that DDX3 is an alternative target of RocA. Developing a RocA derivative with an O-nitrobenzoxadiazole unit (RocA-O-NBD), which can covalently bind to proximate proteins and provide fluorescence to them, we identified DDX3 bound to RocA. As observed in eIF4A, RocA locked the DDX3 protein onto polypurine sequences of RNA in an ATP-independent manner. De novo assembled Aglaia plant transcriptome uncovered the natural amino acid substitutions in Aglaia DDX3 to protect itself from RocA toxicity. Because of the dominant negative effect of RocA, we also proved the protein abundance of eIF4A and DDX3 in cancer cells determines their sensitivity to RocA. Overall, this study discovered DDX3 as another target of RocA and suggests the probability to predict tumor toxicity of RocA by the target abundance.
2020-12-07 | GSE150111 | GEO
Project description:DEAD-box RNA helicases eIF4A and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. eIF4B is a cofactor for eIF4A but might also function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5’UTRs. These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop mRNPs via eIF4F-Pab1 association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eIF4G, the scaffold subunit of eIF4F, preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5’UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly.
2016-05-27 | GSE81966 | GEO
Project description:DEAD-box RNA helicases DDX3 are important developmental regulators of multiple aspects of RNA metabolism of eukaryotes. belle, a single DDX3 ortholog in Drosophila, is essential for fly viability, fertility, and germline stem cell maintenance. Here we showed that RNAi belle knockdown in testis cyst cells caused a disruption of adhesion between germ cells and cyst cells and a generation of tumor-like clusters of stem-like germ cells. Ectopic expression of β-integrin in cyst cells rescued early stages of spermatogenesis in the belle knockdown testes, indicating that integrin adhesion complexes are required for interaction between somatic and germ cells in cyst. To address in details Belle functions in spermatogenesis we performed CLIP-seq analysis and identified multiple mRNAs which interacted with Belle in the testes. A set of Belle targets includes mRNAs of factors that are essential for preventing tumor-like cluster formation of early germ cells and ensuring of sustained gametogenesis.
2020-03-20 | GSE137956 | GEO
Project description:By using ribosome profiling, we demonstrate that catalytic activity of the RNA helicase DDX3 is generally required for mediating translation repression under stress. Intriguingly, however, a cancer-related DDX3 variant DDX3 R534H selectively preserves translation of genes encoding core nucleosome components. Additionally, DDX3 variants also shift ORF usage on select genes, such as RPLP1 and stress-response factors as an added mechanism of translation regulation during stress. Thus, DDX3 through both extensive and selective interactions with RNA and the ribosomal machinery helps to remodel the translational landscape under stress and in cancer.
2016-04-21 | GSE70802 | GEO
Project description:DEAD-box RNA helicases are central players in RNA metabolism, however, their role in translation regulation is largely unexplored in parasitic protozoa. Here, we have investigated the role of DDX3 RNA helicase in ribosome-associated protein quality control in Leishmania. We show that ribosomes move more slowly and de novo polypeptide synthesis is reduced in cells lacking DDX3. In accordance with the slowing of ribosome speed, DDX3 depleted cells exhibit higher levels of ribosome-associated ubiquitination. Especially, ubiquitination of nascent polypeptides is enhanced upon DDX3 loss as determined by the isolation of ribosome-associated nascent chains modified either by HA-Ubiquitin or by endogenous ubiquitin using biotinylated-puromycin labeling. Consistent with increased co-translational ubiquitination, quantitative proteomics analysis revealed higher recruitment of E3 ubiquitin ligases and proteasomal components to DDX3 knockout ribosomes to eliminate aberrant nascent polypeptides. In addition, we show that cells lacking DDX3 accumulate cytosolic aggregates. This along with the higher recruitment of ribosome-associated chaperones and the improvement of translation by increasing HSP70 availability suggests that co-translational control of nascent polypeptides is impaired in the absence of DDX3. Altogether, these results highlight an important role for DDX3 in ribosome-associated quality control by reducing co-translational ubiquitination and proteotoxicity, hence allowing optimal ribosome movement and translation elongation.
2021-07-23 | PXD020667 | Pride
Project description:mTOR regulates mRNA translation. Whereas ribosome-profiling suggested that mTOR exclusively stimulates translation of TOP (containing a 5â-terminal oligopyrimidine [5âTOP] motif) and TOP-like mRNAs, polysome-profiling implied that mTOR also modulates translation of non-TOP mRNAs. We show that ribosome-, but not polysome-profiling, is biased towards identification of TOP mRNAs as differentially translated while obscuring detection of changes in non-TOP mRNA translation. Transcription start site profiling by Nano-Cap Analysis of Gene Expression (nanoCAGE) revealed that many mTOR-sensitive mRNAs do not have 5âTOP motifs. Moreover, nanoCAGE showed that 5â UTR features distinguish two functionally and translationally distinct subsets of mTOR-sensitive mRNAs: i) those with short 5â UTRs enriched for mitochondrial functions such as respiration, that are translated in an eIF4E, but not eIF4A1-dependent manner and ii) mRNAs encoding proliferation- and survival-promoting proteins, that harbor long 5â UTRs, and require both eIF4E and eIF4A1 for their efficient translation. Selective inhibition of translation of mRNAs harboring long 5â UTRs via suppression of eIF4A leads to uncoupling of expression of proteins involved in respiration (e.g. ATP5O) from those protecting mitochondrial integrity (e.g. BCL-2) ultimately resulting in apoptosis. Conversely, simultaneous translational downregulation of both long and short 5â UTR mRNAs by mTOR inhibitors results in suppression of mitochondrial respiration and predominantly cytostatic effects. Therefore, 5â UTR features define differential modes of translation of functionally distinct mTOR-sensitive mRNAs, which explains discrepancies between the effects of mTOR and eIF4A inhibitors on neoplastic cells. Determination of 5'UTR lengths using nanoCAGE in MCF7 cells
2016-03-14 | E-GEOD-77033 | biostudies-arrayexpress
Project description: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.
| S-EPMC3442272 | biostudies-literature