Inhibition Of Translation Initiation Research Articles

Ganoderma lucidum extract decreases inflammatory breast cancer progression via translational control

Inflammatory Breast Cancer is a highly aggressive and chemo/radiation resistant cancer in which cells invade the vascular and lymphatic systems via generation of tumor emboli; a process regulated by selective mRNA translation. Accordingly, our objective is to characterize the role of Ganoderma lucidum extract (GLE) in translational regulation. In IBC, the mammalian target of rapamycin (mTOR) pathway is hyperactivated, while the eukaryotic initiation factor 4G (eIF4GI) is overexpressed. mTOR regulates translation initiation, and together with eIF4G, both regulate resistance to therapy of IBC cells, cancer cell survival and invasion. We previously showed that GLE reduces cap‐dependent mRNA translation in part through the inactivation (dephosphorylation) of mTOR, thereby activating eIF4E sequestration in IBC cells and tumors. GLE also induces cancer cell death, inhibits cancer cell invasion and synergizes with conventional chemotherapy to reduce IBC progression, while not affecting non‐cancerous cells. However the role of GLE on translational control in advanced BC has not been studied. We hypothesize that GLE regulates translation via translation initiation inhibition. Genome‐wide translation and transcription profiling was conducted in SUM‐149 IBC cells treated with vehicle or GLE for 24h. Analysis of actively translated mRNAs revealed that 689 genes showed differential expression by GLE treatment. Ingenuity pathway analysis revealed that the majority of these mRNAs are involved in the eIF2 and mTOR pathways, which control translation. Future directions include performing quantitative PCR arrays to validate the microarray results. This study is relevant to our long‐term goal of contributing to the discovery of therapeutics to reduce mortality of IBC.Support or Funding InformationThis project was sponsored by Title V PPOHA and Title‐V‐Cooperative #P031S130068 US Department of Education #P031M105050, NIH/RCMI #8G12MD007583, NIH/NIGMS #1SC3GM111171 to MMM, NCI #R01CA178509 to RJS, and the Breast Cancer Research Foundation (BCRF‐16–143) to RJS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the supporting agencies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

New tRNA contacts facilitate ligand binding in a Mycobacterium smegmatis T box riboswitch

T box riboswitches are RNA regulatory elements widely used by organisms in the phyla Firmicutes and Actinobacteria to regulate expression of amino acid-related genes. Expression of T box family genes is down-regulated by transcription attenuation or inhibition of translation initiation in response to increased charging of the cognate tRNA. Three direct contacts with tRNA have been described; however, one of these contacts is absent in a subclass of T box RNAs and the roles of several structural domains conserved in most T box RNAs are unknown. In this study, structural elements of a Mycobacterium smegmatis ileS T box riboswitch variant with an Ultrashort (US) Stem I were sequentially deleted, which resulted in a progressive decrease in binding affinity for the tRNAIle ligand. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) revealed structural changes in conserved riboswitch domains upon interaction with the tRNA ligand. Cross-linking and mutational analyses identified two interaction sites, one between the S-turn element in Stem II and the T arm of tRNAIle and the other between the Stem IIA/B pseudoknot and the D loop of tRNAIle These newly identified RNA contacts add information about tRNA recognition by the T box riboswitch and demonstrate a role for the S-turn and pseudoknot elements, which resemble structural elements that are common in many cellular RNAs.

Identification of di-substituted ureas that prevent growth of trypanosomes through inhibition of translation initiation

Some 1,3-diarylureas and 1-((1,4-trans)−4-aryloxycyclohexyl)−3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1–3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.

The mTOR-S6 kinase pathway promotes stress granule assembly

Stress granules are cytoplasmic mRNA-protein complexes that form upon the inhibition of translation initiation and promote cell survival in response to environmental insults. However, they are often associated with pathologies, including neurodegeneration and cancer, and changes in their dynamics are implicated in ageing. Here we show that the mTOR effector kinases S6 kinase 1 (S6K1) and S6 kinase 2 (S6K2) localise to stress granules in human cells and are required for their assembly and maintenance after mild oxidative stress. The roles of S6K1 and S6K2 are distinct, with S6K1 having a more significant role in the formation of stress granules via the regulation of eIF2α phosphorylation, while S6K2 is important for their persistence. In C. elegans, the S6 kinase orthologue RSKS-1 promotes the assembly of stress granules and its loss of function sensitises the nematodes to stress-induced death. This study identifies S6 kinases as regulators of stress granule dynamics and provides a novel link between mTOR signalling, translation inhibition and survival.

Messing up translation from the start: How AtaT inhibits translation initiation in E. coli

ABSTRACTToxin-antitoxin systems (TA) are widespread in bacteria and archea. They are commonly found in chromosomes and mobile genetic elements. These systems move from different genomic locations and bacterial hosts through horizontal gene transfer, using mobile elements as vehicles. Their potential roles in bacterial physiology are still a matter of debate in the field. The mechanisms of action of different toxin families have been deciphered at the molecular level. Intriguingly, the vast majority of these toxins target protein synthesis. They use a variety of molecular mechanisms and inhibit nearly every step of the translation process. Recently, we have identified a novel toxin, AtaT, presenting acetyltransferase activity.1 Our work uncovered the molecular activity of AtaT: it specifically acetylates the methionine moiety on the initiator Met-tRNAfMet. This modification drastically impairs recognition by initiation factor 2 (IF2), thereby inhibiting the initiation step of translation.

Epstein-Barr Virus Protein EB2 Stimulates Translation Initiation of mRNAs through Direct Interactions with both Poly(A)-Binding Protein and Eukaryotic Initiation Factor 4G.

Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could potentially be unfavorably translated compared to cellular spliced mRNAs. To overcome this situation, the virus encodes an RNA-binding protein (RBP) called EB2, which was previously found to both facilitate the export of nuclear mRNAs and increase their translational yield. Here, we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (CBC and eukaryotic initiation factor 4F [eIF4F], respectively) as well as the poly(A)-binding protein (PABP) to enhance translation initiation of a given messenger ribonucleoparticle (mRNP). Interestingly, such an effect can be obtained only if EB2 is initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders translation of these mRNPs less sensitive to translation initiation inhibitors. Taken together, our data suggest that EB2 binds and stabilizes cap-binding complexes in order to increase mRNP translation and furthermore demonstrate the importance of the mRNP assembly process in the nucleus to promote protein synthesis in the cytoplasm.IMPORTANCE Most herpesvirus early and late genes are devoid of introns. However, it is now well documented that mRNA splicing facilitates recruitment on the mRNAs of cellular factors involved in nuclear mRNA export and translation efficiency. To overcome the absence of splicing of herpesvirus mRNAs, a viral protein, EB2 in the case of Epstein-Barr virus, is produced to facilitate the cytoplasmic accumulation of viral mRNAs. Although we previously showed that EB2 also specifically enhances translation of its target mRNAs, the mechanism was unknown. Here, we show that EB2 first is recruited to the mRNA cap structure in the nucleus and then interacts with the proteins eIF4G and PABP to enhance the initiation step of translation.

Protein biosynthesis, a target of sorafenib, interferes with the unfolded protein response (UPR) and ferroptosis in hepatocellular carcinoma cells.

Sorafenib is the first line treatment for advanced hepatocellular carcinoma (HCC). We explored its impact on the proteostasis of cancer cells, i.e. the processes that regulate the synthesis, maturation and turn-over of cellular proteins. We observed that sorafenib inhibits the production of the tumour marker alpha-foetoprotein (AFP) in two different HCC cell lines, an effect that correlated with a radical inhibition of protein biosynthesis. This effect was observed at clinically relevant concentrations of sorafenib and was not related to the effect of sorafenib on the transport of amino acids across the plasma membrane or the induction of the unfolded protein response (UPR). Instead, we observed that sorafenib inhibits translation initiation and the mechanistic target of rapamycin (mTOR) signaling cascade, as shown by the analysis of phosphorylation levels of the protein 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1). We explored the consequences of this inhibition in HCC cells. We observed that overall sorafenib is a weak inducer of the UPR that can paradoxically prevent the UPR induced by tunicamycin. We also found no direct synergistic anticancer effect between sorafenib and various strategies that inhibit the UPR. In agreement with the possibility that translation inhibition might be an adaptive stress response in HCC cells, we noted that it protects cancer cell from ferroptosis, a form of oxidative necrosis. Our findings point to the modulation of protein biosynthesis and mTOR signaling as being important, yet complex determinants of the response of HCC cells to sorafenib.

4EGI-1 represses cap-dependent translation and regulates genome-wide translation in malignant pleural mesothelioma.

Deregulation of cap-dependent translation has been implicated in the malignant transformation of numerous human tissues. 4EGI-1, a novel small-molecule inhibitor of cap-dependent translation, disrupts formation of the eukaryotic initiation factor 4F (eIF4F) complex. The effects of 4EGI-1-mediated inhibition of translation initiation in malignant pleural mesothelioma (MPM) were examined. 4EGI-1 preferentially inhibited cell viability and induced apoptosis in MPM cells compared to normal mesothelial (LP9) cells. This effect was associated with hypophosphorylation of 4E-binding protein 1 (4E-BP1) and decreased protein levels of the cancer-related genes, c-myc and osteopontin. 4EGI-1 showed enhanced cytotoxicity in combination with pemetrexed or gemcitabine. Translatome-wide polysome microarray analysis revealed a large cohort of genes that were translationally regulated upon treatment with 4EGI-1. The 4EGI-1-regulated translatome was negatively correlated to a previously published translatome regulated by eIF4E overexpression in human mammary epithelial cells, which is in agreement with the notion that 4EGI-1 inhibits the eIF4F complex. These data indicate that inhibition of the eIF4F complex by 4EGI-1 or similar translation inhibitors could be a strategy for treating mesothelioma. Genome wide translational profiling identified a large cohort of promising target genes that should be further evaluated for their potential significance in the treatment of MPM.

Depletion of the mRNA translation initiation inhibitor, programmed cell death protein 4 (PDCD4), impairs L6 myotube formation.

The mechanistic (mammalian) target of rapamycin complex 1 (mTORC1) signaling is vital for optimal muscle mass and function. Although the significance of mTORC1 in stimulating muscle growth is unequivocal, evidence in support of its role during muscle regeneration is less clear. Here, we showed that the abundance (protein and mRNA) of the mTORC1/S6K1 substrate, programmed cell death protein 4 (PDCD4), is upregulated at the onset of differentiation of L6 and C2C12 cells. The increase in PDCD4 was not associated with any changes in S6K1 activation, but the abundance of beta transducing repeat‐containing protein (β‐TrCP), the ubiquitin ligase that targets PDCD4 for degradation, increased. Myoblasts lacking PDCD4 showed impaired myotube formation and had markedly low levels of MHC‐1. Analysis of poly (ADP‐ribose) Polymerase (PARP), caspase 7 and caspase 3 indicated reduced apoptosis in PDCD4‐deficient cells. Our data demonstrate a role for PDCD4 in muscle cell formation and suggest that interventions that target this protein may hold promise for managing conditions associated with impaired myotube formation.

EIF2α Phosphorylation Mediates IL24-Induced Apoptosis through Inhibition of Translation.

IL24 is an immunomodulatory cytokine that also displays broad cancer-specific suppressor effects. The tumor-suppressor activities of IL24 include inhibition of angiogenesis, sensitization to chemotherapy, and cancer-specific apoptosis. Supra-physiologic activation and/or overexpression of translation initiation factors are implicated in the initiation and progression of cancer animal models as well as a subset of human cancers. Activation and/or overexpression of translation initiation factors correlate with aggressiveness of cancer and poor prognosis. Two rate-limiting translation initiation complexes, the ternary complex and the eIF4F complex, are regulated by eIF2α and 4E-BP1 phosphorylation, respectively. The work reported here provides direct evidence that IL24 induces inhibition of translation initiation leading to apoptosis in squamous cell carcinoma. A dominant constitutively active mutant of eIF2α, which is resistant to phosphorylation, was used to determine the involvement of eIF2α in IL24-induced apoptosis. Treatment with IL24 resulted in inhibition of protein synthesis, expression of downstream biomarkers of ternary complex depletion such as CHOP, and induction of apoptosis in cancer cells. The constitutively active nonphosphorylatable mutant of eIF2α, eIF2α-S51A, reversed both the IL24-mediated translational block and IL24-induced apoptosis. Intriguingly, IL24 treatment also caused hypophosphorylation of 4E-BP1, which binds to eIF4E with high affinity, thus preventing its association with eIF4G and therefore preventing elF4F complex assembly.Implications: These results demonstrate a previously unrecognized role of IL24 in inhibition of translation, mediated through both phosphorylation of eIF2α and dephosphorylation of 4E-BP1, and provide the first direct evidence for translation control of gene-specific expression by IL24. Mol Cancer Res; 15(8); 1117-24. ©2017 AACR.

Abstract 98: TET suppresses protein translation, differentially effects polysome loading of cell cycle regulatory proteins and inhibits cell growth and proliferation of prostate cancer cells

Abstract Introduction: Translational control is a critical component in development and progression of cancer and could be explored to develop antineoplastic therapies. Results from our laboratory have shown that TET (a derivative of Tetrandrine) inhibits cell growth and promotes apoptosis of prostate cancer (PCa) cells. However, the underlying molecular mechanisms are not clearly understood. We observed that TET inhibited 4EBP phosphorylation, suggesting that it might inhibit translational initiation. In the present study we evaluated the effects of TET on Translation in general and cell cycle related proteins in particular. Methods: Androgen dependent PCa cells LNCaP and Enz resistant CRPCa cells 22Rv1 were maintained in supplemented RPMI as recommended. Where indicated cells were treated with TET (0 or 20µM) for various time points (2h, 8h, 18h and 24h). At the end of the experimental periods cells were pulsed with cycloheximide for 10 minutes in RPMI media. Cytoplasmic RNA was collected by lysing cells in polysome lysis buffer. Cell lysates were centrifuged and the resulting cytosolic supernatant was separated by density (a 10% to 50% sucrose) gradient and centrifugation. Fractions were collected using a TELEDYNE ISCO Density Gradient Fractionation System with continuous monitoring of untranslated mRNAs. Polysome (P)/Monosome (M) ratio were measured. The total RNA in each fraction was extracted using TRIzol LS (Invitrogen) and qRT PCR was done for p21, p27, Cyclin D1 and c-Myc gene for each fraction. Cell viability was measured by MTT assay at various time points. Results: TET treatment resulted in decreased protein synthesis. TET treatment resulted in decrease of RNA associated with Polysomes. There were a significant shift in P/M ratio from 0.82 (control) to 0.48 (24 h. TET) [TET 2hrs (0.75), 8h (0.69) and 18hrs (0.52)] in LNCaP and P/M ratio form 0.91 (control) to 0.32 (8 h TET) [TET 2h (0.78) and 4h (0.53)] in 22Rv1 cells. The decrease of the P/M ratio suggests that TET inhibits translation initiation in prostate cancer cells independent of the androgen dependence. While there was generalized decrease in translation and specific decrease in mRNA for Cyclin D1, c-Myc associated with polysomes following TET treatment. In quite contrast to these findings, we observed an increase in mRNA for p21 and p27 in polysomal fractions of TET treated cells as compare to control at 24 h in LNCaP cells. These data suggest differential effects of TET treatment on polysome loading of cell cycle regulatory proteins. Moreover, TET inhibited growth and proliferation of LNCaP and 22Rv1 cells in dose as well as in time dependent fashion. Conclusions: TET targets translational machinery and differentially effects translation of cell cycle regulatory genes by effecting their polysome loading and effects cell growth and proliferation of androgen dependent and castrate resistant PCa cells. Citation Format: Praveen K. Jaiswal, Sweaty Koul, Qin Dong, Hari Koul. TET suppresses protein translation, differentially effects polysome loading of cell cycle regulatory proteins and inhibits cell growth and proliferation of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 98. doi:10.1158/1538-7445.AM2017-98

Abstract 4489: Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis

Abstract AT-Rich Interaction Domain-containing protein 5a (ARID5A; also known as Modulator Recognition Factor 1(MRF1) ) was first cloned in our laboratory by screening a cDNA library of Tera-2 mRNA with an oligonucleotide probe from the major immediate early gene of human cytomegalo virus. It has been reported that ARID5A forms a complex with androgen receptor (AR) in yeast two hybrid assays and is a cosuppressor of AR in transient transfection assays. In this study, we report that the knockdown of ARID5a expression inhibits proliferation of LNCaP cells on the contrary to the expectation from the transient transfection assay. We found that proliferation of cells stimulated by the treatment with dihydro-testosterone (DHT) was suppressed when ARID5a expression was down-regulated by siRNA technology. Androgen stimulates cell proliferation through activation of mammalian target of rapamycin complex 1 (mTORC1). However, the suppression of ARID5a does not affect androgen-activated mTOR activity. Furthermore, the expression of androgen-inducible and androgen-suppressive genes were not affected by suppression of ARID5a. We concluded that ARID5a is not a cosuppressor of AR. Meanwhile, flow cytometer analysis showed that DHT-stimulated cell cycle was arrested at G1 phase after the knockdown of ARID5a expression. Oil Red O staining assays showed that the inhibition of ARID5a expression led to decreased DHT-induced lipid accumulation. Our data further proved that the knockdown of ARID5a expression inhibited the protein expression of DHT-induced sterol regulatory element binding transcription factor 1 (SREBP-1). In addition, Western blot analysis revealed that the down-regulation of ARID5a decreased the expression of cell survival / proliferation and cell cycle regulation proteins, such as hypoxia inducible factor 1 alpha (HIF1a), cyclin D1 and cyclin D3. Real time PCR data revealed that the knockdown of ARID5a expression did not affect the steady level of HIF1a, cyclin D1 and cyclin D3 mRNA. These suggested that knockdown of ARID5a expression affected protein expression at translational and/or post-translational level. Our data suggested that knockdown of ARID5a expression increased the phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) which inhibited translation initiation. S35 labeling assay confirmed that the knockdown of ARID5a expression suppressed the global protein synthesis. We showed that one of the four eIF2a kinases, general control non-derepressible 2 (GCN2) was activated by the suppression of ARID5a expression. We are currently studying on the mechanisms by which ARID5A regulates GCN2 kinase. Citation Format: Chao Sun, Viktor Chesnokov, Keiichi Itakura. Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4489. doi:10.1158/1538-7445.AM2017-4489

Overexpression of eIF4F components in meningiomas and suppression of meningioma cell growth by inhibiting translation initiation

Meningiomas frequently display activation of the PI3K/AKT/mTOR pathway, leading to elevated levels of phospho-eukaryotic translation initiation factor 4E binding proteins, which enhances protein synthesis; however, it is not known whether inhibition of protein translation is an effective treatment option for meningiomas. We found that human meningiomas expressed high levels of the three components of the eukaryotic initiation factor 4F (eIF4F) translation initiation complex, eIF4A, eIF4E, and eIF4G. The expression of eIF4A and eIF4E was important in sustaining the growth of NF2-deficient benign meningioma Ben-Men-1 cells, as shRNA-mediated knockdown of these proteins strongly reduced cell proliferation. Among a series of 23 natural compounds evaluated, silvestrol, which inhibits eIF4A, was identified as being the most growth inhibitory in both primary meningioma and Ben-Men-1 cells. Silvestrol treatment of meningioma cells prominently induced G2/M arrest. Consistently, silvestrol significantly decreased the amounts of cyclins D1, E1, A, and B, PCNA, and Aurora A. In addition, total and phosphorylated AKT, ERK, and FAK, which have been shown to be important drivers for meningioma cell proliferation, were markedly lower in silvestrol-treated Ben-Men-1 cells. Our findings suggest that inhibiting protein translation could be a potential treatment for meningiomas.

1H, 13C, and 15N backbone chemical shift assignments of 4E-BP144-87 and 4E-BP144-87 bound to eIF4E.

The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)4LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)4LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP144-87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m7GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction.

Translation initiation events on structured eukaryotic mRNAs generate gene expression noise.

Gene expression stochasticity plays a major role in biology, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. We have addressed the lack of knowledge about posttranscriptional contributions to noise by determining cell-to-cell variations in the abundance of mRNA and reporter protein in yeast. Two types of structural element, a stem–loop and a poly(G) motif, not only inhibit translation initiation when inserted into an mRNA 5΄ untranslated region, but also generate noise. The noise-enhancing effect of the stem–loop structure also remains operational when combined with an upstream open reading frame. This has broad significance, since these elements are known to modulate the expression of a diversity of eukaryotic genes. Our findings suggest a mechanism for posttranscriptional noise generation that will contribute to understanding of the generally poor correlation between protein-level stochasticity and transcriptional bursting. We propose that posttranscriptional stochasticity can be linked to cycles of folding/unfolding of a stem–loop structure, or to interconversion between higher-order structural conformations of a G-rich motif, and have created a correspondingly configured computational model that generates fits to the experimental data. Stochastic events occurring during the ribosomal scanning process can therefore feature alongside transcriptional bursting as a source of noise.

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Interferon-induced protein 56 (IFI56) is induced by VHSV infection but not by bacterial infection in olive flounder (Paralichthys olivaceus)

Interferon-inducible protein 56 (IFI56, also known as ISG56/IFIT1, interferon-induced protein with tetratricopeptide repeats 1) is strongly induced in response to interferon and a potent inhibitor of viral replication and translational initiation. Here, we describe the identification of IFI56 (OfIFI56) in olive flounder, its characteristic features, and expression levels in various tissues before and after viral hemorrhagic septicemia virus (VHSV) infection. The full-length OfIFI56 sequence was identified from rapid amplification of cDNA ends PCR. The complete coding sequence of OfIFI56 is 1971 bp in length and encodes 431 amino acids. The putative OfIFI56 protein has multiple tetratricopeptide (TPR) motifs, which regulate diverse biological processes, such as organelle targeting, protein import, and vesicle fusion. Based on sequence analysis, the Larimichthys crocea IFI56 protein (61%) had the highest sequence homology to OfIFI56. In healthy olive flounder, OfIFI56 mRNA expression was detected in many tissues such as intestine, gill, head kidney, heart, spleen, and trunk kidney tissues. After VHSV challenge, OfIFI56 mRNA was significantly up-regulated in these tissues. Additionally, OfIFI56 expression was induced by poly I:C but not by Streptococcus parauberis and S. iniae infection or lipopolysaccharide injection in kidney and spleen tissues of olive flounder. These results demonstrate that piscine OfIFI56 expression is not induced by bacterial infection but is selectively induced by viral infection, especially VHSV, and that OfIFI56 may play an important role in the host response against VHSV infection.

Inhibiting the oncogenic translation program is an effective therapeutic strategy in multiple myeloma.

Multiple myeloma (MM) is a frequently incurable hematological cancer in which overactivity of MYC plays a central role, notably through up-regulation of ribosome biogenesis and translation. To better understand the oncogenic program driven by MYC and investigate its potential as a therapeutic target, we screened a chemically diverse small-molecule library for anti-MM activity. The most potent hits identified were rocaglate scaffold inhibitors of translation initiation. Expression profiling of MM cells revealed reversion of the oncogenic MYC-driven transcriptional program by CMLD010509, the most promising rocaglate. Proteome-wide reversion correlated with selective depletion of short-lived proteins that are key to MM growth and survival, most notably MYC, MDM2, CCND1, MAF, and MCL-1. The efficacy of CMLD010509 in mouse models of MM confirmed the therapeutic relevance of these findings in vivo and supports the feasibility of targeting the oncogenic MYC-driven translation program in MM with rocaglates.

Cap-binding protein 4EHP effects translation silencing by microRNAs

MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.

SRNA154 a newly identified regulator of nitrogen fixation in Methanosarcina mazei strain Gö1

ABSTRACTTrans-encoded sRNA154 is exclusively expressed under nitrogen (N)-deficiency in Methanosarcina mazei strain Gö1. The sRNA154 deletion strain showed a significant decrease in growth under N-limitation, pointing toward a regulatory role of sRNA154 in N-metabolism. Aiming to elucidate its regulatory function we characterized sRNA154 by means of biochemical and genetic approaches. 24 homologs of sRNA154 were identified in recently reported draft genomes of Methanosarcina strains, demonstrating high conservation in sequence and predicted secondary structure with two highly conserved single stranded loops. Transcriptome studies of sRNA154 deletion mutants by an RNA-seq approach uncovered nifH- and nrpA-mRNA, encoding the α–subunit of nitrogenase and the transcriptional activator of the nitrogen fixation (nif)-operon, as potential targets besides other components of the N-metabolism. Furthermore, results obtained from stability, complementation and western blot analysis, as well as in silico target predictions combined with electrophoretic mobility shift-assays, argue for a stabilizing effect of sRNA154 on the polycistronic nif-mRNA and nrpA-mRNA by binding with both loops. Further identified N-related targets were studied, which demonstrates that translation initiation of glnA2-mRNA, encoding glutamine synthetase2, appears to be affected by sRNA154 masking the ribosome binding site, whereas glnA1-mRNA appears to be stabilized by sRNA154. Overall, we propose that sRNA154 has a crucial regulatory role in N-metabolism in M. mazei by stabilizing the polycistronic mRNA encoding nitrogenase and glnA1-mRNA, as well as allowing a feed forward regulation of nif-gene expression by stabilizing nrpA-mRNA. Consequently, sRNA154 represents the first archaeal sRNA, for which a positive posttranscriptional regulation is demonstrated as well as inhibition of translation initiation.