RIP-seq of Saccharomyces cerevisiae to identify RNAs which associate with members of the closed loop complex (eIF4E, eIF4GI, eIF4GII, Pab1p) and inhibtors of the complex (Caf20p, Eap1p)
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ABSTRACT:
INSTRUMENT(S): AB SOLiD 4 System
ORGANISM(S): Saccharomyces Cerevisiae
SUBMITTER: William Rowe
PROVIDER: E-MTAB-2464 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Translation factors eIF4E and eIF4G form eIF4F, which binds to mRNAs to promote ribosome recruitment and translation initiation. We were interested in analysing the effects of stresses on eIF4F interactions with individual mRNAs. We used a RIP-seq approach to assess how mRNA associations with eIF4E, eIF4G1 and eIF4G2 change in response to three stresses: 1) addition of hydrogen peroxide; 2) amino acids withdrawal; and, 3) glucose withdrawal. We find that acute stress leads to changes in eIF4FmRNA interactions that are shared among each factor and across the stresses imposed.
2017-10-11 | E-MTAB-5836 | biostudies-arrayexpress
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
Project description:The unicellular eukaryote Trypanosoma brucei relies heavily on posttranscriptional regulatory mechanisms, as pol II transcription is polycistronic. The parasite has six isoforms of the cap-binding translation initiation factor eIF4E, and five eiF4Gs (PMID: 29077018), which potentially allow for differential mRNA target selection in order to fine-tune translation. EIF4E3 and EIF4E4 appear to be general initiation factors; we are investigating the EIF4E proteins that are presumed to have more specialized functions, i.e., EIF4E1, EIF4E2, EIF4E5, and EIF4E6. EIF4E1 interacts with 4E-interacting protein (4EIP) and not with any EIF4G; they functionally resemble mammalian 4E-HP and GIGYF2. 4EIP suppresses translation and provokes mRNA degradation. 4EIP and EIF4E1 are dispensable in slender “bloodstream forms” (BSFs), which multiply in mammals, but 4EIP is required for translation suppression in the growth-arrested “stumpy” BSF (PMID: 30124912). Meanwhile EIF4E1, but not 4EIP, is required for survival of “procyclic forms” (PCFs), which grow in Tsetse. Tethered EIF4E1 is suppressive only when 4EIP is present. We are investigating whether it can, without an EIF4G, activate translation in BSFs/PCFs, as well as how target mRNAs are selected in absence and presence of 4EIP, based on the proteins it associates with. Like EIF4E1, EIF4E2 does not interact with any EIF4G protein, but was found in association with a homolog of the histone mRNA stem-loop-binding protein, called SLBP2, in PCFs (PMID: 29288414). The protein binding partners in BSFs have not been addressed this far. EIF4E3, EIF4E4, EIF4E5, and EIF4E6 all stimulate expression when tethered. They interact with different EIF4G homologs, and each is essential in at least one life-cycle stage, indicating that each serves a distinct role. We have now evidence that EIF4E6 interacts specifically, not only with EIF4G5, but also with a previously characterized stimulatory complex containing MKT1, PBP1, and LSM12, which we aimed to confirm by this quantitative mass spectrometry approach. The complex is recruited to mRNAs via sequence-specific RNA-binding proteins (PMID: 24470144), offering a novel mechanism for specific translation activation by the EIF4E6-EIF4G5 complex. EIF4E5 on the other hand associates with either EIF4G1 or EIF4G2 to exert its functions. It is dispensable in the BSF, but essential in PCFs, where knock-down results in a motility-related phenotype. Furthermore, previous studies identified several 14-3-3 homologs to be associated with EIF4E5 in complex with either EIF4G1 or EIF4G2 in PCFs (PMID: 24962368). In the course of this study, PTP-tagged versions of EIF4E1+/- 4EIP (BSF, PCF), EIF4E2, EIF4E5, and EIF4E6 (BSF only) were pulled down and bound proteins were analyzed by quantitative mass spectrometry, with EIF4E3-PTP (BSF) and GFP-PTP (BSF, PCF) serving as controls.
2021-05-28 | PXD025913 | Pride
Project description:The eukaryotic translation initiation factor eIF4E, a potent oncogene, is highly regulated. One class of eIF4E regulators, including eIF4G and the 4E-binding proteins (4E-BPs), interact with eIF4E using a conserved YXXXXLPhi-binding site. The structural basis of this interaction and its regulation are well established. Really Interesting New Gene (RING) domain containing proteins, such as the promyelocytic leukemia protein PML and the arenaviral protein Z, represent a second class of eIF4E regulators that inhibit eIF4E function by decreasing eIF4E's affinity for its m(7)G cap ligand. To elucidate the structural basis of this inhibition, we determined the structure of Z and studied the Z-eIF4E complex using NMR methods. We show that Z interacts with eIF4E via a novel binding site, which has no homology with that of eIF4G or the 4E-BPs, and is different from the RING recognition site used in the ubiquitin system. Z and eIF4G interact with distinct parts of eIF4E and differentially alter the conformation of the m(7)G cap-binding site. Our results provide a molecular basis for how PML and Z RINGs reduce the affinity of eIF4E for the m(7)G cap and thereby act as key inhibitors of eIF4E function. Furthermore, our findings provide unique insights into RING protein interactions.
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Project description:The Ribo-seq and TI-seq analysis following i14G1s (eI1-eIF4G1 inhibitors) treatments uncover opposing roles of eIF1-eIF4G1 and eIF4E-eIF4G1 in scanning-dependent and independent translation. Furthermore, i14G1s inhibition of eIF4G1-eIF1 resulted in translation activation of ER/UPR stress-response genes via enhanced ribosome loading, elevated 5’UTR translation at near cognate AUGs, and unexpected concomitant upregulation of coding-region translation.
2022-07-07 | GSE166743 | GEO
Project description:The Ribo-seq and TI-seq analysis following i14G1s (eI1-eIF4G1 inhibitors) treatments uncover opposing roles of eIF1-eIF4G1 and eIF4E-eIF4G1 in scanning-dependent and independent translation. Furthermore, i14G1s inhibition of eIF4G1-eIF1 resulted in translation activation of ER/UPR stress-response genes via enhanced ribosome loading, elevated 5’UTR translation at near cognate AUGs, and unexpected concomitant upregulation of coding-region translation.
2022-07-07 | GSE166742 | GEO
Project description:Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E-Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E-eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation.
| S-EPMC3425778 | biostudies-literature
Project description:Remarkably, tens of thousands of piwi-interacting RNAs (piRNAs) arise from a minimal number of discrete clustered regions in many organisms. However, it remains unclear if organization into these domains contributes to the highly coordinated, germline-specific expression of these small RNAs. Here, we show that SNPC-4, the DNA-binding subunit of the small nuclear RNA activating protein complex (SNAPc), binds piRNA clusters in a germline-specific manner and is required for global piRNA expression in C. elegans. Within piRNA domains, SNPC-4 not only binds at discrete sites through its established sequence motif, but also exhibits widely distributed binding across the region. Intriguingly, discrete peaks are not found at every piRNA, but instead occur frequently at Pol III-occupied tRNA genes, which have been previously implicated in chromatin organization. Given its unique binding pattern, we suggest that SNPC-4 promotes robust piRNA expression by establishing a permissive chromatin environment at clusters specifically in the germ line. This data submission includes a total of 11 ChIP-seq samples and 2 RNA-seq samples. The ChIP-seq samples include six timecourse samples (Emb-YA) and three tissue-specific samples (WA, GL, SO) for one factor called SNPC-4 (previously called GEI-11). The remaining two ChIP-seq samples are for pol II (AMA-1) and pol III (RPC-1) at one developmental stage. All of these samples were done in replicate, with inputs, yielding at least four sequencing files per sample. Some of the ChIP-seq samples already have fastq files submitted to GEO: (SNPC-4 EMB _GSE48744: GSM1183805, GSM1183806, GSM1183807, GSM1183808), (SNPC-4 L1_GSE48702: GSM1183629, GSM1183630, GSM1183631, GSM1183632), (SNPC-4 L2_GSE37809: GSM928364, GSM928365, GSM928366, GSM928367), (SNPC-4 L3_GSE35276: GSM864851, GSM864852, GSM864853, GSM864854), and (AMA-1_GSE15535: GSM389309, GSM389310, GSM389312, GSM389313). We are submitting fastq files for the remaining ChIP-seq datasets: SNPC-4 L4 - 4 files, SNPC-4 YA - 5 files, SNPC-4 WA - 4 files, SNPC-4 GL - 4 files, SNPC-4 SO - 4 files, and RPC-1 WA - 5 files. Thus we are submitting an additional 26 fastq files. Additionally, we are submitting processed files for all 11 datasets, consisting of one normalized file for the combined duplicate IP and one normalized file for the input sequence. In the case of the three tissue-specific samples for SNPC-4, we are submitting an additional processed (but not normalized) file that represents the IP data with input-subtracted. Thus, we are submitting a total of 25 processed files. NOTE: The processed files for the ChIP-seq samples that already have fastq files submitted to GEO (SNPC-4 EMB _GSE48744, etc.) are linked below as supplementary files at the foot of the Series record. The RNA-seq samples represent one control (control_RNAseq) and one treatment (SNPC-4(RNAi)_RNA-seq)and were not duplicated. We are submitting one fastq file for each, as well as one processed data file for each, for a total of 2 fastq and 2 processed data files.
2014-11-17 | E-GEOD-53412 | biostudies-arrayexpress
Project description:Interaction of RNA-binding protein RBM38 with eIF4E on p53 mRNA is known to suppress p53 mRNA translation, which can be disrupted by an 8-amino acid peptide (Pep8-YPYAASPA) derived from RBM38, leading to induction of p53 and tumor suppression. Here, we rationally designed multiple Pep8 derivatives and screened for their binding affinities towards eIF4E in silico. We showed that several key residues within Pep8 are necessary for its structure and function. We identified a shortened 7-amino acid peptide (Pep7-PSAASPV) that has the highest affinity towards eIF4E and is the most potent inducer of p53 expression. We found that iRGD is an effective vehicle to deliver Pep7 inside of cells for induction of p53 expression and growth suppression as compared to other cell penetrating peptides (Penetratin and Pep-1). We found that peptide cyclization enhances Pep8 affinity for eIF4E, induction of p53 and tumor cell growth suppression. We also found that the ability of Pep7 to induce p53 expression and growth suppression is conserved in cells derived from canine osteosarcoma, a spontaneous tumor model frequently used for testing the feasibility of a therapeutic agent for human cancer. Moreover, we showed that both human and canine osteosarcoma cells, which are notoriously resistant to radiation therapy, were sensitized by Pep7 to radiation-induced growth suppression and cell death. Together, our data suggest that Pep7 may be explored to sensitize tumors to radiation therapy.
| S-EPMC9095979 | biostudies-literature
Project description:RNA-seq on HepG2 cells treated with an shRNA knockdown against EIF4G1. (EIF4G1-BGHLV23) For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf
2016-11-08 | GSE88321 | GEO