Mnk is a negative regulator of cap-dependent translation in Aplysia neurons

To investigate the mechanisms underlying regulation of eukaryotic initiation factor 4E (eIF4E) phosphorylation in Aplysia neurons, we have cloned the Aplysia homolog of the vertebrate eIF4E kinases, Mnk1 and -2. Aplysia Mnk shares many conserved regions with vertebrate Mnk, including putative eukaryotic initiation factor 4G binding regions, activation loop phosphorylation sites, and a carboxy-terminal anchoring site for MAP kinases. As expected, purified Aplysia Mnk phosphorylated Aplysia eIF4E at a conserved carboxy-terminal serine and over-expression of Aplysia Mnk in sensory neurons led to increased phosphorylation of endogenous eIF4E. Over-expression of Aplysia Mnk led to strong decreases in cap-dependent translation, while generally sparing internal ribosomal entry site (IRES)-dependent translation. However, decreases in cap-dependent translation seen after expression of Aplysia Mnk could only be partly explained by increases in eIF4E phosphorylation. In Aplysia sensory neurons, phosphorylation of eIF4E is reduced during intermediate memory formation. However, we found that this physiological regulation of eIF4E phosphorylation was independent of changes in Aplysia Mnk phosphorylation. We propose that changes in eIF4E phosphorylation in Aplysia neurons are a consequence of changes in cap-dependent translation that are independent of regulation of Aplysia Mnk.     

SF2/ASF regulates proteomic diversity by affecting the balance between translation initiation mechanisms

Post‐splicing activities have been described for a subset of shuttling serine/arginine‐rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras‐PI 3‐kinase‐Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co‐regulated levels of isoform‐specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5′‐terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap‐independent manner by the action of cis‐acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap‐dependent manner while the other by internal ribosome entry site‐dependent initiation, to show that in vivo over‐expression of SF2/ASF increases the ratio between cap‐dependent and internal ribosome entry site‐dependent translation. Consistently, knocking‐down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor‐2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field. J. Cell. Biochem. 107: 826–833, 2009. © 2009 Wiley‐Liss, Inc.     

The cis-acting RNA trafficking signal from myelin basic protein mRNA and its cognate trans-acting ligand hnRNP A2 enhance cap-dependent translation.

The 21 nucleotide RNA trafficking signal (RTS), originally identified in myelin basic protein mRNA, but also found in a variety of other localized RNAs, is necessary and sufficient for transport of RNA along microtubules in oligodendrocytes. The RTS binds specifically to the RNA binding protein, hnRNP A2. Together, the RTS and hnRNP A2 comprise cis/trans determinants for several steps in the RNA trafficking pathway. Here we show that insertion of the RTS into green fluorescent protein (GFP) RNA enhances translation without affecting stability of microinjected RNA. In dicistronic RNA, the RTS enhances cap-dependent translation without affecting internal ribosome entry site (IRES)-dependent translation. The translation enhancer function of the RTS is position, copy number, and cell type independent, hnRNP A2 dependent, and saturable with increasing amounts of injected RNA. This represents one of the first specific translation enhancer elements identified in a mammalian system.     

HCVIRES介导萤火虫荧光素酶翻译的双顺反子载体的构建与在小鼠体内的表达

将HCVIRES插入双报告基因海肾荧光素酶 (Rluc)基因和萤火虫荧光素酶 (Fluc)基因之间 ,建立了“依赖帽子的扫描机制”翻译表达Rluc ,HCVIRES调控Fluc翻译的双顺反子表达载体pCI Rluc HCVIRES Fluc ,通过酶切反应及转染HepG2细胞鉴定双荧光素酶瞬间表达活性等试验 ,证实获得了表达双荧光素酶的双顺反子载体 .并应用水压转染法将双顺反子表达质粒导入小鼠体内 ,在小鼠肝脏检测到高水平表达的Rluc和Fluc .该研究成功构建一种HCVIRES介导萤火虫荧光素酶基因表达的双顺反子载体 ,并在HepG2细胞及小鼠体内进行了瞬时表达 ,为进一步建立稳定评价靶向HCVIRES药物作用的细胞及小动物模型研究奠定了基础     

Structural scaffold for eIF4E binding selectivity of 4E‐BP isoforms: crystal structure of eIF4E binding region of 4E‐BP2 and its comparison with that of 4E‐BP1

To clarify the higher eukaryotic initiation factor 4E (eIF4E) binding selectivity of 4E‐binding protein 2 (4E‐BP2) than of 4E‐BP1, as determined by Trp fluorescence analysis, the crystal structure of the eIF4E binding region of 4E‐BP2 in complex with m⁷GTP‐bound human eIF4E has been determined by X‐ray diffraction analysis and compared with that of 4E‐BP1. The crystal structure revealed that the Pro47‐Ser65 moiety of 4E‐BP2 adopts a L ‐shaped conformation involving extended and α‐helical structures and extends over the N‐terminal loop and two different helix regions of eIF4E through hydrogen bonds, and electrostatic and hydrophobic interactions; these features were similarly observed for 4E‐BP1. Although the pattern of the overall interaction of 4E‐BP2 with eIF4E was similar to that of 4E‐BP1, a notable difference was observed for the 60–63 sequence in relation to the conformation and binding selectivity of the 4E‐BP isoform, i.e. Met‐Glu‐Cys‐Arg for 4E‐BP1 and Leu‐Asp‐Arg‐Arg for 4E‐BP2. In this paper, we report that the structural scaffold of the eIF4E binding preference for 4E‐BP2 over 4E‐BP1 is based on the stacking of the Arg63 planar side chain on the Trp73 indole ring of eIF4E and the construction of a compact hydrophobic space around the Trp73 indole ring by the Leu59‐Leu60 sequence of 4E‐BP2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant

Translation initiation factors are universal determinants of plant susceptibility to RNA viruses, but the underlying mechanisms are poorly understood. Here, we show that a sequence in the 3' untranslated region (3'-UTR) of a viral genome that is responsible for overcoming plant eIF4E-mediated resistance (virulence determinant) functions as a 3' cap-independent translational enhancer (3'-CITE). The virus/plant pair studied here is Melon necrotic spot virus (MNSV) and melon, for which a recessive resistance controlled by melon eIF4E was previously described. Chimeric viruses between virulent and avirulent isolates enabled us to map the virulence and avirulence determinants to 49 and 26 nucleotides, respectively. The translational efficiency of a luc reporter gene flanked by 5'- and 3'-UTRs from virulent, avirulent and chimeric viruses was analysed in vitro, in wheatgerm extract, and in vivo, in melon protoplasts, showing that: (i) the virulence determinant mediates the efficient cap-independent translation in vitro and in vivo; (ii) the avirulence determinant was able to promote efficient cap-independent translation in vitro, but only when eIF4E from susceptible melon was added in trans, and, coherently, only in protoplasts of susceptible melon, but not in the protoplasts of resistant melon; (iii) these activities required the 5'-UTR of MNSV in cis. Thus, the virulence and avirulence determinants function as 3'-CITEs. The activity of these 3'-CITEs was host specific, suggesting that an inefficient interaction between the viral 3'-CITE of the avirulent isolate and eIF4E of resistant melon impedes the correct formation of the translation initiation complex at the viral RNA ends, thereby leading to resistance.     

Suppression of translation during in vitro maturation of pig oocytes despite enhanced formation of cap-binding protein complex eIF4F and 4E-BP1 hyperphosphorylation

In this study, we document that the overall rate of protein synthesis decreases during in vitro maturation (IVM) of pig oocytes despite enhanced formation of the 5' cap structure eIF4F. Within somatic/interphase cells, formation of the eIF4F protein complex correlates very well with overall rates of protein translation, and the formation of this complex is controlled primarily by the availability of the 5' cap binding protein eIF4E. We show that the eIF4E inhibitory protein, 4E-BP1, becomes phosphorylated during IVM, which results in gradual release of eIF4E from 4E-BP1, as documented by immunoprecipitation analyses. Isoelectric focusing and Western blotting experiments show conclusively that eIF4E becomes gradually phosphorylated with a maximum at metaphase II (M II). The activity of eIF4E and its ability to bind mRNA also increases during oocyte maturation as documented in experiments with m7-methyl GTP-Sepharose, which mimics the cap structure of mRNA. Complementary analysis of flow-through fraction for 4E-BP1, and eIF4G proteins additionally provides evidence for enhanced formation of cap-binding protein complex eIF4F. Altogether, our results bring new insights to the regulation of translation initiation during meiotic division, and more specifically clarify that 4E-BP1 hyper-phosphorylation is not the cause of the observed suppression of overall translation rates.     

Role of amino acids in translational mechanisms governing milk protein synthesis in murine and ruminant mammary epithelial cells

The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were 2.1- or 3.1-fold higher, respectively, than in their bovine L-1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase (S6K1) on Thr(389) but not on Ser(411) was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E-BP1 or S6K1 phosphorylation than deprivation of all AA. In AA-deprived CID-9 cells, Leu specifically restored BLG synthesis from pre-existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E-BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu- and AA-stimulated BLG translation initiation in a dose-dependent manner. A further reduction was observed for Leu-treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells.     

4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig

The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25‐kDa, mRNA cap‐binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E‐BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E‐BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E‐, eIF4G‐, and 4E‐BP1‐specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E‐BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E‐BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E‐BP1 and binding dynamic of eIF4E/4E‐BP1 in distinct forms of implantation. Mol. Reprod. Dev. 78:895–905, 2011. © 2011 Wiley Periodicals, Inc.     

Elongation factor 2 diphthamide is critical for translation of two IRES-dependent protein targets,XIAP and FGF2, under oxidative stress conditions

Elongation factor-2 (eEF2) catalyzes the movement of the ribosome along the mRNA. A single histidine residue in eEF2 (H715) is modified to form diphthamide. A role for eEF2 in the cellular stress response is highlighted by the fact that eEF2 is sensitive to oxidative stress and that it must be active to drive the synthesis of proteins that help cells to mitigate the adverse effects of oxidative stress. Many of these proteins are encoded by mRNAs containing a sequence called an “internal ribosomal entry site” (IRES). Under high oxidative stress conditions diphthamide-deficient cells were significantly more sensitive to cell death. These results suggest that diphthamide may play a role in protection against the degradation of eEF2. This protection is especially important in those situations in which eEF2 is necessary for the reprogramming of translation from global to IRES synthesis. Indeed, we found that the expression of X-linked inhibitor of apoptosis (XIAP) and fibroblast growth factor 2 (FGF2), two proteins synthesized from mRNAs with IRESs that promote cell survival, is deregulated in diphthamide-deficient cells. Our findings therefore suggest that eEF2 diphthamide controls the selective translation of IRES-dependent protein targets XIAP and FGF2, critical for cell survival under conditions of oxidative stress.     

E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.     

Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit

Caenorhabditis elegans is a powerful in vivo model in which transgenesis is highly developed. However, while the analysis of biological phenomena often require the expression of more than one protein of interest, no reliable tool exists to ensure efficient concomitant and equivalent expression of more than two polypeptides from a single promoter. We report the use of viral 2A peptides, which trigger a “ribosomal-skip” or “STOP&GO” mechanism during translation, to express multiple proteins from a single vector in C. elegans. Although none of the viruses known to infect C. elegans contain 2A-like sequences, our results show that 2A peptides allow the production of separate functional proteins in all cell types and at all developmental stages tested in the worm. In addition, we constructed a toolkit including a 2A-based polycistronic plasmid and reagents to generate 2A-tagged fosmids. 2A peptides constitute an important tool to ensure the delivery of multiple polypeptides in specific cells, enabling several novel applications such as the reconstitution of multi-subunit complexes.     

Cell‐Cycle‐Dependent Regulation of Translation: New Interpretations of Old Observations in Light of New Approaches

It is a long‐standing view that global translation varies during the cell cycle and is much lower in mitosis than in other cell‐cycle phases. However, the central papers in the literature are not in agreement about the extent of downregulation in mitosis, ranging from a dramatic decrease to only a marginal reduction. Herein, it is argued that the discrepancy derives from technical challenges. Cell‐cycle‐dependent variations are most conveniently studied in synchronized cells, but the synchronization methods by themselves often evoke stress responses that, in turn, affect translation rates. Further, it is argued that previously reported cell‐cycle‐dependent changes in the global translation rate to a large extent reflect responses to the synchronization methods. Recent findings strongly suggest that the global translation rate is not regulated in a cell‐cycle‐dependent manner. Novel techniques allowing a genome‐wide analysis of translational profiles suggest that the extent and importance of selective translational regulation associated with cell‐cycle transitions have been underestimated. Therefore, the main question is which messenger RNAs (mRNAs) are translated, rather than whether the global translation rate is decreased.     

Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.     

小鼠胚胎干细胞分化心肌细胞血管紧张素Ⅱ 1型和2型受体的表达特征

胚胎干细胞(ESC)具有无限增殖和分化为体内3个胚层来源的各种类型组织细胞的潜能,经过体外诱导能够分化为心肌细胞,亦称为胚胎干细胞分化心肌细胞(ESCM).本研究探讨了ESC诱导分化心肌细胞过程中血管紧张素Ⅱ受体(ATR)的亚型AT1R和AT2R的表达特征.10-4mol/L维生素C体外诱导小鼠R1胚胎干细胞分化为自发搏动的心肌细胞,用免疫荧光法检测分化后的细胞表达心肌细胞特异性标志物α辅肌动蛋白.小鼠胚胎干细胞在诱导分化为心肌细胞以后,逆转录聚合酶链反应(RT-PCR)和实时定量RT-PCR(Real-timeRT-PCR)方法检测到ESCM表达AT1R,并且呈时间依赖性逐渐增加的特点,在第14d达到高峰.Western印记法检测AT1R表达特征与RT-PCR结果相符.Western印记法的结果显示,血管紧张素Ⅱ(10-6mol/L)可作为AT1R激动剂激活AT1R,并使其下游的细胞外信号调节激酶(ERK1/2)磷酸化水平上调,预孵育AT1R抑制剂Losartan(10-6mol/L),此作用被抑制.RT-PCR方法显示,与新生小鼠心室肌细胞相比,ESCM的AT2R表达水平较低.