Translation of intronless RNAs is strongly stimulated by the Epstein–Barr virus mRNA export factor EB2

The Epstein–Barr virus protein (EB2) allows the nuclear export of a particular subset of early and late viral RNAs derived from intronless genes. EB2 is conserved among most herpesvirus members and its presence is essential for the production of infectious particles. Here we show that, besides its role as a nuclear export factor, EB2 strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation. Interestingly, this effect can be reversed by the addition of an intron within the gene. The spliced mRNA is then efficiently exported and translated even in the absence of EB2. Moreover, we show that EB2 associates with translating ribosomes and increases the proportion of its target RNA in the polyribosomal fraction. Finally, testing of EB2 homolog proteins derived from EBV-related herpesviruses, shows that, even if they play similar roles within the replication cycle of their respective virus, their mechanisms of action are different.     

Translation of Virus mRNA: Synthesis of Bacteriophage Qβ Proteins in a Cell-Free Extract from Wheat Embryo

The RNA from bacteriophage Qbeta can be translated by cell-free extracts from wheat embryos. This translation, by 80S ribosomes, occurs at a low magnesium ion concentration. Three products are synthesized which coelectrophorese with Qbeta proteins synthesized in Escherichia coli extracts. The smallest of these has been identified as coat protein. Although the polycistronic bacteriophage message is translated with fidelity, the efficiency is much less than when the monocistronic brome mosaic virus coat protein message is translated.     

Thermodynamics of 7-Methylguanosine Cation Stacking with Tryptophan upon mRNA 5′ Cap Binding to Translation Factor eIF4E

Abstract

All eukaryotic nuclear transcribed mRNAs possess the cap structure, consisting of 7-methylguanosine linked by the 5′-5′ triphosphate bridge to the first nucleoside. The goal of the present study is to dissect the enthalpy and entropy changes of association of the mRNA 5′ cap with eIF4E into contributions originating from the interaction of 7-methylguanosine with tryptophan. The model results are discussed in the context of the thermodynamic parameters for the association of eIF4E with synthetic cap analogues.     

Translation inhibition reveals interaction of 2′-deoxy and 2′-O-methyl molecular beacons with mRNA targets in living cells

Understanding the interaction between oligonucleotide probes and RNA targets in living cells is important for biological and clinical studies of gene expression in vivo. Here, we demonstrate that starvation of cells and translation inhibition by blocking the mTOR or PI-3 kinase pathway could significantly reduce the fluorescence signal from 2′-deoxy molecular beacons (MBs) targeting K-ras and GAPDH mRNAs in living cells. However, the intensity and localization of fluorescence signal from MBs targeting nontranslated 28S rRNA remained the same in normal and translation-inhibited cells. We also found that, in targeting K-ras and GAPDH mRNAs, the signal level from MBs with 2′-O-methyl backbone did not change when translation was repressed. Taken together, our findings suggest that MBs with DNA backbone hybridize preferentially with mRNAs in their translational state in living cells, whereas those with 2′-O-methyl chemistry tend to hybridize to mRNA targets in both translational and nontranslational states. This work may thus provide a significant insight into probe design for detection of RNA molecules in living cells and RNA biology.     

Evolutionary Relationship Between Translation Initiation Factor eIF-2γ and Selenocysteine-Specific Elongation Factor SELB: Change of Function in Translation Factors

Eubacterial and eukaryotic translation initiation systems have very little in common, and therefore the evolutionary events that gave rise to these two disparate systems are difficult to ascertain. One common feature is the presence of initiation, elongation, and release factors belonging to a large GTPase superfamily. One of these initiation factors, the γ subunit of initiation factor 2 (eIF-2γ), is found only in eukaryotes and archaebacteria. We have sequenced eIF-2γ gene fragments from representative diplomonads, parabasalia, and microsporidia and used these new sequences together with new archaebacterial homologues to examine the phylogenetic position of eIF-2γ within the GTPase superfamily. The archaebacterial and eukaryotic eIF-2γ proteins are found to be very closely related, and are in turn related to SELB, the selenocysteine-specific elongation factor from eubacteria. The overall topology of the GTPase tree further suggests that the eIF-2γ/SELB group may represent an ancient subfamily of GTPases that diverged prior to the last common ancestor of extant life. Received: 2 January 1998 / Accepted: 1 June 1998     

A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397-1399 and AUG1415-1417, at 5′-terminus of the open reading frame (ORF,nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent in-frame AUGs at 5′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397-1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5′-ORF-AUG1397-1399 as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that astable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.     

真核生物mRNA非翻译区结构特征与mRNA翻译

真核生物中蛋白质合成通常在翻译水平受到调控,而mRNA非翻译区与mRNA翻译之间关系密切。真核生物mRNA非翻译区长度、mRNA二级结构、GC含量、顺式作用元件与反式作用因子、mRNA帽端结构和多聚腺苷酸尾等结构对翻译具有重要的调控作用。本文就真核生物mRNA非翻译区结构特征对其翻译的影响做一综述。     

mRNA展示技术

mRNA展示技术是一种新兴的体外筛选多肽和蛋白质的有力工具.在筛选过程中,mRNA与其编码的多肽或蛋白质共价结合,形成mRNA-蛋白质融合体,能在大容量的多肽文库(1013～1015)中筛选具有特定生物学功能的多肽和蛋白质.目前,mRNA展示技术主要应用于各种靶分子的多肽和蛋白质适体的发现以及蛋白质相互作用机制的阐明和分析.由于其自身的巨大发展潜力,mRNA展示技术具有更为广阔的应用前景.     

mRNA差异显示

分离差异表达的基因是研究生命调节过程的重要手段,mRNA差异显示技术是一种能成功分离差异表达基因的方法,文章对该方法的基本原理、方法步骤及其应用作了较为详细的介绍。     

mRNA药物新时代

<正>2023年诺贝尔生理学或医学奖授予了卡塔琳·考里科(Katalin Karikó)和德鲁·韦斯曼(Drew Weissman),以表彰其“在核苷碱基修饰方面的发现,使得开发有效的针对COVID-19的m RNA疫苗成为可能”.他们的研究工作,从根本上改变了人们对m RNA如何与免疫系统相互作用的理解,使疫苗研发达到前所未有的速度.这两位科学家的突破性发现对迎接未来的健康挑战产生了深远的影响.     

DNA Damage Regulates Translation through β-TRCP Targeting of CReP

The Skp1-Cul1-F box complex (SCF) associates with any one of a number of F box proteins, which serve as substrate binding adaptors. The human F box protein βTRCP directs the conjugation of ubiquitin to a variety of substrate proteins, leading to the destruction of the substrate by the proteasome. To identify βTRCP substrates, we employed a recently-developed technique, called Ligase Trapping, wherein a ubiquitin ligase is fused to a ubiquitin-binding domain to “trap” ubiquitinated substrates. 88% of the candidate substrates that we examined were bona fide substrates, comprising twelve previously validated substrates, eleven new substrates and three false positives. One βTRCP substrate, CReP, is a Protein Phosphatase 1 (PP1) specificity subunit that targets the translation initiation factor eIF2α to promote the removal of a stress-induced inhibitory phosphorylation and increase cap-dependent translation. We found that CReP is targeted by βTRCP for degradation upon DNA damage. Using a stable CReP allele, we show that depletion of CReP is required for the full induction of eIF2α phosphorylation upon DNA damage, and contributes to keeping the levels of translation low as cells recover from DNA damage.     

Translation attenuation regulation of chloramphenicol resistance in bacteria — a review

The chloramphenicol (Cm)-inducible cat and cmlA genes are regulated by translation attenuation, a regulatory device that modulates mRNA translation. In this form of gene regulation, translation of the Cm^R coding sequence is prevented by mRNA secondary structure that sequesters its ribosome-binding site (RBS). A translated leader of nine codons precedes the secondary structure, and induction results when a ribosome becomes stalled at a specific site in the leader. Here we demonstrate that the site of ribosome stalling in the leader is selected by a cis effect of the nascent leader peptide on its translating ribosome.     

Host Subunit of Qβ Replicase is Translation Control Factor i

THE tetrameric phage Qβ replicase is composed of three pre-existing E. coli proteins in addition to the phage coded synthetase^1,2. The host subunits also appear to form part of the f2 replicase³. We have found that the cistron specific factor i⁴ cross reacts immunologically and coelectrophoresis on SDS-acrylamide gel with the largest replicase subunit.     

非编码的mRNA

非编码的mRNA是近年发现的一类不含典型ORF的mRNA.目前已发现或克隆的这类基因主要有：H19基因,XIST基因,XLSIRT基因,His-1基因,bic基因,rox1和rox2基因等.它们与胚胎发育,肿瘤发生及X染色体失活密切相关.