Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1049869 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27132528_1.fastq.gz | Fastqsanger.gz | |||
| SRR27132528_2.fastq.gz | Fastqsanger.gz | |||
| SRR27132529_1.fastq.gz | Fastqsanger.gz | |||
| SRR27132529_2.fastq.gz | Fastqsanger.gz | |||
| SRR27132530_1.fastq.gz | Fastqsanger.gz |
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Project description:Translational dysregulation is an emerging hallmark of cancer, and increased activity of the mRNA helicase eIF4A is associated with poor survival in malignancies. This is believed to be due to the unwinding of secondary structures within the 5’UTRs of oncogenic mRNAs, with studies showing that in general eIF4A-dependent mRNAs have longer 5’UTRs with more stable secondary structures, yet our ability to predict eIF4A-dependency from 5’UTR properties alone remains poor. We therefore used Structure-seq 2 to measure transcriptome-wide changes in RNA structure in MCF7 cells, following eIF4A inhibition with hippuristanol. This technique measures the single-strandedness of RNA by specific and rapid methylation of single-stranded adenosines and cytosines with Dimethyl Sulphate (DMS). When paired with polysome profiling data to identify which mRNAs are most translationally repressed, we can identify the structural determinants of eIF4A-dependency. Upon eIF4A inhibition, both 5’UTRs and CDSs become generally more structured, while overall this was not observed for 3’UTRs. This was most pronounced in CDSs, supporting recent findings that the ribosome sculpts RNA structure in this region. 5’UTRs are generally more structured at their 5’ ends and highly translated mRNAs are less structured just upstream of the CDS. Following eIF4A inhibition, the 5’UTR is remodelled. eIF4A-dependent mRNAs have greater localised gains of structure. The degree of these structural changes is strongly correlated with 5’UTR length, explaining why eIF4A-dependent mRNAs have longer 5’UTRs. Crucially, in eIF4A-dependent mRNAs these highly-structured elements are located predominantly at the 3’ end of the 5’UTR, suggesting that increased structure just upstream of the CDS is most inhibitory to translation following eIF4A inhibition and is a key determinant of eIF4A-dependency.
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Project description:We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S pre-initiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5’-untranslated regions (5’UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at start codons in 5’UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5’UTRs.
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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.
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2023-01-17 | PXD034343 | Pride
Project description:We use ribosome footprinting to assess the gene expression changes occuring after 10 minutes of heat stress at either 40°C or 42°C compared to non-stressed yeast cells (30°C). We find that between 40°C and 42°C there is a shift in gene expression from the expression of genes harboring long and structured 5'UTRs to the expression of genes harboring thranscripts with short and unstructure 5'UTRs. We performed these experiments for WT yeast cells as well as for mutant yeast cells in which the RNA helicase Ded1 condensates at lower temperatures (Ded1-IDRm). We find upon comparing mutant and WT cells at 42°C, that Ded1-IDRm has increased expression of genes harboring unstructured 5'UTRs and decreased expression of genes harboring structured 5'UTRs.
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Project description:Sex differences influence congenital heart disease (CHD) development, yet underlying molecular mechanisms remain largely unclear. We demonstrate that the X-linked RNA helicase DDX3X associates in the heart with ribosomal subunit proteins, and eCLIP mapping reveals its preferential binding to cardiac mRNAs with long, structured 5′ untranslated regions (5′UTRs) that can hinder translation. Using a cardiomyocyte-specific mouse Ddx3x knockout model, we show that female embryos lacking Ddx3x die at mid-gestation from heart failure due to impaired translation of key cardiac regulators, whereas male littermates survive. Ribosome profiling and proteomics demonstrate that DDX3X is required for efficient translation of female-differential cardiac mRNAs. Reporter assays confirm that translation of essential cardiac genes such as Srf and Rcor2 depends on their 5′ UTRs and requires DDX3X. These findings uncover a sex-specific post-transcriptional mechanism by which DDX3X safeguards female heart development through selective mRNA translation, providing insight into how X-linked dosage-sensitive regulators contribute to CHD.
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Project description:Sex differences influence congenital heart disease (CHD) development, yet underlying molecular mechanisms remain largely unclear. We demonstrate that the X-linked RNA helicase DDX3X associates in the heart with ribosomal subunit proteins, and eCLIP mapping reveals its preferential binding to cardiac mRNAs with long, structured 5′ untranslated regions (5′UTRs) that can hinder translation. Using a cardiomyocyte-specific mouse Ddx3x knockout model, we show that female embryos lacking Ddx3x die at mid-gestation from heart failure due to impaired translation of key cardiac regulators, whereas male littermates survive. Ribosome profiling and proteomics demonstrate that DDX3X is required for efficient translation of female-differential cardiac mRNAs. Reporter assays confirm that translation of essential cardiac genes such as Srf and Rcor2 depends on their 5′ UTRs and requires DDX3X. These findings uncover a sex-specific post-transcriptional mechanism by which DDX3X safeguards female heart development through selective mRNA translation, providing insight into how X-linked dosage-sensitive regulators contribute to CHD.
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