eIF4E作用及调节机制

eIF4E作为翻译启动因子复合体eIF4-F的关键部分,调节细胞的蛋白质合成的限速步骤,ras基因加强eIF4E的磷酸化,c-myc基因增加eIF4E mRNA水平,eIF4E活性随其本身磷酸化的加强而加强,随其结合蛋白的磷酸化加强而减弱。eIF4E是新近发现的原癌,这量的eIF4E选择性地加强原癌基因等“弱mRNA”的翻译：可以通过加强蛋白质合成的速度,也可以加速mRNA由细胞核由胞浆转移。     

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。     

Multiple elements in the eIF4G1 N-terminus promote assembly of eIF4G1•PABP mRNPs in vivo

eIF4G is the scaffold subunit of the eIF4F complex, whose binding domains for eIF4E and poly(A)-binding protein (PABP) are thought to enhance formation of activated eIF4F•mRNA•PABP complexes competent to recruit 43S pre-initiation complexes. We found that the RNA-binding region (RNA1) in the N-terminal domain (NTD) of yeast eIF4G1 can functionally substitute for the PABP-binding segment to rescue the function of an eIF4G1-459 mutant impaired for eIF4E binding. Assaying RNA-dependent PABP–eIF4G association in cell extracts suggests that RNA1, the PABP-binding domain, and two conserved elements (Box1 and Box2) between these segments have overlapping functions in forming native eIF4G•mRNA•PABP complexes. In vitro experiments confirm the role of RNA1 in stabilizing eIF4G–mRNA association, and further indicate that RNA1 and Box1 promote PABP binding, in addition to RNA binding, by the eIF4G1 NTD. Our findings indicate that PABP–eIF4G association is only one of several interactions that stabilize eIF4F•mRNA complexes, and emphasize that closed-loop mRNP formation via PABP–eIF4G interaction is non-essential in vivo. Interestingly, two other RNA-binding regions in eIF4G1 have critical functions downstream of eIF4F•mRNA assembly.     

Interaction Between Yeast Eukaryotic Initiation Factor eIF4E and mRNA 5′ Cap Analogues Differs from That for Murine eIF4E

Abstract

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (K_as) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5′ cap than those of the murine and human proteins. The observed differences in the K_as values and the enthalpy changes of the association (ΔH°) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.     

我国辣椒种质资源eIF4E基因多态性分析

利用国家蔬菜种质资源库的1904份辣椒资源材料,采用测序技术获得eIF4E(eukaryotic translation initiation 4E)基因exon1序列,研究e IF4E基因多样性及我国辣椒种质资源群体多样性。结果表明:在1904份材料中共发现17个单倍型,14个有义多态性位点,其中9个为新的位点,位点大多集中在eIF4E蛋白表面环上;8个地理群体的平均单倍型多样性(Hd)和平均核苷酸多样性(Pi)分别为0.519和0.00210;群体间分化指数(Fst值)及基因流(Nm)表明不同群体间表现差异的分化程度;AMOVA分析表明总变异主要来源于群体内个体间的变异(97.23%),只有2.77%变异发生在群体间。本研究将有助于了解我国辣椒eIF4E基因多样性,为抗PVY育种提供更多抗源材料。     

DAP5 associates with eIF2β and eIF4AI to promote Internal Ribosome Entry Site driven translation

Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5′UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2β and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.     

本生烟eIF4Es基因克隆及其酵母双杂交诱饵载体构建

由木薯褐色条斑病毒(Cassava brown steak virus, CBSV)和乌干达木薯褐色条斑病毒(Uganda Cassava brown steak virus, UCBSV)引起的木薯褐色条斑病毒病,是危害木薯的主要病害之一。目前尚无有效防治方法,并且特别是缺乏CBSV/UCBSV抗性育种材料。真核翻译起始因子4E (eukaryotie translation initiation factor4E, eIF4E)不仅参与蛋白质的翻译起始,并且Potyviruses的复制和翻译也依赖于eIF4E与病毒基因组连接蛋白(virus genome linked protein, VPg)的相互作用,目前发现的植物隐性抗病基因大部分都是eIF4E家族的等位基因。本研究利用生物信息学方法和RT-PCR技术获得本生烟(Nicotiana benthamiana) eIF4E家族的8个基因,聚类分析显示它们分别编码eIF4E、eIF4E的异构体和类似帽结合的蛋白。为筛选与CBSV/UCBSV相互作用的本生烟eIF4E家族蛋白,本研究进一步构建了这8个基因的Lex A-酵母双杂交系统的诱饵载体,并导入酵母细胞EGY48 (p8op-LacZ)进行细胞毒性和自激活检测。结果显示导入重组质粒的EGY48 (p8op-LacZ)酵母细胞在SD/-His/-Ura缺陷型培养基上生长良好,在SD/-Trp/-Ura缺陷型平板上不生长,并且在SD/Gal/Raf/-His/-Ura/X-Gal培养基上不显蓝色,说明重组质粒表达产物不仅对该酵母细胞无毒性,而且对下游报告基因无自激活作用,可用于该系统的互作蛋白筛选研究。本研究为利用酵母双杂交系统发现与CBSV/UCBSV相互作用的本生烟eIF4E基因,探索通过基因编辑与CBSV/UCBSV互作的寄主因子进行抗CBSV/UCBSV分子育种提供科学依据。     

DEAGGREGATION OF eIF4E INDUCED BY mRNA 5′ CAP BINDING

All eukaryotic mRNAs contain a 5′ terminal cap structure, which consists of 7-methylguanosine linked by a 5′-5′ triphosphate bridge to the first transcribed nucleoside (m⁷GpppN). Specific recognition of the cap by the eukaryotic initiation factor eIF4E plays a key role in regulation of translation initiation as a rate-limiting step. Using dynamic light scattering (DLS), the apo-form of murine eIF4E (33–217) was shown to aggregate. After addition of m⁷GTP, progressive deaggregation with the time of incubation in the presence of the cap analogue has been observed.     

The binding mechanism of eIF2β with its partner proteins, eIF5 and eIF2Bε

The eukaryotic translation initiation factor eIF2 delivers Met-tRNAiMet to the ribosomal small subunit in GTP-bound form associated with eIF1, eIF1A, eIF3 and eIF5, and dissociates together with eIF5 as eIF5-eIF2-GDP complex from the ribosomal small subunit after formation of start codon-anticodon base pairing between Met-tRNAiMet and mRNA. The inactive form eIF2-GDP is then exchanged for the active form eIF2-GTP by eIF2B for further initiation cycle. Previous studies showed that the C-terminal domains of eIF5 (eIF5-CTD) and eIF2Bε (eIF2Bε-CTD) have a common eIF2β-binding site for interacting with an N-terminal region of eIF2β (eIF2β-NTD). Here we have reconstructed the complexes of (eIF5-CTD)-(eIF2β-NTD) and (eIF2Bε-CTD)-(eIF2β-NTD) in vitro, and investigated binding mechanism by circular dichroism spectroscopy and small angle X-ray scattering in solution. The results showed the conformation of eIF2β-NTD was changed when bound to partner proteins, whereas the structures of eIF5-CTD and eIF2Bε-CTD were similar in both isolated and complex states. We propose that eIF2β-NTD works as an intrinsically disordered domain which is disorder in the isolated state, but folds into a definite structure when bound to its partner proteins. Such flexibility of eIF2β-NTD is expected to be responsible for its binding capability.     

Mnk mediates integrin α6β4-dependent eIF4E phosphorylation and translation of VEGF mRNA

It was previously shown that integrin α6β4 contributes to translation of cancer-related mRNAs such as VEGF via initiation factor eIF4E. In this study, we found that integrin α6β4 regulates the activity of eIF4E through the Ser/Thr kinase Mnk. Although a role for Mnk in various aspects of cancer progression has been established, a link between integrin and Mnk activity has not. Here we show that Mnk1 is a downstream effector of integrin α6β4 and mediates the α6β4 signaling, important for translational control. Integrin α6β4 signals through MEK and p38 MAPK to increase phosphorylation of Mnk1 and eIF4E. Inhibition of Mnk1 activity by CGP57380 or downregulation by shRNA blocks α6β4-dependent translation of VEGF mRNA. Our studies suggest that Mnk1 could be a therapeutic target in cancers where the integrin α6β4 level is high.     

eIF4A1与TrkA相互作用后抑制TrkA的泛素化

神经生长因子（NGF）结合细胞表面受体p75NTR(p75神经营养素受体)和TrkA（酪氨酸蛋白激酶A）后介导了细胞分化、细胞生存、凋亡、增殖和侵袭等多个重要的生理病理过程. TrKA能与细胞内多个蛋白质相互作用,但是由于NGF信号通路的复杂性,现在仍有必要发现与之相互作用的蛋白质以更准确地了解NGF信号通路. 本研究中我们通过酵母双杂交的方法筛选到了一个新的与TrKA相互作用的蛋白质——真核生物翻译起始因子4A1（eIF4A1）,然后通过谷胱甘肽巯基转移酶融合蛋白沉降实验（GST-pull-down）和免疫共沉淀实验（Co-IP）证明了TrkA和eIF4A1的相互作用. 此外NGF能够增强TrkA和eIF4A1的相互作用. 在鉴定相互作用位点实验中,我们发现eIF4A1的氨基端结构域和TrkA的TK结构域参与了相互作用. TrkA和eIF4A1共定位在细胞膜上. NGF能够引起TrkA与泛素蛋白63位的赖氨酸连接,而eIF4A1与TrkA相互作用后能够抑制TrkA与泛素蛋白63位的赖氨酸连接. 综上,得出结论 eIF4A1通过与TrkA相互作用抑制其泛素化调控NGF信号通路.     

eIF4A在帽依赖和IRES介导的翻译起始中的作用

生物体内翻译起始机制分为两类：帽依赖性翻译起始和内部核糖体进入位点(internal ribosome entry sites, IRES)介导的翻译起始。真核生物的翻译起始为经典的帽依赖性翻译起始模型,而大多数正链RNA病毒选择依赖于IRES的翻译机制。真核翻译起始因子4A (eukaryotic initiation factor 4A, eIF4A)是DEAD-box RNA解旋酶家族的成员,具有依赖于RNA的ATP酶活性和RNA解旋酶活性,而e IF4A具体的解旋机制至今仍不清晰。同时,eIF4A与其他翻译因子有着复杂而紧密的联系,在帽依赖性与IRES介导的翻译起始过程中扮演着至关重要的角色。本文主要对eIF4A的功能、结构以及eIF4A在帽依赖性与IRES介导的翻译起始过程中的机制作一综述。     

应用套叠PCR技术构建番木瓜eIF4E和eIFiso4E基因的hpRNA植物表达载体

植物真核翻译起始因子4E(eIF4E)在蛋白质合成的起始中发挥重要作用,参与植物-病毒互作,影响病毒的侵染过程.为了研究eIF4E在植物病毒侵染中的功能,建立了一种快速的套叠PCR新方法,成功构建了番木瓜eIF4E和eIFiso4E基因的hpRNA结构,并将其连接到改造的植物表达载体pBI121上,为利用RNA干扰技术研究番木瓜eIF4E和eIFiso4E基因在病毒侵染中的作用奠定了基础.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

shRNA干扰eIF4A1基因对胶质瘤细胞体内外生物学特征的影响

胶质瘤是颅内常见的肿瘤,其中恶性脑胶质瘤成弥漫性生长,尽管给予手术、放疗或化疗等综合治疗,仍极易复发,迫切需要探索新的治疗方法.但是胶质瘤的治疗靶点匮乏,因此探索有效的靶点对其治疗具有重要的意义.本研究中首先利用中国脑胶质瘤基因组图谱计划数据库(Chinese glioma genome altas, CGGA)分析了真核生物翻译起始因子中eIF4A1与脑胶质瘤的关系.生物信息学分析显示, eIF4A与胶质瘤病理分级相关并且在胶质瘤细胞中高表达. eIF4A1的抑制剂Silvestrol明显抑制胶质瘤细胞的增殖能力.利用慢病毒感染胶质瘤细胞建立shRNA干扰eIF4A1基因的胶质瘤细胞株,进行了一系列体内外生物学特征的实验,研究结果发现, shRNA干扰eIF4A1基因后能够有效抑制胶质瘤细胞的增殖、克隆形成、细胞侵袭和迁移.因此,本实验研究证实eIF4A1是胶质瘤治疗的有效靶点,为胶质瘤的临床治疗提供可靠的理论基础.     

Sequential Eukaryotic Translation Initiation Factor 5 (eIF5) Binding to the Charged Disordered Segments of eIF4G and eIF2β Stabilizes the 48S Preinitiation Complex and Promotes Its Shift to the Initiation Mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.     

水稻eIF3大亚基（eIF3a）编码基因的克隆及其表达模式分析

真核生物翻译起始因子（eIFs)在蛋白合成中起关键作用,在已经鉴定 的13个因子中eIF3（由8个或更多的亚基组成）是分子量最大的一个并在翻译起始过程中起着核心作用。eIF3a是eIF3中最大的亚基并介导了eIF3的大多数的功能的实现。利用荧光－差异显示PCR法对生长素处理后的水稻材料进行分析发现eIF3a的表达可被生长素诱导,进一步通过筛选cDNA文库分离出水稻编码eIF3a的全长cDNA（命名为OseIF3a1),OseIF3a1含3459碱基（含5‘和3‘非翻译区）并编码一个986氨基酸的蛋白,与基因组序列比较表明OseIF3a1基因存在有12个内含子。OseIF3a1与玉米和烟草的同源蛋白一致性分别为82．4％和70．1％并与其他真核生物eIF3a蛋白具较高一致性。以组织材料进行的RT－PCR结果表明OseIF3a1在根、幼苗、幼穗、茎和叶中表达,启动子－报告基因的转基因结果进一步表明OseIF3a1在根尖及幼嫩叶片表达较高,进一步的RT－PCR分析确证了生长素对OseIF3a1的诱导,表明生长素在调控植物生长时可能涉及了翻译水平上的调节。     

Does phosphorylation of the cap-binding protein eIF4E play a role in translation initiation?

Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E can interact either with the scaffold protein eIF4G or with repressor proteins termed eIF4E-binding proteins (4E-BPs). High levels of expression can disrupt cellular growth control and are associated with human cancers. A fraction of the cellular eIF4E is found in the nucleus where it may play a role in the transport of certain mRNAs to the cytoplasm. eIF4E undergoes regulated phosphorylation (at Ser209) by members of the Mnk group of kinases, which are activated by multiple MAP kinases (hence Mnk = MAP-kinase signal integrating kinase). The functional significance of its phosphorylation has been the subject of considerable interest. Recent genetic studies in Drosophila point to a key role for phosphorylation of eIF4E in growth and viability. Initial structural data suggested that phosphorylation of Ser209 might allow formation of a salt bridge with a basic residue (Lys159) that would clamp eIF4E onto the mRNA and increase its affinity for ligand. However, more recent structural data place Ser209 too far away from Lys159 to form such an interaction, and biophysical studies indicate that phosphorylation actually decreases the affinity of eIF4E for cap or capped RNA. The implications of these studies are discussed in the light of other, in vitro and in vivo, investigations designed to address the role of eIF4E phosphorylation in mRNA translation or its control.     

敲除eIF4B基因对小鼠胚胎肝脏细胞凋亡的影响北大核心CSCD

真核翻译起始因子4B(eukaryotic translation initiation factor 4B,eIF4B)在mRNA翻译起始、细胞存活和增殖过程中发挥着关键作用,然而其在生物体内的生物学功能仍存在许多疑问。本研究利用eIF4B基因敲除小鼠模型,结合苏木精和伊红(hematoxylin-eosin,HE)染色、流式细胞术、Western blotting和免疫组化等一系列实验技术手段,探究了eIF4B在胚胎发育过程中的功能及作用机理。结果显示,eIF4B敲除小鼠胚胎的肝脏呈现严重病理损伤,主要表现为胚肝细胞的凋亡和坏死,而小鼠胚胎的肺脏、脑、胃和胰腺则发育正常。进一步研究发现,在eIF4B敲除小鼠的胚胎肝脏中活化型caspase 3(cleaved-caspase 3)的表达水平显著升高。此外,eIF4B敲除小鼠的胚胎成纤维细胞和胚胎肝脏中mTOR通路下游信号分子p70S6K的表达和磷酸化水平以及4EBP1的磷酸化水平显著升高。综上所述,eIF4B敲除导致cleaved-caspase 3表达增加和mTOR信号通路过度活化,促进胚肝细胞的凋亡,致使小鼠胚肝发育异常,最终引发小鼠胚胎死亡。本研究揭示了eIF4B在胚胎发育过程中的重要作用,为深入了解eIF4B在生物体内的生物学功能提供了新的科学依据。