Ribosome profiling analysis of Denr and Eif2d depleted cells reveals the absence of re-initiation function of EIF2D
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ABSTRACT:
PROVIDER: PRJNA1100515 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28696239.fastq.gz | Fastqsanger.gz | |||
| SRR28696240.fastq.gz | Fastqsanger.gz | |||
| SRR28696241.fastq.gz | Fastqsanger.gz | |||
| SRR28696242.fastq.gz | Fastqsanger.gz | |||
| SRR28696243.fastq.gz | Fastqsanger.gz |
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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. MCTS1-DENR is the first re-initiation-specific factor that has been identified. Its homolog, EIF2D, is assumed to act in uORF REI as well, yet formal proof of EIF2D-mediated REI has remained scarce. In our study, we present our in vivo analyses of DENR and EIF2D in translation re-initiation in mammalian cells. At the transcriptome-wide scale, ribosome profiling clearly identifies DENR-dependent REI, yet disproves the assumption that EIF2D also functions as a REI-factor.
2024-10-30 | GSE263991 | GEO
Project description:Although eukaryotic initiation factor 2D (eIF2D) is implicated in translation initiation, re-initiation, and ribosome recycling, its precise role remains unclear. Here, we show that eIF2D promotes 40S ribosome recycling during intrinsic ribosome destabilization (IRD), a process in which ribosomes stochastically destabilize while translating proteins with consecutive acidic amino acids at their NH2-terminus. Unrecycled 40S ribosomes accumulate in eIF2D-deficient cells, leading to 80S ribosome stalling. Selective translation complex profiling (TCP-seq) reveals that eIF2D preferentially associates with IRD-prone regions. The winged-helix (WH) domain, unique to eIF2D but absent in MCTS1–DENR, enhances its binding to 40S subunits, but likely clashes with ABCE1 during stop codon-associated recycling. Loss of eIF2D reduces the expression of IRD-inducing proteins, including splicing factors. Together, these findings define a previously unappreciated role for eIF2D in 40S recycling and clarify its mechanistic divergence from the MCTS1–DENR complex.
2025-10-24 | GSE288755 | GEO
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
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
Project description:The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we developed an enhanced 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.
2021-03-01 | GSE145904 | GEO
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
Project description:Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) recycle post-termination 40S subunits in vivo
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Project description:The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.
| S-EPMC7495428 | biostudies-literature
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
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.
2014-06-30 | GSE54625 | GEO