Free initiation factors eIF4A and eIF4B are dispensable for translation initiation on uncapped mRNAs

The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex.     

Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent.

Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N-terminal part, and binds eIF3 and eIF4A through the central and C-terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs. Although this stimulation is cap-independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of eIF4G-mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.     

Translational control of eIF5A in various diseases

Translational control is a crucial component in the development and progression of different diseases. Translational control may involve selective translation of specific mRNAs, which promote cell proliferation or lead to alterations in translation factor levels and activities. Eukaryotic initiation factor 5A (eIF5A) is the only known protein to contain the unusual amino acid hypusine [N (ε)-(4-amino-2-hydroxybutyl)-lysine], which is formed from the polyamine spermidine by two catalytic steps. eIF5A is involved in translation, elongation and stimulating peptide bond formation. Hypusination of eIF5A is essential for its activity in promoting cell proliferation. Meanwhile, there is evidence that eIF5A is a key protein in the pathogenicity of different diseases, such as diabetes, several human cancers, malaria and HIV-1 infections. Hitherto, the available data suggest that eIF5A has a role of a cell context-dependent function being more proliferative in the case of several human cancers and being involved under stress conditions in diabetes. Secondly, in HIV-1 infections and in diabetes, eIF5A also has a nuclear function by its sequence-specific binding of mRNAs as an mRNA-shuttle in conjunction with nuclear membrane export proteins. This binding may also influence the half-lives of mRNAs or their sequestration. Based on these data, there is a considerable therapeutic interest in eIF5A as a selective target for drug development through inhibition of hypusination.     

Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer''s disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer''s Disease Research Center (ADRC) and Alzheimer''s Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10⁻¹⁰) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.     

eIF4A1-dependent mRNAs employ purine-rich 5’UTR sequences to activate localised eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation

Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5’UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5’UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.     

Synergistic activation of eIF4A by eIF4B and eIF4G

eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants K_D of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A–eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A–eIF4B–RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.     

eIF4B,eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle

Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5''-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation.     

B 淋巴细胞刺激因子（BLyS）调控相关蛋白的研究进展

B淋巴细胞刺激因子是肿瘤坏死因子超家族的成员之一,它同自身免疫疾病的发生发展密切相关,BLyS过度表达会促使B淋巴细胞增殖并分泌自身抗体。BLyS也是治疗自身免疫疾病的重要靶点。关于BLyS的调控涉及了许多蛋白。本文主要就G-CSF、TGF-β1、IFN-γ、IL-10和Poly(I:C)等对BLyS的调控作用作一综述。     

eIF4B and eIF4G Jointly Stimulate eIF4A ATPase and Unwinding Activities by Modulation of the eIF4A Conformational Cycle

Eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box protein that participates in translation initiation. As an ATP-dependent RNA helicase, it is thought to resolve secondary structure elements from the 5′-untranslated region of mRNAs to enable ribosome scanning. The RNA-stimulated ATPase and ATP-dependent helicase activities of eIF4A are enhanced by auxiliary proteins, but the underlying mechanisms are still largely unknown. Here, we have dissected the effect of eIF4B and eIF4G on eIF4A RNA-dependent ATPase- and RNA helicase activities and on eIF4A conformation. We show for the first time that yeast eIF4B, like its mammalian counterpart, can stimulate RNA unwinding by eIF4A, although it does not affect the eIF4A conformation. The eIF4G middle domain enhances this stimulatory effect and promotes the formation of a closed eIF4A conformation in the presence of ATP and RNA. The closed state of eIF4A has been inferred but has not been observed experimentally before. eIF4B and eIF4G jointly stimulate ATP hydrolysis and RNA unwinding by eIF4A and favor the formation of the closed eIF4A conformer. Our results reveal distinct functions of eIF4B and eIF4G in synergistically stimulating the eIF4A helicase activity in the mRNA scanning process.     

Synthesis and Phosphorylation of Maize Acidic Ribosomal Proteins : Implications in Translational Regulation

The objective of this research was to determine the role of acidic ribosomal protein (ARP) phosphorylation in translation. Ribosomes (Rbs) from germinated maize (Zea mays L.) axes had four ARP bands within 4.2 to 4.5 isoelectric points when analyzed by isoelectric focusing. Two of these bands disappeared after alkaline phosphatase hydrolysis. During germination a progressive change from nonphosphorylated (0 h) to phosphorylated ARP (24 h) forms was observed in the Rbs; a free cytoplasmic pool of nonphosphorylated ARPs was also identified by immunoblot and isoelectric focusing experiments. De novo ARP synthesis initiated very slowly early in germination, whereas ARP phosphorylation occurred rapidly within this period. ARP-phosphorylated versus ARP-nonphosphorylated Rbs were tested in an in vitro reticulocyte lysate translation system. Greater in vitro mRNA translation rates were demonstrated for the ARP-phosphorylated Rbs than for the non-ARP-phosphorylated ones. Rapamycin application to maize axes strongly inhibited S6 ribosomal protein phosphorylation, but did not interfere with the ARP phosphorylation reaction. We conclude that ARP phosphorylation does not depend on ARP synthesis or on ARP assembly into Rbs. Rather, this process seems to be part of a translational regulation mechanism.     

20S proteasome differentially alters translation of different mRNAs via the cleavage of eIF4F and eIF3

The molecular basis for coordinated regulation of protein synthesis and degradation is not understood. Here we report that the 20S proteasome endoproteolytically cleaves the translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3. The cleavage of eIF4G or eIF3a differentially affects the assembly of ribosomal preinitiation complexes on different cellular and viral mRNAs in an in vitro system containing pure components. Inhibition of proteolytic activity of the 20S proteasome with specific inhibitors prevents cleavage of both factors in vitro and in vivo, restores assembly of ribosomal complexes in vitro, and differentially affects translation of different mRNAs in vivo. These studies demonstrate the importance of the endoproteolytic activity of proteasomes in regulation of cellular processes and suggest a link between protein synthesis and degradation.     

Evidence for Variation in the Optimal Translation Initiation Complex: Plant eIF4B,eIF4F,and eIF(iso)4F Differentially Promote Translation of mRNAs

Expansins are cell wall proteins associated with the process of plant growth. However, investigations in which expansin gene expression has been manipulated throughout the plant have often led to inconclusive results. In this article, we report on a series of experiments in which overexpression of expansin was targeted to specific phases of leaf growth using an inducible promoter system. The data indicate that there is a restricted window of sensitivity when increased expansin gene expression leads to increased endogenous expansin activity and an increase in leaf growth. This phase of maximum expansin efficacy corresponds to the mid phase of leaf growth. We propose that the effectiveness of expansin action depends on the presence of other modulating factors in the leaf and we suggest that it is the control of expression of these factors (in conjunction with expansin gene expression) that defines the extent of leaf growth. These data help to explain some of the previously observed variation in growth response following manipulation of expansin gene expression and highlight a potential linkage of the expression of modifiers of expansin activity with the process of exit from cell division.Expansins were initially identified as cell wall proteins that had the ability to promote the extension of plant tissue in vitro (McQueen-Mason et al., 1992). Further work on these proteins and the genes encoding them has revealed a picture in which, although a general correlation with growth has often been substantiated, it is clear that control of growth is a much more complex process than the control of expression of a single protein type (for review, see Cosgrove, 2000; Lee et al., 2001; Li et al., 2003). In addition, although it is clear that expansins play a role in many growth processes, there are a number of open questions about exactly how expansins contribute to these processes. First, we still have a very limited understanding of the molecular mechanism of expansin action. Efforts to identify classical enzymatic activities associated with expansins have proven fruitless (McQueen-Mason and Cosgrove, 1995; Li and Cosgrove, 2001) and the remaining, somewhat speculative, interpretation is that expansins intercalate within carbohydrate matrices in the cell wall, leading to transient loosening of noncovalent interactions and, thus, the ability of these matrices to move relative to each other (McQueen-Mason and Cosgrove., 1994). In addition, by unlocking aspects of the molecular architecture of the cell wall, expansins may allow access of other cell wall proteins/enzymes to particular substrates. Depending on the nature of these other proteins/enzymes, expansin activity could thus be associated not only with growth processes, but also with cell wall modifications linked with differentiation. Such a mechanism would help to explain observations (described below) that the effectiveness of expansin action appears to be context dependent and is not only associated with changes in plant growth but also with differentiation.Various analyses have revealed that expansins are present in a wide range of plants, including bryophytes, ferns, angiosperms, and conifers (Hutchison et al., 1999; Kim et al., 2000; Schipper et al., 2002). Moreover, they are generally encoded by relatively large gene families whose members often show distinct patterns of gene expression (Kende et al., 2004). Some of these expression patterns correlate with growth processes, such as root growth (Wu et al., 1996), internode growth (Cho and Kende, 1997), leaf growth (Muller et al., 2007), and cotton (Gossypium hirsutum) fiber growth (Ruan et al., 2001), whereas others correlate with events of differentiation, such as fruit ripening (Rose et al., 1997; Brummell et al., 1999b), grass tiller formation (Reidy et al., 2001), and endosperm breakdown (Chen and Bradford, 2000). In addition, some novel nonplant expansin activities have been identified that suggest that pathogens may induce altered cell wall structure via an expansin-mediated mechanism (Qin et al., 2004). Since in vitro assays have suggested that the activities of expansins extracted from different sources tend to be similar (Cosgrove, 2000), it has been proposed that this tissue, organ, and environmental specificity of expression pattern reflects a specialized role for expansins in specific contexts rather than any major difference in activity of the protein. As stated above, this specific function may depend on the presence (or absence) of tissue-specific cofactors, the nature of which is as yet unclear.In addition to biochemical approaches to understanding expansin function, numerous groups have undertaken transgenic experiments to alter expansin gene expression in plants to observe the outcome on plant phenotype. Although some successes with antisense strategies have been reported (Brummell et al., 1999a; Cho and Cosgrove, 2000), the encoding of expansin by large gene families means that genetic redundancy poses a significant problem for such approaches (e.g. Schipper et al., 2002). Simple overexpression strategies to alter expansin activity may also be difficult to interpret. For example, when expansins were constitutively overexpressed throughout Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), and rice (Oryza sativa) plants, the outcomes tended to be pleiotropic, including a decrease in overall plant growth (Cho and Cosgrove, 2000; Rochange et al., 2001; Choi et al., 2003). However, when altered expansin expression was targeted more specifically to a particular tissue or organ, then more easily interpretable results were obtained. For example, when altered expansin expression was directed to the developing leaf petiole in Arabidopsis, altered leaf growth was observed (Cho and Cosgrove, 2000), consistent with the idea that expansins promote growth, and when inducible expression of expansin was targeted throughout rice plants, quantitative changes in growth were observed (Choi et al., 2003). The results of these experiments indicate that expansin gene expression can be used as a tool to modulate growth, but that the timing and spatial extent of expression can have a significant influence on the phenotype observed. Again, these data support the hypothesis that the effectiveness of expansin in promoting specific growth or differentiation events is dependent on the presence of particular tissue- or developmental-specific cofactors. So far, little progress has been made on the identification and characterization of these cofactors.In previous work, we reported on the characterization of transgenic lines of tobacco (Nicotiana tabacum) in which a cucumber (Cucumis sativus) expansin (CsEXP1) could be induced by application of a chemical inducer (anhydrotetracycline [Ahtet]). In these experiments, we targeted expansin overexpression to localized regions of either the shoot apical meristem or very young leaf primordia, which led to localized promotion of growth (Pien et al., 2001), consistent with the idea that expansins play a role in the endogenous mechanism of leaf initiation (Reinhardt et al., 1998). However, when inductions were performed throughout the plant the resulting phenotypes were variable and difficult to interpret (S. Pien and A. Fleming, unpublished data), in line with other reports (Rochange et al., 2001). To investigate the possibility that this variable response reflected a differential sensitivity to expansin in different tissues at different stages of development, we performed a series of experiments (reported here) in which overexpression of expansin was targeted to specific stages of leaf growth. Our data indicate that the efficacy of expansin action depends on the presence of other factors that are present in a developmentally controlled fashion, so that increased expansin gene expression is only effective in promoting leaf growth during a specific developmental period of leaf growth. This period corresponds to the inflection point of relative growth rate (RGR) and, thus, to the phase of maximum leaf growth rate. An intriguing article by Cookson et al. (2005) reported on potential correlations between various parameters of leaf growth and final leaf size. They found that the best predictor of final leaf size was the maximum value of absolute leaf growth rate. Thus, the experiments reported here identify a novel, developmental control of expansin efficacy in the regulation of leaf growth, investigate the reported correlation between maximal leaf expansion rate and leaf size, and provide an insight into potential means of controlling leaf growth.     

Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.

Eukaryotic initiation factor (eIF) 4A is a DEAD box RNA helicase that works in conjunction with eIF4B, eIF4H, or as a subunit of eIF4F to unwind secondary structure in the 5'-untranslated region of mRNA, which facilitates binding of the mRNA to the 40 S ribosomal subunit. This study demonstrates how the helicase activity of eIF4A is modulated by eIF4B, eIF4H, or as a subunit of eIF4F. Results indicate that a linear relationship exists between the initial rate or amplitude of unwinding and duplex stability for all factor combinations tested. eIF4F, like eIF4A, behaves as a non-processive helicase. Either eIF4B or eIF4H stimulated the initial rate and amplitude of eIF4A-dependent duplex unwinding, and the magnitude of stimulation is dependent on duplex stability. Furthermore, eIF4A (or eIF4F) becomes a slightly processive helicase in the presence of eIF4B or eIF4H. All combinations of factors tested indicate that the rate of duplex unwinding is equivalent in the 5' --> 3' and 3' --> 5' directions. However, the optimal rate of unwinding was dependent on the length of the single-stranded region of the substrate when different combinations of factors were used. The combinations of eIF4A, eIF4A + eIF4B, eIF4A + eIF4H, and eIF4F showed differences in their ability to unwind chemically modified duplexes. A simple model of how eIF4B or eIF4H affects the duplex unwinding mechanism of eIF4A is proposed.     

Role of p70S6K1-mediated Phosphorylation of eIF4B and PDCD4 Proteins in the Regulation of Protein Synthesis

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5′-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.     

eIF4E抑制性蛋白翻译调控机制

真核生物mRNA的翻译调控,通常发生在起始阶段。异源三聚体复合物eIF4F中的eIF4E与mRNA5'端帽子结构的结合是该阶段的核心,而eIF4E抑制性蛋白正是通过与eIF4E的相互作用而调控着翻译起始过程,进而调控着翻译的速率。eIF4E抑制性蛋白对翻译的这种调控作用对细胞的生长、发育、癌症以及神经生物学方面有巨大影响,现主要就eIF4E抑制性蛋白的翻译调控机制进行综述。