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Polysomes from DENR knockdown cells

ABSTRACT: The aim was to identify transcripts that are poorly translated upon knockdown of DENR. Lysates from control (GFP) and DENR knockdown S2 cells were run on polysome gradients. RNA from the (80S peak + polysome peaks) was isolated and analyzed by microarrays. In parallel, total RNA from the cells was also profiled as a normalization control and profiled. The 'translation score' of translated mRNA to total RNA was then calculated for control and DENR knockdown cells.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE54625 | GEO | 2014/06/30

SECONDARY ACCESSION(S): PRJNA237227

REPOSITORIES: GEO

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Polysomes from DENR knockdown cells

Project description:The aim was to identify transcripts that are poorly translated upon knockdown of DENR. Lysates from control (GFP) and DENR knockdown S2 cells were run on polysome gradients. RNA from the (80S peak + polysome peaks) was isolated and analyzed by microarrays. In parallel, total RNA from the cells was also profiled as a normalization control and profiled. The 'translation score' of translated mRNA to total RNA was then calculated for control and DENR knockdown cells. Two biological replicates for DENR knockdown and GFP knockdown were performed. For each sample, (80S+polysomes) and total RNA were profiled, leading to 8 samples in total.

2014-06-30 | E-GEOD-54625 | biostudies-arrayexpress

Master stress response gene ATF4 & other oncogenes are translated by DENR-dependent reinitiation

Project description:Translation efficiency varies 1000-fold between different mRNAs, thereby strongly impacting protein expression. Translation of the master stress response gene ATF4 increases in response to stress, but the molecular mechanisms are not well understood. We discover here that translation initiation factors DENR, MCTS1 and eIF2D are absolutely required to induce ATF4 translation upon stress, by promoting translation reinitiation on the ATF4 5’UTR. Hence DENR and MCTS1 are important players in the cellular Integrated Stress Response. We find DENR and MCTS1 promote reinitiation after long uORFs with specific penultimate codons, due to the tRNA that remains attached to 40S ribosomes after translation termination. This provides a model for how DENR and MCTS1 promote translation reinitiation. Since cancer cells are exposed to many stresses, they require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner. This explains in part why DENR and MCTS1 are oncogenes.

2020-07-18 | GSE140084 | GEO

DENR controls JAK2 translation to induce PD-L1 expression for tumor immune evasion

Project description:To identify the potential signaling pathway involved in DENR KO cells upon PD-L1 production, we harvested IFNγ-treated control and DENR KO cells for RNA-seq. Bioinformatic analysis of the RNA-seq data showed that 114 genes, including PD-L1, were significantly downregulated in DENR KO cells, and multiple classical signaling pathways, including JAK-STAT, and PD-L1, were enriched.

2022-02-28 | GSE183980 | GEO

Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) recycle post-termination 40S subunits in vivo

Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation.

2018-08-30 | GSE108942 | GEO

DENR controls JAK2 translation to induce PD-L1 expression for tumor immune evasion [CRISPR screen]

Project description:RNA-binding proteins (RBPs) can recognize thousands of RNAs that help to maintain cell homeostasis, and RBP dysfunction is frequently observed in various cancers. However, whether specific RBPs are involved in tumor immune evasion by regulating programmed death ligand-1 (PD-L1) is unclear. We performed targeted RBP CRISPR/Cas9 screening and identified density regulated re-initiation and release factor (DENR) as a PD-L1 regulator. DENR-depleted cancer cells exhibited reduced PD-L1 expression in vitro and in vivo. DENR depletion significantly suppressed tumor growth and enhanced the tumor-killing activity of CD8+ T cells. Mechanistically, DENR antagonized the translational repression of three consecutive upstream open reading frames (uORFs) upstream of Janus kinase 2 (JAK2); thus, DENR deficiency impaired JAK2 translation and the IFNγ-JAK-STAT signaling pathway, resulting in reduced PD-L1 expression in tumors. Overall, we discovered that the RBP DENR is a novel regulator in PD-L1 expression and highlighted the potential of DENR as a therapeutic target for immunotherapy.

2022-02-28 | GSE184048 | GEO

Identification of DENR-interacting proteins in NIH3t3 cells using a co-IP MS-MS approach on endogenously tagged DENR.

Project description:Among the transcript features that regulate translation rate, upstream open reading frames (uORFs) have emerged as a major class of inhibitory elements which limit how many scanning ribosomes reach the start codon of the main protein coding sequence (CDS). Two processes are implicated in ensuring that CDS translation of uORF-containing transcripts can take place nevertheless: first, leaky-scanning, which involves the bypassing of uORF start codons by the scanning 40S ribosome, and second, re-initiation (REI), which allows the 40S subunit of the ribosome that terminated on the uORF stop codon to resume scanning to reach a downstream start codon. REI has remained particularly enigmatic, as it is at odds with generally accepted principles of conventional translation initiation, termination and post-termination subunit recycling. The heterodimer MCTS1-DENR is the first re-initiation-specific factor that has been identified. MCTS1 has been described as the only obligatory partner of DENR. Nevertheless, MCTS1 depletion phenotypes do not always copy the phenotype of DENR depletion in mammalian cells, suggesting that another partner could compensate for the lack of MCTS1. We performed co-immunoprecipitation followed by MS-MS analysis of endogenously FLAG-tagged DENR and identified MCTS2 as a new protein partner. MCTS2 is a homolog and retrogene copy of MCTS1. Downstream analyses demonstrated that MCTS2-DENR was able to promote re-initiation in vitro and showed that Mcts2 mRNA was highly abundant and the protein at least as efficiently synthesized as MCTS1 in NIH3t3 cells, implying that MCTS2 might be the main expressed variant in certain cell types.

2024-11-01 | PXD051482 | Pride

mRNA loading on polysomes in skeletal muscle of Eif6 heterozygous mice

Project description:Gastrocnemius muscle from Eif6 heterozygous mice and wild-type mice was used in a polysome profiling experiment. Total RNA was extracted from the whole muscle and from monosome/polysome fractions and profiled using Agilent gene expression arrays.

2018-07-07 | GSE116715 | GEO

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

Project description:The non-canonical initiation factor DENR promotes translation reinitiation on uORF-containing mRNAs. Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting that uORF usage and reinitiation regulate clock function. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We found 240 transcripts with altered translation rate, and used linear regression analysis to extract uORF features predictive of DENR dependance. Among core clock genes, we identified Clock as a DENR target. Using Clock 5'UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments identified an alternative downstream start codon, which likely represents the true CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.

2019-04-01 | GSE116221 | GEO

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock

2019-04-01 | GSE124790 | GEO

Polysomes

Project description:Translational profiles of wild type yeast strains were identified by microarray analysis by isolating polysomes using sucrose gradients to recover polysome-associated RNA and comparing polysome-associated RNA vs total RNA

2013-04-05 | GSE45797 | GEO

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