eIF5A Promotes Translation Elongation,Polysome Disassembly and Stress Granule Assembly

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.     

Spatio-temporal Dynamics and Mechanisms of Stress Granule Assembly

Stress granules (SGs) are non-membranous cytoplasmic aggregates of mRNAs and related proteins, assembled in response to environmental stresses such as heat shock, hypoxia, endoplasmic reticulum (ER) stress, chemicals (e.g. arsenite), and viral infections. SGs are hypothesized as a loci of mRNA triage and/or maintenance of proper translation capacity ratio to the pool of mRNAs. In brain ischemia, hippocampal CA3 neurons, which are resilient to ischemia, assemble SGs. In contrast, CA1 neurons, which are vulnerable to ischemia, do not assemble SGs. These results suggest a critical role SG plays in regards to cell fate decisions. Thus SG assembly along with its dynamics should determine the cell fate. However, the process that exactly determines the SG assembly dynamics is largely unknown. In this paper, analyses of experimental data and computer simulations were used to approach this problem. SGs were assembled as a result of applying arsenite to HeLa cells. The number of SGs increased after a short latent period, reached a maximum, then decreased during the application of arsenite. At the same time, the size of SGs grew larger and became localized at the perinuclear region. A minimal mathematical model was constructed, and stochastic simulations were run to test the modeling. Since SGs are discrete entities as there are only several tens of them in a cell, commonly used deterministic simulations could not be employed. The stochastic simulations replicated observed dynamics of SG assembly. In addition, these stochastic simulations predicted a gamma distribution relative to the size of SGs. This same distribution was also found in our experimental data suggesting the existence of multiple fusion steps in the SG assembly. Furthermore, we found that the initial steps in the SG assembly process and microtubules were critical to the dynamics. Thus our experiments and stochastic simulations presented a possible mechanism regulating SG assembly.     

Inhibition of lacZ Gene Translation Initiation in trp-lac Fusion Strains

Different levels of beta-galactosidase are found in various trp-lac fusion strains. These levels of beta-galactosidase fall within a 60-fold range. The amount of thiogalactoside transacetylase activity detected in these same strains only varies 10-fold and is found in amounts greater than those predicted from the beta-galactosidase levels. The observation that the beta-galactosidase and thiogalactoside transacetylase levels are not directly proportional, that the lacZ messenger ribonucleic acid (mRNA) levels are not proportional to the beta-galactosidase activity, that, at least for the one fusion strain tested, the SuA polarity suppressor does not affect the beta-galactosidase level, and that, in all but one strain, the beta-galactosidase activity appears to reside in normal beta-galactosidase molecules suggests that the disproportionately low production of beta-galactosidase is due to a decrease in the frequency of translation initiation of lacZ mRNA in these strains. Several mechanisms are proposed to explain this decrease. Some possible bases for the disproportional production of beta-galactosidase and thiogalactoside transacetylase are also described. The preferred explanation for these disproportional enzyme levels is that only a fraction of the full complement of ribosomes need initiate translation at lacZ for the functional synthesis of lac mRNA to occur and that once the lac ribonucleic acid is made a full complement of ribosomes can bind at internal translation initiation sites at Y and A.     

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.     

YB-1 Structure/Function Relationship in the Packaging of mRNPs and Consequences for Translation Regulation and Stress Granule Assembly in Cells

Biochemistry (Moscow) - From their synthesis in the nucleus to their degradation in the cytoplasm, all mRNAs have the same objective, which is to translate the DNA-stored genetic information into...     

Global Brain Ischemia and Reperfusion: Modifications in Eukaryotic Initiation Factors Associated with Inhibition of Translation Initiation

Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF-4E, eIF-4G, and eIF-2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF-4E. However, we observed in all 90-min-reperfused samples eIF-4G fragments that also bound eIF-4E. The amount of eIF-2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated eIF-2α in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated eIF-2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF-2α in these samples. The serine phosphorylation of eIF-2α and partial fragmentation of eIF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.     

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK

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不依赖甲硫氨酸的翻译起始

Ｓａｓａｋｉ等在ＰｒｏｃＮａｔｌＡｃａｄＳｃｉＵＳＡ 2 0 0 0年第 4期上报道了一种不依赖甲硫氨酸的翻译起始方式[1] 。ＰＳＩＶ (Ｐｌａｕｔｉａｓｔａｌｉｉｎｔｅｓｔｉｎｅｖｉｒｕｓ)是一种昆虫ＲＮＡ病毒 ,属蟋蟀麻痹样病毒组 (Ｃｒｉｃｋｅｔｐａｒａｌｙｓｉｓ ｌｉｋｅｖｉｒｕｓｅｓ) ,为正链ＲＮＡ病毒。属于该组的还有ＤＣＶ、ＲｈＰＶ、ＨｉＰＶ等。该组病毒的理化性质与哺乳动物的小ＲＮＡ病毒 (ｐｉｃｏｒｎａｖｉｒｕｓ)相似 ,但基因图 1　 (Ａ)ＰＳＩＶ基因组结构 ;(Ｂ)预测的外壳蛋白编码区上游的茎环结构组织不同。…     

Conditional Knockout of Tumor Overexpressed Gene in Mouse Neurons Affects RNA Granule Assembly,Granule Translation,LTP and Short Term Habituation

In neurons, specific RNAs are assembled into granules, which are translated in dendrites, however the functional consequences of granule assembly are not known. Tumor overexpressed gene (TOG) is a granule-associated protein containing multiple binding sites for heterogeneous nuclear ribonucleoprotein (hnRNP) A2, another granule component that recognizes cis-acting sequences called hnRNP A2 response elements (A2REs) present in several granule RNAs. Translation in granules is sporadic, which is believed to reflect monosomal translation, with occasional bursts, which are believed to reflect polysomal translation. In this study, TOG expression was conditionally knocked out (TOG cKO) in mouse hippocampal neurons using cre/lox technology. In TOG cKO cultured neurons granule assembly and bursty translation of activity-regulated cytoskeletal associated (ARC) mRNA, an A2RE RNA, are disrupted. In TOG cKO brain slices synaptic sensitivity and long term potentiation (LTP) are reduced. TOG cKO mice exhibit hyperactivity, perseveration and impaired short term habituation. These results suggest that in hippocampal neurons TOG is required for granule assembly, granule translation and synaptic plasticity, and affects behavior.     

Processing Body and Stress Granule Assembly Occur by Independent and Differentially Regulated Pathways in Saccharomyces cerevisiae

A variety of ribonucleoprotein (RNP) granules form in eukaryotic cells to regulate the translation, decay, and localization of the encapsulated messenger RNA (mRNAs). The work here examined the assembly and function of two highly conserved RNP structures, the processing body (P body) and the stress granule, in the yeast Saccharomyces cerevisiae. These granules are induced by similar stress conditions and contain translationally repressed mRNAs and a partially overlapping set of protein constituents. However, despite these similarities, the data indicate that these RNP complexes are independently assembled and that this assembly is controlled by different signaling pathways. In particular, the cAMP-dependent protein kinase (PKA) was found to control P body formation under all conditions examined. In contrast, the assembly of stress granules was not affected by changes in either PKA or TORC1 signalling activity. Both of these RNP granules were also detected in stationary-phase cells, but each appears at a distinct time. P bodies were formed prior to stationary-phase arrest, and the data suggest that these foci are important for the long-term survival of these quiescent cells. Stress granules, on the other hand, were not assembled until after the cells had entered into the stationary phase of growth and their appearance could therefore serve as a specific marker for the entry into this quiescent state. In all, the results here provide a framework for understanding the assembly of these RNP complexes and suggest that these structures have distinct but important activities in quiescent cells.EUKARYOTIC cells contain a number of membrane-bound compartments that partition the cytoplasm into distinct functional units. Proteins that act in similar pathways are often localized to the same compartment whereas those with competing activities are sequestered within different environments. Interestingly, recent data suggest that particular proteins and RNAs are also concentrated in what can be thought of as nontraditional compartments that lack a boundary membrane. These ribonucleoprotein (RNP) complexes, or granules, are more dynamic in nature and are found in both the nucleus and the cytoplasm of the cell (Anderson and Kedersha 2006; Mao et al. 2011; Weber and Brangwynne 2012). The formation of these granules can be induced by a variety of cues, including an exposure to stress or specific developmental transitions. In some cases, the underlying reasons for this reorganization of protein and RNA are known. For example, the polar granules present in germ cells store maternal mRNAs that are translated following fertilization (Schisa et al. 2001; Leatherman and Jongens 2003). However, for most RNP granules, the physiological role of the larger aggregate-like structures remains unclear. Nonetheless, the prevalence and evolutionary conservation of these complexes suggests that they serve important functions in the cell.Two of the better-characterized cytoplasmic RNPs are the processing bodies (P bodies) and stress granules that form in response to a variety of stress conditions. These particles contain translationally repressed messenger RNA (mRNAs) and a partially overlapping set of protein constituents (Kedersha and Anderson 2002; Anderson and Kedersha 2009; Balagopal and Parker 2009). Since a number of factors important for protein translation are also found in stress granules, these structures have been suggested to be sites of mRNA storage (Yamasaki and Anderson 2008). In contrast, P bodies were originally identified as cytoplasmic foci containing proteins important for mRNA decay (Sheth and Parker 2003; Eulalio et al. 2007a). Although this initially led to speculation that these foci were sites of mRNA turnover, more recent studies have found that this decay proceeds normally in cells lacking the larger P body complexes (Stoecklin et al. 2006; Decker et al. 2007; Eulalio et al. 2007b). As a result, the biological functions associated with P body foci remain unclear. However, an intriguing possibility has been suggested by studies demonstrating that P bodies contain proteins that do not appear to have a direct role in mRNA decay. These proteins include the phosphatase, calcineurin, and the catalytic subunits of the cAMP-dependent protein kinase (PKA) (Tudisca et al. 2010; Kozubowski et al. 2011). P bodies may therefore carry out specific functions that are dictated by the particular proteins present within these cytoplasmic structures. These functions may vary depending upon the particular cell type and stress condition used to induce the foci.A significant body of work has linked the induction of both P bodies and stress granules to the inhibition of protein synthesis, but less is known about the mechanisms regulating the subsequent formation of the larger aggregate-like assemblies (Franks and Lykke-Andersen 2008). These latter structures appear to form by a self-assembly process that involves the prion-like domains present in a number of granule proteins (Gilks et al. 2004; Decker et al. 2007; Reijns et al. 2008). Some insight into the regulation of this latter process was provided by a recent study of the P bodies that form in response to glucose deprivation in Saccharomyces cerevisiae (Ramachandran et al. 2011). This work showed that the inactivation of the PKA signaling pathway was both a necessary and a sufficient condition for P body foci formation. PKA directly phosphorylates Pat1, a conserved core constituent of these RNP structures, and thereby disrupts Pat1 interactions with a number of P body components, including the RNA helicase Dhh1 (Ramachandran et al. 2011). In contrast, defects in other nutrient-sensing pathways, including those involving the TORC1 or Snf1 protein kinases, did not have a significant effect upon P body formation. This work also suggested that P body foci were important for the long-term survival of cells that had entered into the stationary phase of growth. In particular, mutants that were defective for foci formation lost viability more rapidly during this period of quiescence (Ramachandran et al. 2011). This latter result is of interest in light of other work indicating that as much as 20% of the yeast proteome might relocalize to cytoplasmic foci when cells enter into this G₀-like resting state (Narayanaswamy et al. 2009; Noree et al. 2010). Since this phenomenon might not be restricted to yeast (An et al. 2008; Noree et al. 2010), the concentration of material at discrete sites in the cytoplasm may be generally important for the biology of the quiescent cell. Determining the underlying reasons for this relocalization of protein will therefore be critical for a complete understanding of the physiology of the eukaryotic cell.In S. cerevisiae, PKA is an essential component of one of the key signaling pathways responsible for coordinating cell growth with nutrient availability (Bahn et al. 2007; Zaman et al. 2008). This pathway also involves the GTP-binding Ras proteins Ras1 and Ras2 and is thought to respond, either directly or indirectly, to the levels of glucose present within cells (Santangelo 2006; Slattery et al. 2008; Zaman et al. 2009). The active, GTP-bound forms of the Ras proteins interact with the adenylyl cyclase Cyr1 and stimulate the production of cAMP (Field et al. 1990; Suzuki et al. 1990). This cyclic nucleotide is then bound by Bcy1, the regulatory subunit of the PKA enzyme, leading to the subsequent release of the active catalytic subunits; the basal state of PKA is an inactive heterotetramer made up of two catalytic and two regulatory subunits (Uno et al. 1982; Toda et al. 1987a; Taylor et al. 2008). These catalytic subunits are then free to phosphorylate their respective targets and thereby influence cell growth (Budovskaya et al. 2005). The existing genetic data suggest that this Ras/PKA pathway might play an important role in regulating the entry into stationary phase. For example, mutants that inactivate this pathway result in a growth arrest that resembles stationary phase (Iida and Yahara 1984; Schneper et al. 2004). Conversely, cells with constitutively elevated levels of PKA activity fail to arrest normally in stationary phase when nutrients are limiting (Broek et al. 1985; Broach 1991). The above results with Pat1 suggest that the PKA-mediated control of P body formation is one important component of this regulation of stationary-phase biology.In this study, we examined the regulation of P body and stress granule assembly in response to a variety of environmental cues, including several that can induce both of these RNP foci. This work demonstrated that the PKA pathway has a general role in the regulation of P body foci formation as mutants with constitutive PKA signaling were defective for P body assembly in all conditions tested. In contrast, stress granule formation was not influenced by changes in either PKA or TORC1 signalling activity. The results here also demonstrate that both types of RNP foci are present in stationary-phase cells and provide further support for a role for P bodies in the long-term survival of these quiescent cells. Finally, we show that P bodies and stress granules form at different times during batch culture growth and that stress granules in particular appear only after cells enter into stationary phase. Therefore, stress granule formation could serve as a useful marker for cell entry into this quiescent state. In all, this work indicates that P bodies and stress granules form independently of one another and that each assembly pathway is regulated by distinct signaling mechanisms.     

真核翻译起始因子与肿瘤

真核翻译起始因子(eukaryotic translation initiation factors,eIFs)是一类在蛋白质翻译起始的过程中发挥各自不同作用的蛋白质。近年来的研究发现,eIFs除了在蛋白质翻译起始中起作用外,还具有其他的作用,而且多种eIFs均与肿瘤的发生和进展相关。现就eIFs、eIFs与肿瘤的相关性及其在肿瘤治疗方面的应用等研究进展作一综述。     

Arsenite-Activated JNK Signaling Enhances CPEB4-Vinexin Interaction to Facilitate Stress Granule Assembly and Cell Survival

Stress granules (SGs) are compartmentalized messenger ribonucleoprotein particles (mRNPs) where translationally repressed mRNAs are stored when cells encounter environmental stress. Cytoplasmic polyadenylation element-binding protein (CPEB)4 is a sequence-specific RNA-binding protein and translational regulator. In keeping with the results obtained from the study of other RNA-binding proteins, we found CPEB4 localized in SGs in various arsenite-treated cells. In this study, we identified that Vinexin, a CPEB4-interacting protein, is a novel component of SGs. Vinexin is a SH3-domain-containing adaptor protein and affects cell migration through its association with Vinculin to localize at focal adhesions (FAs). Unexpectedly, Vinexin is translocated from FAs to SGs under arsenite-induced stress. The recruitment of Vinexin to SGs depends on its interaction with CPEB4 and influences SG formation and cell survival. Arsenite-activated c-Jun N-terminal kinase (JNK) signaling enhances the association between CPEB4 and Vinexin, which consequently facilitates SG localization of Vinexin. Taken together, this study uncovers a novel interaction between a translational regulator and an adaptor protein to influence SG assembly and cell survival.     

真核翻译起始因子3与肿瘤

真核翻译起始因子3是由多个亚基组成的,在真核翻译起始中发挥重要作用,近年来的研究表明其多个亚基在多种肿瘤细胞中存在异常表达的现象且与肿瘤的侵袭性、转移能力、分化程度及预后相关,使其有望成为肿瘤治疗的新靶点。     

Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.     

真核生物蛋白质翻译的内部起始机制

蛋白质翻译过程中,翻译的起始步骤是非常重要的．真核生物的翻译起始主要是通过依赖帽子结构的扫描机制进行的．近几年在翻译的研究工作中发现,在一些动物病毒中,蛋白质合成通过一种不同于扫描机制的内部起始机制起始翻译．用内部起始机制翻译的mRNA的5′端非翻译区有一个相对保守的结构,它在内部起始过程中具有重要作用,一些特异的蛋白质因子能够促进在特定位点起始翻译．     

mRNA翻译起始区的结构改变对几个外源基因翻译的影响

为观察ｍＲＮＡ翻译起始区结构与基因表达的关系,利用密码子的简并性,在不改变表达产物氨基酸序列的前提下定点突变几个外源基因的５′端若干位点,使基佤表达载体重组后转录形成的ｍＲＮＡ翻译起始区结构发生改变。经ＳＤＳ－ＰＡＧＥ等分析证实这些改变大大提高了外源基因的表达水平,ＲＮＡｄｏｔｂｌｏｔ表明突变与非突变基因转录水平差别不大,表达水平的提高主要由于翻译效率的提高,ｍＲＮＡ翻译起始区二级结构预测提示其生