肽链释放因子和核糖体蛋白L11在八肋游仆虫细胞中的定位

原生动物纤毛虫是一类单细胞真核生物,其蛋白质合成终止过程中密码子使用的特殊性使其成为研究蛋白质合成终止机制的一个经典模型。为了能够有效地分析生物大分子在该细胞中的功能作用位点,本研究根据该生物染色体结构的特征,构建了含有红色荧光蛋白基因的大核人工染色体EoMAC_R,并与之前构建的含绿色荧光蛋白基因的大核染色体EoMAC_G一起,对蛋白质合成终止有关的3个重要因子核糖体大亚基蛋白L11、多肽链释放因子eRF1和eRF3在八肋游仆虫细胞中进行了荧光共定位分析。结果显示,在八肋游仆虫细胞中,蛋白质翻译过程主要位于"C"形大核内侧区域。构建的人工染色体能够作为一种有效的工具,对目的蛋白质在八肋游仆虫细胞中进行定位分析。     

真核翻译因子与蛋白质生物合成

真核mRNA在80S核糖体上翻译成蛋白质是一个复杂的过程,需要多步反应及多种因子参与,文章就真核蛋白质的生物合成机制简要综述翻译起始、延伸和终止因子的结构、功能和性质及其在肽链合成过程中的作用研究新进展.     

真核翻译起始因子5A在蛋白质合成中的功能及机制

在蛋白质合成过程中,除核糖体、氨酰 tRNA和mRNA外,还有多种翻译因子参与其中。真核翻译起始因子5A（eukaryotic translation initiation factor 5A, eIF5A）是维持细胞活性必不可少的翻译因子,在进化上高度保守。eIF5A是真核细胞中唯一含有羟腐胺赖氨酸（hypusine）的蛋白质,该翻译后修饰对eIF5A的活性至关重要。1978年,人们首次鉴定出eIF5A,认为它在翻译起始阶段促进第1个肽键的形成。直到2013年才证实它主要在翻译延伸阶段调控含多聚脯氨酸基序蛋白质的翻译。在经过四十多年研究后,人们对eIF5A的功能有了新的认识。近期基于核糖体图谱数据的分析表明,eIF5A能够缓解翻译延伸过程中核糖体在多种基序处的停滞,并不局限于多聚脯氨酸基序,并且它还能够通过促进肽链的释放增强翻译终止。此外,eIF5A还可以通过调控某些蛋白质的翻译,间接影响细胞内的各种生命活动。本文综述了eIF5A的多种翻译后修饰、在蛋白质合成和细胞自噬过程中的调控作用以及与人类疾病的关系,并与细菌及古细菌中的同源蛋白质进行了比较,探讨了该因子在进化中的保守性,以期为相关领域的研究提供一定的理论基础。     

八肋游仆虫第二类肽链释放因子核定位信号分析

蛋白质合成终止过程中肽链释放因子负责终止密码子的识别.真核生物第二类肽链释放因子（eRF3）是一类GTP酶,协助第一类肽链释放因子（eRF1）识别终止密码子和水解肽酰 tRNA酯键.之前的研究表明,两类肽链释放因子在细胞核中发挥功能,参与蛋白质合成和纺锤体的组装.本研究根据软件预测结果,构建了一系列八肋游仆虫eRF3的截短型突变体,分析在其N端是否存在引导eRF3的核定位信号.结果表明,在eRF3的N端有两个区域（NLS1:23-36 aa 和 NLS2: 236-272 aa）可以引导eRF3进入细胞核中,而且这两个区域具有典型的核定位信号的氨基酸序列特征. eRF3的核定位与其作为一种穿梭蛋白的功能相一致,即参与细胞有丝分裂纺锤体的形成和无义介导的mRNA降解途径.     

蛋白质生物合成的第四步：翻译终止后复合物的解体

蛋白质合成过程一般被归纳为由合成的起始、肽链的延伸和合成的终止组成的三步曲 . 然而,随着对核糖体再循环因子 (ribosome recycling factor , RRF) 在蛋白质合成过程中作用的深入研究,人们提出了蛋白质生物合成应是四步曲, 这第四步就是翻译终止后核糖体复合物的解体 , 也就是通常说的核糖体循环再利用 . 简要地介绍了翻译终止后复合物解体的可能机制：核糖体再循环因子和蛋白质合成延伸因子 G 在核糖体上协同作用催化这一过程的完成 .     

肽链释放因子识别终止密码子的机制

肽链释放因子(polypeptide release factor, RF)是参与细胞内蛋白质合成终止过程中新生肽链释放的一组重要的蛋白质,包括两类,即第一类肽链释放因子(classⅠrelease factor, RFⅠ)和第二类肽链释放因子(classⅡrelease factor, RFⅡ).关于第一类肽链释放因子识别终止密码子的机制和功能位点是目前分子细胞生物学领域的一个研究热点,第二类肽链释放因子作为一类GTP酶,在第一类肽链释放因子识别终止密码子和肽链释放过程中的协同作用也备受关注.近些年来,通过构建体内和体外的测活体系,对第一类肽链释放因子识别终止密码子的机制的研究取得了一些进展,提出了多种假说和模型,尤其是对第一类肽链释放因子的晶体结构及两类肽链释放因子复合体的空间结构的研究,为揭示真核生物细胞内蛋白质合成终止机制提供了直接的证据.     

蛋白质合成肽链释放因子的多功能性

肽链释放因子在蛋白质合成终止过程中,对新生肽链从核糖体上释放起重要作用。第一类肽链释放因子识别终止密码子,水解肽酰-tRNA酯键;第二类肽链释放因子是一类依赖于第一类肽链释放因子和核糖体的GTP酶,促进第一类肽链释放因子发挥肽链释放的功能。最近的研究表明,肽链释放因子不仅在细胞内蛋白质合成终止过程中起重要的作用,其在细胞的骨架形成,尤其是细胞有丝分裂过程中对纺锤体的形成起重要的作用。两类肽链释放因子还与其他功能蛋白质相互作用,表现出多功能蛋白质的特征。     

转录因子X-box结合蛋白1相互作用蛋白的筛选和鉴定

X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.     

原生动物八肋游仆虫cDNA文库的构建

细胞内蛋白质合成过程是一个由多种蛋白质相互作用参与调节的开放系统,形成了复杂的mRNA代谢和蛋白质翻译为核心的基因表达调控的网络和信号转导途径。【目的】为了获得更多参与调节蛋白质合成终止过程的蛋白质种类和功能信息,进一步了解其中的网络和信号转导途径,本研究构建了原生动物八肋游仆虫的cDNA文库。【方法】构建过程严格遵循Clontech公司的BD MatchmakerTM Library ConstructionScreening kit提供的方案进行文库构建和筛选.【结果】首次得到了可用于筛选功能基因的原生动物纤毛虫的cDNA文库,文库滴度为2.437×107cfu/mL。利用第二类肽链释放因子为诱饵,筛选得到了一些可能与之相互作用的蛋白质,其中包括一个可能编码RNA解旋酶的基因序列。该文库为进一步筛选和研究八肋游仆虫功能基因提供了便利的平台。     

翻译延伸因子1A的研究进展

翻译延伸因子1A(EF1α)是一个主要的翻译因子,EF1α.GTP催化氨酰tRNA结合到核糖体的A位点。EF1α不仅仅是翻译必须的蛋白,而且是一个重要的多功能蛋白。EF1α参与许多重要的细胞过程和疾病,包括信号传导、翻译控制、凋亡、细胞骨架组成、病毒复制及癌基因转化等。     

Cytoplasmic hGle1A regulates stress granules by modulation of translation

When eukaryotic cells respond to stress, gene expression pathways change to selectively export and translate subsets of mRNAs. Translationally repressed mRNAs accumulate in cytoplasmic foci known as stress granules (SGs). SGs are in dynamic equilibrium with the translational machinery, but mechanisms controlling this are unclear. Gle1 is required for DEAD-box protein function during mRNA export and translation. We document that human Gle1 (hGle1) is a critical regulator of translation during stress. hGle1 is recruited to SGs, and hGLE1 small interfering RNA–mediated knockdown perturbs SG assembly, resulting in increased numbers of smaller SGs. The rate of SG disassembly is also delayed. Furthermore, SG hGle1-depletion defects correlate with translation perturbations, and the hGle1 role in SGs is independent of mRNA export. Interestingly, we observe isoform-specific roles for hGle1 in which SG function requires hGle1A, whereas mRNA export requires hGle1B. We find that the SG defects in hGle1-depleted cells are rescued by puromycin or DDX3 expression. Together with recent links of hGLE1 mutations in amyotrophic lateral sclerosis patients, these results uncover a paradigm for hGle1A modulating the balance between translation and SGs during stress and disease.     

An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export

Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.     

The mRNA export factor human Gle1 interacts with the nuclear pore complex protein Nup155

The protein Gle1 is required for export of mRNAs from the nucleus to the cytoplasm in both lower and higher eukaryotic cells. In human (h) cells, shuttling of hGle1 between the nucleus and cytoplasm is essential for bulk mRNA export. To date, no hGle1-interacting proteins have been reported and the mechanism by which hGle1 interacts with the nuclear pore complex (NPC) and mediates export is unknown. To identify proteins that can interact with hGle1, a genome-wide yeast two-hybrid screen was performed. Three potential hGle1-interacting partners were isolated, including clones encoding the C-terminal region of the NPC protein hNup155. This interaction between hGle1 and full-length hNup155 was confirmed in vitro, and deletion analysis identified the N-terminal 29 residues of hGle1 as the hNup155-binding domain. Experiments in HeLa cells confirmed that the nuclear rim localization of the major hGle1 protein variant (hGle1B) was dependent on the presence of these 29 N-terminal residues. This suggests that this domain of hGle1 is necessary for targeting to the NPC. This work also characterizes the first domain in hNup155, a 177 C-terminal amino acid span that binds to hGle1. The mutual interaction between hGle1 and the symmetrically distributed nuclear pore protein Nup155 suggests a model in which hGle1's association with hNup155 may represent a step in the Gle1-mediated mRNA export pathway.     

Interaction between the shuttling mRNA export factor Gle1 and the nucleoporin hCG1: a conserved mechanism in the export of Hsp70 mRNA

Translocation of messenger RNAs through the nuclear pore complex (NPC) requires coordinated physical interactions between stable NPC components, shuttling transport factors, and mRNA-binding proteins. In budding yeast (y) and human (h) cells, Gle1 is an essential mRNA export factor. Nucleocytoplasmic shuttling of hGle1 is required for mRNA export; however, the mechanism by which hGle1 associates with the NPC is unknown. We have previously shown that the interaction of hGle1 with the nucleoporin hNup155 is necessary but not sufficient for targeting hGle1 to NPCs. Here, we report that the unique C-terminal 43 amino acid region of the hGle1B isoform mediates binding to the C-terminal non-FG region of the nucleoporin hCG1/NPL1. Moreover, hNup155, hGle1B, and hCG1 formed a heterotrimeric complex in vitro. This suggested that these two nucleoporins were required for the NPC localization of hGle1. Using an siRNA-based approach, decreased levels of hCG1 resulted in hGle1 accumulation in cytoplasmic foci. This was coincident with inhibition of heat shock-induced production of Hsp70 protein and export of the Hsp70 mRNA in HeLa cells. Because this closely parallels the role of the hCG1 orthologue yNup42/Rip1, we speculate that hGle1-hCG1 function in the mRNA export mechanism is highly conserved.     

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells

The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA‐dependent DEAD‐box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP₆)‐bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure‐function analysis in S. cerevisiae and human (h) cells, and find that the high‐affinity Nup42‐Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy‐terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1‐Dbp5/hDDX19B interaction site. A nup42‐CTD/gle1‐CTD/Dbp5 trimeric complex forms in the presence of IP₆. Deletion of NUP42 abrogates Gle1‐Dbp5 interaction, and disruption of the Nup42 or IP₆ binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42‐CTD and IP₆ stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42‐hGle1B interaction.      

The mRNA export factor Gle1 and inositol hexakisphosphate regulate distinct stages of translation

Gene expression requires proper messenger RNA (mRNA) export and translation. However, the functional links between these consecutive steps have not been fully defined. Gle1 is an essential, conserved mRNA export factor whose export function is dependent on the small molecule inositol hexakisphosphate (IP(6)). Here, we show that both Gle1 and IP(6) are required for efficient translation termination in Saccharomyces cerevisiae and that Gle1 interacts with termination factors. In addition, Gle1 has a conserved physical association with the initiation factor eIF3, and gle1 mutants display genetic interactions with the eIF3 mutant nip1-1. Strikingly, gle1 mutants have defects in initiation, whereas strains lacking IP(6) do not. We propose that Gle1 functions together with IP(6) and the DEAD-box protein Dbp5 to regulate termination. However, Gle1 also independently mediates initiation. Thus, Gle1 is uniquely positioned to coordinate the mRNA export and translation mechanisms. These results directly impact models for perturbation of Gle1 function in pathophysiology.     

Functions of Gle1 are governed by two distinct modes of self-association

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis.     

Control of mRNA Export and Translation Termination by Inositol Hexakisphosphate Requires Specific Interaction with Gle1

The unidirectional translocation of messenger RNA (mRNA) through the aqueous channel of the nuclear pore complex (NPC) is mediated by interactions between soluble mRNA export factors and distinct binding sites on the NPC. At the cytoplasmic side of the NPC, the conserved mRNA export factors Gle1 and inositol hexakisphosphate (IP₆) play an essential role in mRNA export by activating the ATPase activity of the DEAD-box protein Dbp5, promoting localized messenger ribonucleoprotein complex remodeling, and ensuring the directionality of the export process. In addition, Dbp5, Gle1, and IP₆ are also required for proper translation termination. However, the specificity of the IP₆-Gle1 interaction in vivo is unknown. Here, we characterize the biochemical interaction between Gle1 and IP₆ and the relationship to Dbp5 binding and stimulation. We identify Gle1 residues required for IP₆ binding and show that these residues are needed for IP₆-dependent Dbp5 stimulation in vitro. Furthermore, we demonstrate that Gle1 is the primary target of IP₆ for both mRNA export and translation termination in vivo. In Saccharomyces cerevisiae cells, the IP₆-binding mutants recapitulate all of the mRNA export and translation termination defects found in mutants depleted of IP₆. We conclude that Gle1 specifically binds IP₆ and that this interaction is required for the full potentiation of Dbp5 ATPase activity during both mRNA export and translation termination.     

The effect of eukaryotic release factor depletion on translation termination in human cell lines

Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome. Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide. Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension. The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs. Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines. Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells. The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems. We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells. These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner.