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Identifying DAP5 mRNA translation and direct-binding targets in human embryonic stem cells (hESCs) using Ribo-seq and CLIP-seq.

ABSTRACT: The eIF4G1 DAP5 homolog is involved in several non-canonical translation initiation mechanisms. We applied ribosome profiling and found that DAP5 translationally activated mRNAs are enriched with translated uORFs. We also performed a genome-wide UV-cross linking immunoprecipitation screen (CLIP-seq) for identifying mRNAs that directly interact with DAP5 in hESCs. We found 959 bound mRNAs common to both Abs (out of 1073 and 2152 enriched mRNAs identified by CS and MBL antibodies, respectively).

ORGANISM(S): Homo sapiens

PROVIDER: GSE193115 | GEO | 2022/07/28

REPOSITORIES: GEO

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DAP5 enables main ORF translation on mRNAs with structured and uORF-containing 5’ leaders

Project description:Half of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here, we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is required for translation initiation on select transcripts. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-dependent translation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates main CDS, but not uORF, translation suggesting a role for DAP5 in translation re-initiation. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

2022-11-07 | GSE155854 | GEO

Identification of mRNAs with reduced ribosomal loading upon knock-down of translation factor DAP5 from hESCs.

Project description:We have generated stable human ESCs (H9) expressing control or DAP5-targeting shRNA. Polysome profiles reveal no major changes in overall translation. PolyA+ RNA and RNA accociated with heavy polysomal fractions were purified in biological duplicates and sequenced using Illumina HiSeq 2000 instrument. We identified 122 potential mRNA targets of DAP5 translation that display reduced ribosomal loading, and hence reduced translation, in the absence of DAP5.

2016-09-14 | GSE80467 | GEO

eIF4E/mTOR-independent mRNA translation plasticity conferred by DAP5/eIF3d is required for breast cancer cell mesenchymal transition and metastasis

Project description:We report the application of polysome profiling sequencing technology for high-throughput transcriptomics and translatomics in 4T1 cells. We compare reduction of Dap5 to control in a BALB/c mouse breast cancer cell line in transcription and polysome enriched translation using RNA sequencing. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factors and their mRNA targets involved in EMT, angiogenesis, DNA repair and translation initiation, among other mRNAs. Metastatic potential of cells is significanlty decreased upon Dap5 silencing as confirmed by different in vivo models.

2023-06-01 | GSE188733 | GEO

Proteomic investigation of the effect of DAP5 knock-down on human embryonic stem cells

Project description:We performed a deep mass-spec analysis to determine DAP5 proteomic signature on human embryonic stem cells (hESCs) by evaluating the steady state proteins level on DAP5-KD and NT control cells.

2022-08-10 | PXD030717 | Pride

Ribo-seq data from NIH 3T3 cells lacking eIF4G2

Project description:Eukaryotic translation initiation factor (eIF) 4G2 is a long-known homologue of the canonical eIF4G1. However, unlike the latter, it does not bind eIF4E or PABP, thus it remains unclear what and when brings eIF4G2 onto a ribosome. Here we report results of ribosome profiling performed in NIH 3T3 eIF4G2 (DAP5) -/- cells.

2020-10-19 | GSE158136 | GEO

DAP5 enables main ORF translation on mRNAs with structured and uORF-containing 5’ leaders

Project description:DAP5 enables main ORF translation on mRNAs with structured and uORF-containing 5’ leaders

| PRJNA655835 | ENA

Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

Project description:Translation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ~12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.

2020-06-25 | GSE139391 | GEO

Yeast eIF4G homogenic whole cell oxidative stress LC-MSMS

Project description:Translation initiation factor 4G (eIF4G) is an integral component of the eIF4F complex which is key to translation initiation for most eukaryotic mRNAs. Many eIF4G isoforms have been described in diverse eukaryotic organisms but we currently have a poor understanding of their functional roles and whether they regulate translation in an mRNA specific manner. The yeast Saccharomyces cerevisiae expresses two eIF4G isoforms, eIF4G1 and eIF4G2, that have previously been considered as functionally redundant with any phenotypic differences arising due to alteration in eIF4G expression levels. Using homogenic strains that express eIF4G1 or eIF4G2 as the sole eIF4G isoforms at comparable expression levels to total eIF4G, we show that eIF4G1 is specifically required to mediate the translational response to oxidative stress. We use quantitative proteomics to show that eIF4G1 promotes oxidative stress-specific proteome changes.

2023-10-24 | PXD041682 | Pride

DNA damage and eIF4G1 in breast cancer cells reprogram translation for survival and DNA repair mRNAs

Project description:The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis (Survivin, HIF1α, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1, BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers, in response to DNA damage caused by ionizing radiation,

2012-10-17 | GSE41627 | GEO

Transcription profiling of Arabidopsis eif3h mutants vs. wild type

Project description:Microarray comparisons of transcript level in wild-type Arabidopsis and eif3h mutant plants. Goal:; To detect any change in transcript level between WT and eif3h mutant. BACKGROUND: The eukaryotic translation initiation factor eIF3 has multiple roles during the initiation of translation of cytoplasmic mRNAs. However, the contributions of individual subunits of eIF3 to the translation of specific mRNAs remain poorly understood. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana it was demonstrated that the h subunit of; eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5â leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant plants revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. Independently, eIF3h also boosted polysome loading of mRNAs with long coding sequences. Moreover, the lesion in eIF3h revealed a concerted upregulation of translation for specific functional subgroups of mRNAs, including ribosomal proteins and proteins involved in photosynthesis. CONCLUSIONS: The intact eIF3h protein contributes to efficient translation initiation on 5â leader sequences harboring multiple uORFs, although mRNA features independent of uORFs were also implicated. Moreover, our data suggest that regulons of translational control can be revealed by mutations in generic translation initiation factors. Experiment Overall Design: Total RNA samples were isolated from 10-day-old wild-type and eif3h mutant plants

2008-06-14 | E-GEOD-6025 | biostudies-arrayexpress

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