Yeast eIF2A plays a minimal role in translation initiation in vivo
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ABSTRACT:
PROVIDER: PRJNA1008247 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25735500.fastq.gz | Fastqsanger.gz | |||
| SRR25735501.fastq.gz | Fastqsanger.gz | |||
| SRR25735502.fastq.gz | Fastqsanger.gz | |||
| SRR25735503.fastq.gz | Fastqsanger.gz | |||
| SRR25735504.fastq.gz | Fastqsanger.gz |
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Project description:Earlier investigations have associated mammalian eIF2A with Met-tRNAi binding to the 40S subunit and its recruitment to specialized mRNAs in a GTP-independent manner. Additionally, eIF2A has been implicated in non-AUG start codon initiation, particularly under conditions where eIF2 function is attenuated by phosphorylation of its α-subunit during stress or starvation. However, the precise role of eIF2A in vivo translation remains unclear. Moreover, it's uncertain if the conserved ortholog in budding yeast can functionally substitute for eIF2 during stress. To address these questions, we conducted ribosome profiling on a yeast deletion mutant lacking eIF2A, alongside isogenic wild-type (WT) cells, both in the presence or absence of eIF2α phosphorylation induced by amino acid starvation.
2024-01-03 | GSE241473 | GEO
Project description:We apply ribosome profiling here to assess the role of eIF2A in translation initiation. For this we test the change in translation efficiency between HeLa control and eIF2A-KO cells, however we do not find any transcript to depend on eIF2A. Since eIF2A is thought to take over the function of eIF2 when eIF2 is inhibited, we also test conditions where the integrated stress response is activated, thereby leading to eIF2 inactivation. In none of our assays, however, could we detect a role of eIF2A in translation initiation.
2024-11-29 | GSE282509 | GEO
Project description:Human eIF2A has a minimal role in translation initiation and in uORFmediated translational control
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Project description:Many positive-strand RNA viruses, including all known coronaviruses, employ programmed –1 ribosomal frameshifting (–1 PRF) to regulate the translation of polycistronic viral RNAs. However, only a few host factors have been shown to regulate –1 PRF. Through a reporter-based genome-wide CRISPR/Cas9 knockout screen, we identified several host factors that either suppressed or enhanced –1 PRF of SARS-CoV-2. One of these factors is eukaryotic translation initiation factor 2A (eIF2A), which specifically and directly enhanced –1 PRF in vitro and in cells. Consistent with the crucial role of efficient –1 PRF in transcriptase/replicase expression, loss of eIF2A reduced SARS-CoV-2 replication in cells. Transcriptome-wide analysis of eIF2A-interacting RNAs showed that eIF2A primarily interacted with 18S ribosomal RNA near the contacts between the SARS-CoV-2 frameshift-stimulatory element (FSE) and the ribosome. Thus, our results revealed an unexpected role for eIF2A in modulating the translation of specific RNAs independent of its previously described role during initiation.
2024-07-11 | GSE223690 | GEO
Project description:Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) helps decide b cell survival in diabetes. The alternative eukaryotic initiation factor 2A (EIF2A) has been proposed to mediate EIF2S1-independent translation during cellular stress and viral infection, but its role in b cells is unknown. EIF2A abundance is high in human and mouse islets relative to other tissues, and both thapsigargin and palmitate significantly increased EIF2A mRNA and EIF2A protein levels in MIN6 cells, mouse islets and human islets. Knockdowns of EIF2A, the related factor EIF2D, or both EIF2A and EIF2D, were not sufficient to cause apoptosis. On the other hand, transient or stable EIF2A over-expression protected MIN6 cells, primary mouse islets, and human islets from ER stress-induced, caspase-3-dependent apoptosis. Mechanistically, EIF2A overexpression decreased ERN1 (also known as IRE1a) expression in thapsigargin-treated MIN6 cells or human islets. In vivo, b cell specific EIF2A viral overexpression reduced ER stress, improved insulin signaling, and abrogated hyperglycemia in Ins2Akita/WT mice. EIF2A overexpression significantly increased expression of genes involved protein translation and reduced expression of pro-apoptotic genes (e.g. ALDH1A3). Remarkably, the decrease in global protein synthesis during UPR was prevented by EIF2A, despite ER stress-induced EIF2S1 phosphorylation. The protective effects of EIF2A were additive to those of ISRIB, a drug that counteracts the effects of EIF2S1 phosphorylation. Cells overexpressing EIF2A showed higher expression of translation factor EIF2B5, which may contribute to the lack of translational inhibition in these cells. We conclude that EIF2A is a novel target for b cell protection and the circumvention of EIF2S1-mediated translational repression.
2022-08-31 | GSE166829 | GEO
Project description:Translation initiation is a complex and highly regulated process that represents an important mechanism, controlling gene expression. eIF2A was proposed as an alternative initiation factor, however, its role and biological targets remain to be discovered. To further gain insight into the function of eIF2A in Saccharomyces cerevisiae, we identified mRNAs associated with the eIF2A complex and showed that 24% of the most enriched mRNAs encode proteins related to cell wall biogenesis and maintenance. In agreement with this result, we showed that an eIF2A deletion sensitized cells to cell wall damage induced by calcofluor white. eIF2A overexpression led to a growth defect, correlated with decreased synthesis of several cell wall proteins. In contrast, no changes were observed in the transcriptome, suggesting that eIF2A controls the expression of cell wall-related proteins at a translational level. The biochemical characterization of the eIF2A complex revealed that it strongly interacts with the RNA binding protein, Ssd1, which is a negative translational regulator, controlling the expression of cell wall-related genes. Interestingly, eIF2A and Ssd1 bind several common mRNA targets and we found that the binding of eIF2A to some targets was mediated by Ssd1. Surprisingly, we further showed that eIF2A is physically and functionally associated with the exonuclease Xrn1 and other mRNA degradation factors, suggesting an additional level of regulation. Altogether, our results highlight new aspects of this complex and redundant fine-tuned regulation of proteins expression related to the cell wall, a structure required to maintain cell shape and rigidity, providing protection against harmful environmental stress.
2023-09-15 | PXD043985 | Pride
Project description:Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)-/- and PKR-like endoplasmic reticulum kinase (PERK)-/- cells, with more substantial reductions of CAGG-encoded QAGR in PKR-/- cells. Experiments using mutant eIF2α-S51A HEK293T cells show that p-eIF2α is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNAs. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNAs that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2α is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.
| S-EPMC8170841 | biostudies-literature
Project description:BackgroundEIF2A is an unconventional translation factor required for initiation of protein synthesis from non-AUG codons from a variety of transcripts, including oncogenes and stress related transcripts in mammalian cells. Its function in multicellular organisms has not been reported.ResultsHere, we identify and characterize mutant alleles of the CG7414 gene, which encodes the Drosophila EIF2A ortholog. We identified that CG7414 undergoes sex-specific splicing that regulates its male-specific expression. We characterized a Mi{Mic} transposon insertion that disrupts the coding regions of all predicted isoforms and is a likely null allele, and a PBac transposon insertion into an intron, which is a hypomorph. The Mi{Mic} allele is homozygous lethal, while the viable progeny from the hypomorphic PiggyBac allele are male sterile and female fertile. In dEIF2A mutant flies, sperm failed to individualize due to defects in F-actin cones and failure to form and maintain cystic bulges, ultimately leading to sterility.ConclusionsThese results demonstrate that EIF2A is essential in a multicellular organism, both for normal development and spermatogenesis, and provide an entrée into the elucidation of the role of EIF2A and unconventional translation in vivo.
| S-EPMC10885012 | biostudies-literature
Project description:Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A, and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.
2021-07-01 | GSE174329 | GEO
Project description:The major heat shock protein Hsp70 has been shown to form a complex with a scaffold protein Bag3, linking it to multiple signaling pathways. Via these interactions, the Hsp70-Bag3 module functions as a proteotoxicity sensor that controls cell signaling. Here, as a tool to identify signaling pathways regulated by this complex, we utilized JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3. Gene expression profiling followed by the pathway analysis indicated that a set of signaling pathways including the unfolded protein response (UPR) was activated by JG-98. Surprisingly, only the translation initiation factor eIF2a-associated branch of the UPR was activated under these conditions, while other UPR branches mediating induction of ER chaperones were not induced, suggesting that the response was not related to ER proteotoxicity and thus to ER-associated kinase PERK1. Indeed, induction of the UPR genes under these conditions was dependent on activation of a distinct cytoplasmic eIF2a kinase, HRI. We demonstrated that the Hsp70-Bag3 complex directly interacted with HRI and regulated phosphorylation of eIF2a upon induction of cytoplasmic proteotoxicity. Therefore, we uncovered a novel signaling response, which regulates cell death upon the buildup of abnormal protein species in cytoplasm via an Hsp70-Bag3-HRI-eIF2a axis.
2021-04-14 | GSE171440 | GEO