Inhibition Of Translation Initiation Research Articles

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5′ cap

Initiation is the rate-limiting step during mRNA 5′ cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.

Abstract C132: Targeting heat shock factor 1 improves the antitumor efficiency of hyperthermia.

Abstract The heat-shock response is a powerful transcriptional program which acts genome-wide, not only to restore normal protein folding through the induction of heat shock proteins (HSP), but to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by the Heat Shock Factor 1 (HSF1) transcription factor. We have previously shown that HSF1 plays a fundamental role in tumorigenesis, by promoting the survival and malignance of tumor cells, both in tissue culture and in mouse models of cancer [1]. HSF1 exerts its role by activating a unique transcriptional program in the cancer cells [2]. Indeed, increased HSF1 levels, as well as activation of its transcriptional signature, are associated with reduced survival in breast, lung and colon cancer patients [3]. Cancer cells are exquisitely dependent on HSF1 for survival. Exposure to additional stress, such as heat, further increases their dependency on HSF1. Recently we described how translation is linked to HSF1 activation using a derivative of the natural compound rocaglamide [4]. We found that this drug-like inhibitor of translation-initiation inhibits HSF1 and leads to tumor regression in hematopoietic malignancies. Here we combine this compound, or genetic inhibition of HSF1 expression, with focal heat therapy delivered via gold nano rods. We find that inhibiting HSF1 in solid tumors increases the efficiency of hyperthermia as an anticancer treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C132. Citation Format: Ruth Scherz-Shouval, Alexander F. Bagley, Luke Whitesell, Sangeeta N. Bhatia, Susan Lindquist. Targeting heat shock factor 1 improves the antitumor efficiency of hyperthermia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C132.

Abstract B109: Novel orally bioavailable triple-negative breast cancer drug candidates that prevent the development of cancer associated fibroblasts in the tumor stroma

Abstract One in 8 women in the US will develop breast cancer during her lifetime. Breast cancer is the second most commonly diagnosed form of cancer and the second leading cause of cancer deaths in women. The main cause of breast cancer death is metastasis. Although the survival rate is 99% for localized disease, it is only 23% for distant stage disease. Changes in the tumor stroma create an environment that provides support for tumor growth, progression, invasion and metastasis. The most prominent cell type in the tumor stroma is an activated form of fibroblasts, known as cancer (or carcinoma) associated fibroblast (CAF). In breast cancer, as much as 80% of stromal fibroblasts acquire the CAF phenotype. Triple negative (estrogen receptor, progesterone receptor, and HER2 negative) breast cancer (TNBC) has a low detection rate by mammography, and TNBC tumors generally have a higher mean size and grade at diagnosis than other forms of breast cancer. TNBC can be particularly aggressive with a high rate of recurrence and metastasis and a less favorable prognosis than other subtypes (64% vs. 81%, respectively). Unlike other forms of breast cancer (Tamoxifen for ER+ tumors, Herceptin for tumors with HER2/Neu), no targeted therapy exists for TNBC. We have previously described small molecule inhibitors of translation initiation with safety and efficacy in animal models for the treatment of TNBC. One of these compounds, NM891, showed a durable response after treatment cessation even though plasma and tumor tissue concentrations were negligible. Recent data indicate that this may be a result of NM891 acting on the stroma that supports the growth and progression of the tumor. In preliminary studies, NM891 and NM922 (a related analog) were shown to prevent the TGF-β dependent activation of fibroblasts using alpha smooth muscle actin (α-SMA) as a biomarker of CAF. Studies to determine the effect of NM891 and NM922 on pathways (such as TGF-β) that drive the development of CAF are ongoing. In addition, both NM891 and NM922 have recently been demonstrated to be orally bioavailable (20.5% and 19.6%, respectively, after oral gavage compared to i.v. administration in mice) with reasonable serum half-lives (1.5 and 2.4 hours, respectively), making them attractive not only for initial treatment, but also as part of a maintenance regimen. Future studies include the development of formulations to further enhance oral bioavailability and serum half-life as well as determination of optimum dosing range and schedule in support of an IND. NM891 and NM922 are promising orally bioavailable clinical candidates that preferentially kill tumor cell and also prevent the transition of stimulated fibroblasts to the CAF phenotype, thus potentially decreasing recurrence and metastasis and increasing survival rates in TNBC. Citation Format: Leah Fung, Kyle W.H. Chan, Robert W. Sullivan, Sabine Ottilie, Cathy A. Swindlehurst. Novel orally bioavailable triple-negative breast cancer drug candidates that prevent the development of cancer associated fibroblasts in the tumor stroma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B109.

Synthesis and SAR study of novel 3,3-diphenyl-1,3-dihydroindol-2-one derivatives as potent eIF2·GTP·Met-tRNAiMet ternary complex inhibitors

The growing recognition of inhibition of translation initiation as a new and promising paradigm for mechanism-based anti-cancer therapeutics is driving the development of potent, specific, and druggable inhibitors. The 3,3-diaryloxindoles were recently reported as potential inhibitors of the eIF2·GTP·Met-tRNAiMet ternary complex assembly and 3-{5-tert-butyl-2-hydroxyphenyl}-3-phenyl-1,3-dihydro-2H-indol-2-one #1181 was identified as the prototypic agent of this chemotype. Herein, we report our continuous effort to further develop this chemotype by exploring the structural latitude toward different polar and hydrophobic substitutions. Many of the novel compounds are more potent than the parent compound in the dual luciferase ternary complex reporter assay, activate downstream effectors of reduced ternary complex abundance, and inhibit cancer cell proliferation in the low μM range. Moreover, some of these compounds are decorated with substituents that are known to endow favorable physicochemical properties and as such are good candidates for evaluation in animal models of human cancer.

Aspergillus oryzae AoSO Is a Novel Component of Stress Granules upon Heat Stress in Filamentous Fungi

Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi.The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.

Synthesis, Pharmacophores, and Mechanism Study of Pyridin‐2(1H)‐one Derivatives as Regulators of Translation Initiation Factor 3A

Twenty-seven 1,5-disubstituted-pyridin-2(1H)-one derivatives were synthesized and evaluated for their anti-cancer and anti-fibrosis activity by A549 and NIH3T3 cell viability assays, respectively. To study the selectivity between the cancer and fibrosis cell lines, pharmacophore models (F1-F4) were built in advance for compounds with pyridin-2(1H)-one scaffold, which revealed the relationship between the occupation of the aromatic sub-site F4 and potent anti-cancer activity. The relationship between structure and anti-cancer activity for all target compounds is also reported herein: 1-Phenyl-5-((m-tolylamino)methyl)pyridine-2(1H)-one (22) displayed both potency and selectivity (IC50=0.13 mM) toward the A549 cell line through the inhibition of translation initiation, especially by eIF3a suppression, and can be treated as a lead for the design of novel eIF3a regulators and anti-lung cancer agents.

Small-molecule targeting of translation initiation for cancer therapy

Translation initiation plays a critical role in the regulation of cell growth and tumorigenesis. We report here that inhibiting translation initiation through induction of eIF2α phosphorylation by small-molecular-weight compounds restricts the availability of the eIF2.GTP.Met-tRNAi ternary complex and abrogates the proliferation of cancer cells in vitro and tumor growth in vivo. Restricting the availability of the ternary complex preferentially down-regulates the expression of growth-promoting proteins and up-regulates the expression of ER stress response genes in cancer cells as well as in tumors excised from either animal models of human cancer or cancer patients. These findings provide the first direct evidence for translational control of gene-specific expression by small molecules in vivo and indicate that translation initiation factors are bona fide targets for development of mechanism-specific anti-cancer agents.

Tight coordination of protein translation and HSF1 activation supports the anabolic malignant state.

The ribosome is centrally situated to sense metabolic states, but whether its activity, in turn, coherently rewires transcriptional responses is unknown. Here, through integrated chemical-genetic analyses, we found that a dominant transcriptional effect of blocking protein translation in cancer cells was inactivation of heat shock factor 1 (HSF1), a multifaceted transcriptional regulator of the heat-shock response and many other cellular processes essential for anabolic metabolism, cellular proliferation, and tumorigenesis. These analyses linked translational flux to the regulation of HSF1 transcriptional activity and to the modulation of energy metabolism. Targeting this link with translation initiation inhibitors such as rocaglates deprived cancer cells of their energy and chaperone armamentarium and selectively impaired the proliferation of both malignant and premalignant cells with early-stage oncogenic lesions.

Modifying chemotherapy response by targeted inhibition of eukaryotic initiation factor 4A

Translation is regulated predominantly at the initiation phase by several signal transduction pathways that are often usurped in human cancers, including the PI3K/Akt/mTOR axis. mTOR exerts unique administration over translation by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex responsible for recruiting 40S ribosomes (and associated factors) to mRNA 5′ cap structures. Hence, there is much interest in targeted therapies that block eIF4F activity to assess the consequences on tumor cell growth and chemotherapy response. We report here that hippuristanol (Hipp), a translation initiation inhibitor that selectively inhibits the eIF4F RNA helicase subunit, eIF4A, resensitizes Eμ-Myc lymphomas to DNA damaging agents, including those that overexpress eIF4E—a modifier of rapamycin responsiveness. As Mcl-1 levels are significantly affected by Hipp, combining its use with the Bcl-2 family inhibitor, ABT-737, leads to a potent synergistic response in triggering cell death in mouse and human lymphoma and leukemia cells. Suppression of eIF4AI using RNA interference also synergized with ABT-737 in murine lymphomas, highlighting eIF4AI as a therapeutic target for modulating tumor cell response to chemotherapy.

The sRNA RyhB Regulates the Synthesis of the Escherichia coli Methionine Sulfoxide Reductase MsrB but Not MsrA

Controlling iron homeostasis is crucial for all aerobically grown living cells that are exposed to oxidative damage by reactive oxygen species (ROS), as free iron increases the production of ROS. Methionine sulfoxide reductases (Msr) are key enzymes in repairing ROS-mediated damage to proteins, as they reduce oxidized methionine (MetSO) residues to methionine. E. coli synthesizes two Msr, A and B, which exhibit substrate diastereospecificity. The bacterial iron-responsive small RNA (sRNA) RyhB controls iron metabolism by modulating intracellular iron usage. We show in this paper that RyhB is a direct regulator of the msrB gene that encodes the MsrB enzyme. RyhB down-regulates msrB transcripts along with Hfq and RNaseE proteins since mutations in the ryhB, fur, hfq, or RNaseE-encoded genes resulted in iron-insensitive expression of msrB. Our results show that RyhB binds to two sequences within the short 5′UTR of msrB mRNA as identified by reverse transcriptase and RNase and lead (II) protection assays. Toeprinting analysis shows that RyhB pairing to msrB mRNA prevents efficient ribosome binding and thereby inhibits translation initiation. In vivo site directed-mutagenesis experiments in the msrB 5′UTR region indicate that both RyhB-pairing sites are required to decrease msrB expression. Thus, this study suggests a novel mechanism of translational regulation where a same sRNA can basepair to two different locations within the same mRNA species. In contrast, expression of msrA is not influenced by changes in iron levels.

Double-stranded RNA binding by the human cytomegalovirus PKR antagonist TRS1

Protein Kinase R (PKR) inhibits translation initiation following double-stranded RNA (dsRNA) binding and thereby represses viral replication. Human cytomegalovirus (HCMV) encodes two noncanonical dsRNA binding proteins, IRS1 and TRS1, and the expression of at least one of these PKR antagonists is essential for HCMV replication. In this study, we investigated the role of dsRNA binding by TRS1 in PKR inhibition. We found that purified TRS1 binds specifically to dsRNA with an affinity lower than that of PKR. Point mutants in the TRS1 dsRNA binding domain that were deficient in rescuing the replication of vaccinia virus lacking its PKR antagonist E3L were unable to bind to dsRNA but retained the ability bind to PKR. Thus TRS1 binding to dsRNA and to PKR are separable. Overall, our results are most consistent with a model in which TRS1 binds simultaneously to both dsRNA and PKR to inhibit PKR activation.

Abstract 3186: Characterizing the antitumor effects of inhibiting translation initiation in glioblastoma multiforme.

Abstract Oncogene addiction is the process by which a tumor cell becomes increasingly dependent on the expression of a particular gene for the maintenance of its tumorigenicity. Alleviating this dependence via genetic or pharmacological means may profoundly inhibit tumor growth. However many cancers are not dependent on a single gene for survival. Glioblastoma Multiforme (GBM) presents itself with many genetic aberrations contributing to its aggressive phenotype. These include loss of PTEN, amplification of CDK4 and EGFR among others, making targeting a single genetic node ineffective where other oncogenic networks may buffer any therapeutic benefit. Over the last 10 years it has been shown that targeting protein translation initiation results in the simultaneous targeting of many oncogenic pathways. Translation initiation is highly implicated in tumorigenesis with over 10 initiation factors acting as either proto-oncogenes or tumor suppressors, the most well described being the eIF4E. The overexpression of eIF4E in many cancers results in messenger RNA discrimination giving rise to an increased translation of a subset of oncogenic mRNAs with highly structured 5’UTRs. However the precise role translation initiation plays in maintaining tumorigenicity in GBM and the subsequent anti-neoplastic properties of its inhibition are not known. Here, we characterize the anti-tumor effects of targeting protein translation in three glioblastoma cell lines U87MG, U251N and the highly aggressive U87ΔEGFR, using a small molecule inhibitor of eIF4E, 4EGI-1. We show that 4EGI-1 severely impairs cell survival over a 72 hour time interval, in a dose-dependent manner. This is marked by an arrest of cell proliferation starting at 24 hours, induction of apoptosis at 48 hours with increased annexin V staining and a decrease in cell motility. We found these effects coincide with a decrease in protein expression of several oncogenes with highly structured 5’UTRs after treatment with 4EGI-1 including cell proliferation proteins c-Myc and Cyclin D1 which are completely lost by 48 hours and regulators of apoptosis such as Bcl-xL, Mcl-1 and Survivin which decrease and are lost by 72 hours, while not affecting overall transcription of these genes. Our results demonstrate the sensitivity of gliomas towards inhibition of translation initiation. This further highlights translation regulation control as a strong force in cancer therapy, particularly in glioblastoma where options are limiting. Citation Format: Arjuna K. Rajakumar, Josephine Nalbantoglu. Characterizing the antitumor effects of inhibiting translation initiation in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3186. doi:10.1158/1538-7445.AM2013-3186

Widespread regulation of translation by elongation pausing in heat shock

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation. Heat shocked cells globally inhibit protein synthesis, however the extent of translation initiation inhibition cannot explain the degree of translational repression observed.Exploring translational control during heat stress in mammalian cells using ribosome footprinting, we discovered that severe heat shock triggers elongation pausing of ribosomes after translating ~70 codons on the vast majority of mRNAs. The genome‐wide nature of the phenomenon, its location and the features of protein N‐termini suggested the involvement of ribosome‐associated chaperones.Reduced association of Hsp70 chaperones with ribosomes was seen following severe heat shock. Pre‐treatment with mild heat stress or overexpression of Hsp70 protected cells from the elongation pause induced by severe heat shock, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Finally, rewiring of the Hsp70‐translational machinery interactome during heat stress hinted at a mechanism that involves interactions with nascent peptides and the exit tunnel. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of the cellular stress response.

In vitro structure/activity relationship studies of second generation derivatives of the translation initiation inhibitor desmethyl, desamino‐Pateamine A

The natural product Pateamine A (PatA) from the marine sponge Mycale hentscheli has been found to be a highly potent antiproliferative agent with potential as a novel anti‐neoplastic agent. PatA binds to the eukaryotic translation initiation factor 4A (eIF4A) and prevents translation initiation. Previous structure/activity relationship (SAR) studies identified a rigid binding region on the molecule consisting of the “eastern” portion of the macrolide ring and a side chain terminating in a N,N‐dimethyl tertiary amine. The “western” portion of the macrolide serves as a flexible scaffold domain. Further SAR studies are presented here with a second generation of derivatives of the simplified Pateamine A analog desmethyl,desamino‐PatA (DMDA‐PatA). In vitro translation assays, and enzyme assays with purified recombinant eIF4AI were performed. The results presented here confirm the findings of the first SAR analysis and further clarify the structural requirements for activity. Rigidity of the binding domain was found to be critical for activity, and the tertiary amine found at the end of the side chain of the binding domain may serve as a potential site for future modifications to improve activity and specificity. Source of Funding, St. John's University.

Native Tertiary Structure and Nucleoside Modifications Suppress tRNA’s Intrinsic Ability to Activate the Innate Immune Sensor PKR

Interferon inducible protein kinase PKR is an essential component of innate immunity. It is activated by long stretches of dsRNA and provides the first line of host defense against pathogens by inhibiting translation initiation in the infected cell. Many cellular and viral transcripts contain nucleoside modifications and/or tertiary structure that could affect PKR activation. We have previously demonstrated that a 5′-end triphosphate–a signature of certain viral and bacterial transcripts–confers the ability of relatively unstructured model RNA transcripts to activate PKR to inhibit translation, and that this activation is abrogated by certain modifications present in cellular RNAs. In order to understand the biological implications of native RNA tertiary structure and nucleoside modifications on PKR activation, we study here the heavily modified cellular tRNAs and the unmodified or the lightly modified mitochondrial tRNAs (mt-tRNA). We find that both a T7 transcript of yeast tRNAPhe and natively extracted total bovine liver mt-tRNA activate PKR in vitro, whereas native E. coli, bovine liver, yeast, and wheat tRNAPhe do not, nor do a variety of base- or sugar-modified T7 transcripts. These results are further supported by activation of PKR by a natively folded T7 transcript of tRNAPhe in vivo supporting the importance of tRNA modification in suppressing PKR activation in cells. We also examine PKR activation by a T7 transcript of the A14G pathogenic mutant of mt-tRNALeu, which is known to dimerize, and find that the misfolded dimeric form activates PKR in vitro while the monomeric form does not. Overall, the in vitro and in vivo findings herein indicate that tRNAs have an intrinsic ability to activate PKR and that nucleoside modifications and native RNA tertiary folding may function, at least in part, to suppress such activation, thus serving to distinguish self and non-self tRNA in innate immunity.

Global translational pausing in response to stress

The ability of mammalian cells to respond rapidly to stress through global suppression of translation has mainly been attributed to inhibition of translation initiation. However, these papers show that pausing of ribosomes during elongation has an important role. These studies used deep sequencing of ribosome-protected mRNAs or ribosomal footprinting in cells that were subjected to heat shock or proteotoxic stress. In response to both types of stress, the authors found widespread pausing of ribosomes early in elongation and showed that chaperone proteins are involved in regulating this pausing.

Treatment of Breast and Lung Cancer Cells with a N-7 Benzyl Guanosine Monophosphate Tryptamine Phosphoramidate Pronucleotide (4Ei-1) Results in Chemosensitization to Gemcitabine and Induced eIF4E Proteasomal Degradation

The development of cancer and fibrotic diseases has been shown to be highly dependent on disregulation of cap-dependent translation. Binding protein eIF4E to N(7)-methylated guanosine capped mRNA has been found to be the rate-limiting step governing translation initiation, and therefore represents an attractive target for drug discovery. Our group has found that 7-benzyl guanosine monophosphate (7Bn-GMP) is a potent antagonist of eIF4E cap binding (K(d) = 0.8 μM). Recent X-ray crystallographic studies have revealed that the cap-dependent pocket undergoes a unique structural change in order to accommodate the benzyl group. Unfortunately, 7Bn-GMP is not cell permeable. Recently, we have prepared a tryptamine phosphoramidate prodrug of 7Bn-GMP, 4Ei-1, and shown that it is a substrate for human histidine triad nucleotide binding protein (hHINT1) and inhibits eIF4E initiated epithelial-mesenchymal transition (EMT) by Zebra fish embryo cells. To assess the intracellular uptake of 4Ei-1 and conversion to 7Bn-GMP by cancer cells, we developed a sensitive assay using LC-ESI-MS/MS for the intracellular quantitation of 4Ei-1 and 7Bn-GMP. When incubated with the breast cancer cell line MDA-231 or lung cancer cell lines H460, H383 and H2009, 4Ei-1 was found to be rapidly internalized and converted to 7Bn-GMP. Since oncogenic mRNAs are predicted to have the highest eIF4E requirement for translation, we carried out chemosensitization studies with 4Ei-1. The prodrug was found to chemosensitize both breast and lung cancer cells to nontoxic levels of gemcitabine. Further mechanistic studies revealed that the expressed levels of eIF4E were substantially reduced in cells treated with 4Ei-1 in a dose-dependent manner. The levels of eI4E could be restored by treatment with the proteasome inhibitor MG-132. Taken together, our results demonstrate that 4Ei-1 is likely to inhibit translation initiation by eIF4E cap binding by both antagonizing eIF4E cap binding and initiating eIF4E proteasomal degradation.

Cotranslational Response to Proteotoxic Stress by Elongation Pausing of Ribosomes

Translational control permits cells to respond swiftly to a changing environment. Rapid attenuation of global protein synthesis under stress conditions has been largely ascribed to the inhibition of translation initiation. Here we report that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected messenger RNA (mRNA) fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and cotranslational folding. Our results further support the chaperone "trapping" mechanism in promoting the passage of nascent chains. Our study reveals that translating ribosomes fine tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a cotranslational stress response to maintain the intracellular protein homeostasis.

Widespread Regulation of Translation by Elongation Pausing in Heat Shock

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70's interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses.

Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

BackgroundA large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC).ResultsExploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = −4.4 kcal/mol) inhibited Cdr1p expression by ~50%.ConclusionWe have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.