Identification of DDX3X-dependent translation targets in the E11.5 mouse cortex
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ABSTRACT:
PROVIDER: PRJNA838490 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19217700.fastq.gz | Fastqsanger.gz | |||
| SRR19217701.fastq.gz | Fastqsanger.gz | |||
| SRR19217702.fastq.gz | Fastqsanger.gz | |||
| SRR19217703.fastq.gz | Fastqsanger.gz | |||
| SRR19217704.fastq.gz | Fastqsanger.gz |
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Project description:To identify DDX3X-dependent translation targets in the developing mouse cortex, we used E11.5 cortices from both sexes of wildtype and conditional Ddx3x knockout mice (driven by Emx1-Cre). We performed RNAseq and Riboseq on these samples in parallel.
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Project description:Whole-genome sequencing recently identified recurrent missense mutations in the RNA helicase DDX3X in pediatric medulloblastoma (MB) and other tumors. The normal function of DDX3X is poorly understood, and the consequences of its cancer-associated mutations have not been explored. Here we used genomic, biochemical, cell biological, and animal modeling approaches to investigate normal DDX3X function and the impact of cancer-associated DDX3X mutations. Cross-linking immunoprecipitation–high-throughput sequencing (CLIPseq) analyses revealed that DDX3X binds primarily to ~1000 mature mRNA targets at binding sites spanning the full mRNA length; their enrichment in the coding regions suggests that DDX3X plays a role in translational elongation. The association of wild-type DDX3X with polysomes is consistent with this observation. Cancer-associated mutations result in loss of DDX3X from polysomes and accumulation of mutant DDX3X in stress granules (cytoplasmic accumulations of translationally arrested mRNAs). Mutation-dependent redistribution of DDX3X to stress granules is also observed in a Drosophila model system and in MB tumor cells from patients carrying DDX3X mutations. Importantly, mRNAs targeted by DDX3X are enriched in translation factors, suggesting that DDX3X regulates translation both directly and indirectly. Indeed, depletion of DDX3X by RNAi or over-expression of mutant DDX3X significantly impairs global protein synthesis. Ribosome profiling confirmed this observation and showed a 5’ bias in ribosomal occupancy, further confirming the role of DDX3X in translational elongation. Together, our data show that DDX3X is a key regulator of translation and that this function is impaired by cancer-associated mutations. Finally, we found that medulloblastoma-related mutant DDX3X can efficiently bind the wild-type form suggesting that mutant DDX3X could exert a dominant negative effect in vivo.
2016-05-20 | GSE59095 | GEO
Project description:Whole-genome sequencing recently identified recurrent missense mutations in the RNA helicase DDX3X in pediatric medulloblastoma (MB) and other tumors. The normal function of DDX3X is poorly understood, and the consequences of its cancer-associated mutations have not been explored. Here we used genomic, biochemical, cell biological, and animal modeling approaches to investigate normal DDX3X function and the impact of cancer-associated DDX3X mutations. Cross-linking immunoprecipitation–high-throughput sequencing (CLIPseq) analyses revealed that DDX3X binds primarily to ~1000 mature mRNA targets at binding sites spanning the full mRNA length; their enrichment in the coding regions suggests that DDX3X plays a role in translational elongation. The association of wild-type DDX3X with polysomes is consistent with this observation. Cancer-associated mutations result in loss of DDX3X from polysomes and accumulation of mutant DDX3X in stress granules (cytoplasmic accumulations of translationally arrested mRNAs). Mutation-dependent redistribution of DDX3X to stress granules is also observed in a Drosophila model system and in MB tumor cells from patients carrying DDX3X mutations. Importantly, mRNAs targeted by DDX3X are enriched in translation factors, suggesting that DDX3X regulates translation both directly and indirectly. Indeed, depletion of DDX3X by RNAi or over-expression of mutant DDX3X significantly impairs global protein synthesis. Ribosome profiling confirmed this observation and showed a 5’ bias in ribosomal occupancy, further confirming the role of DDX3X in translational elongation. Together, our data show that DDX3X is a key regulator of translation and that this function is impaired by cancer-associated mutations. Finally, we found that medulloblastoma-related mutant DDX3X can efficiently bind the wild-type form suggesting that mutant DDX3X could exert a dominant negative effect in vivo.
2016-05-20 | GSE59094 | GEO
Project description:Whole-genome sequencing recently identified recurrent missense mutations in the RNA helicase DDX3X in pediatric medulloblastoma (MB) and other tumors. The normal function of DDX3X is poorly understood, and the consequences of its cancer-associated mutations have not been explored. Here we used genomic, biochemical, cell biological, and animal modeling approaches to investigate normal DDX3X function and the impact of cancer-associated DDX3X mutations. Cross-linking immunoprecipitation–high-throughput sequencing (CLIPseq) analyses revealed that DDX3X binds primarily to ~1000 mature mRNA targets at binding sites spanning the full mRNA length; their enrichment in the coding regions suggests that DDX3X plays a role in translational elongation. The association of wild-type DDX3X with polysomes is consistent with this observation. Cancer-associated mutations result in loss of DDX3X from polysomes and accumulation of mutant DDX3X in stress granules (cytoplasmic accumulations of translationally arrested mRNAs). Mutation-dependent redistribution of DDX3X to stress granules is also observed in a Drosophila model system and in MB tumor cells from patients carrying DDX3X mutations. Importantly, mRNAs targeted by DDX3X are enriched in translation factors, suggesting that DDX3X regulates translation both directly and indirectly. Indeed, depletion of DDX3X by RNAi or over-expression of mutant DDX3X significantly impairs global protein synthesis. Ribosome profiling confirmed this observation and showed a 5’ bias in ribosomal occupancy, further confirming the role of DDX3X in translational elongation. Together, our data show that DDX3X is a key regulator of translation and that this function is impaired by cancer-associated mutations. Finally, we found that medulloblastoma-related mutant DDX3X can efficiently bind the wild-type form suggesting that mutant DDX3X could exert a dominant negative effect in vivo.
2016-05-20 | GSE59093 | GEO
Project description:In eukaryotes, regulation of mRNA translation initiation greatly impacts gene expression, and is critical for cellular stress responses. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in 5´ UTR scanning and start codon decoding in non-stressed and stressed cells is still elusive. Here we show that DDX3X engages with thousands of mRNAs as part of the 48S scanning complex, simultaneously acting to promote or suppress translation of select mRNAs in non-stressed conditions, and switches this regulation in opposite directions to establish a stress response translational program upon acute ER stress. We find distinct DDX3X binding patterns of differentially regulated mRNAs, which lead us to identify N4-acetylation of cytidines surrounding the start codon as a crucial feature for DDX3X-mediated selective regulation. Our findings highlight a novel co-dependence between an RNA helicase and a post-transcriptional modification in regulating mRNA translation
2025-06-09 | GSE282245 | GEO
Project description:In eukaryotes, regulation of mRNA translation initiation greatly impacts gene expression, and is critical for cellular stress responses. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in 5´ UTR scanning and start codon decoding in non-stressed and stressed cells is still elusive. Here we show that DDX3X engages with thousands of mRNAs as part of the 48S scanning complex, simultaneously acting to promote or suppress translation of select mRNAs in non-stressed conditions, and switches this regulation in opposite directions to establish a stress response translational program upon acute ER stress. We find distinct DDX3X binding patterns of differentially regulated mRNAs, which lead us to identify N4-acetylation of cytidines surrounding the start codon as a crucial feature for DDX3X-mediated selective regulation. Our findings highlight a novel co-dependence between an RNA helicase and a post-transcriptional modification in regulating mRNA translation
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Project description:In eukaryotes, regulation of mRNA translation initiation greatly impacts gene expression, and is critical for cellular stress responses. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in 5´ UTR scanning and start codon decoding in non-stressed and stressed cells is still elusive. Here we show that DDX3X engages with thousands of mRNAs as part of the 48S scanning complex, simultaneously acting to promote or suppress translation of select mRNAs in non-stressed conditions, and switches this regulation in opposite directions to establish a stress response translational program upon acute ER stress. We find distinct DDX3X binding patterns of differentially regulated mRNAs, which lead us to identify N4-acetylation of cytidines surrounding the start codon as a crucial feature for DDX3X-mediated selective regulation. Our findings highlight a novel co-dependence between an RNA helicase and a post-transcriptional modification in regulating mRNA translation
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Project description:The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes we identified the microphtalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5’ untranslated region. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential in vivo and response to targeted therapy. Together these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas.
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Project description:Heterozygous mutations in the X-linked RNA helicase DDX3X cause DDX3X syndrome, a rare neurodevelopmental disorder associated with cortical malformation and autism. Amongst the ~200 known de novo DDX3X variants, half are missense while the others are predicted loss-of-function (LoF). LoF mouse models reveal DDX3X loss impairs progenitors’ ability to generate excitatory neurons. Yet, how missense mutations impact corticogenesis in vivo is unknown. Here, we generated a conditional mouse model of Ddx3xT532M, a clinically severe and recurrent DDX3X syndrome mutation found in females. Using Cre-mediated expression of Ddx3xT532M in cortical progenitors and their progeny, we show this mutation alters corticogenesis and translation. Ddx3xT532M conditional knock-in (KI) males have severe microcephaly and apoptosis. In contrast, Ddx3xT532M conditional heterozygous (cHet) females exhibit only mild reductions in cortical size and neurogenesis. Using polysome fractionation of Ddx3xT532M and Ddx3xLoF cHet female cortices, we discover that Ddx3xT532M affects translation of key targets, with qualitative differences from Ddx3xLoF cHet females. Collectively, these cellular and molecular findings suggest that, the clinically severe Ddx3xT532M variant in cHet females is phenotypically similar to, but molecularly distinct from, Ddx3x haploinsufficiency in corticogenesis. Our study establishes a new model for understanding how missense mutations may cause DDX3X syndrome.
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