细胞内部核糖体进入位点研究进展

细胞内部核糖体进入位点(IRES)mRNA5’端非编码区的一段特殊的序列,它允许核糖体不从mRNA的5’到3’端阅读而直接在此序列处结合mRNA并起始翻译。本综述了IRES的发现、IRES的识别及细胞IRES的特征、作用机理、生物学意义及其生物学应用等方面的研究进展。     

Mapping of the minimal internal ribosome entry site element in the human embryonic stem cell gene OCT4B mRNA

The OCT4 gene is an important regulator of self-renewal in embryonic stem cells and can generate three spliced variants, OCT4A, OCT4B, and OCT4B1. In OCT4B, the single mRNA can generate at least three protein isoforms, OCT4B-164, OCT4B-190, and OCT4B-265, using alternative translation initiation. OCT4B-164 and OCT4B-190 can be translated by an internal ribosome entry site (IRES)-mediated mechanism. Our work previously demonstrated that nucleotides (nt) 102-326 contained an IRES. We have mapped a 30-nt sequence (nt 201-231), which is sufficient to promote internal initiation of translation of OCT4B mRNA. The minimal element contains a sequence unique to OCT4B as well as a sequence common to OCT4A and OCT4B, and the two are essential for IRES activity. Like other cellular IRESs, the IRES activity of the minimal element shows significant variation in different cell lines. The minimal element is also functional under oxidative stress.     

Specific interference between two unrelated internal ribosome entry site elements impairs translation efficiency

Internal ribosome entry site (IRES) elements allow simultaneous synthesis of multiple proteins in eukaryotic cells. Here, two unrelated IRESs that perform efficiently in bicistronic constructs, the picornavirus foot-and-mouth disease virus (FMDV) and the cellular immunoglobulin heavy chain binding protein (BiP) IRES, were used to generate a tricistronic vector. Functional analysis of the tricistronic RNA evidenced that the efficiency of protein synthesis under the control of BiP IRES was lower than that of the FMDV IRES, relative to the efficiency measured in bicistronic vectors. A specific competition between these elements was verified using two separate mono- or bicistronic constructs in vivo and in vitro. In contrast, no interference was detected with the hepatitis C virus (HCV) IRES. The interference effect of FMDV IRES was observed in cis and trans, in support of competition for common transacting factors different than those used in cap- and HCV-dependent initiation.     

RNA病毒翻译调控元件—内部核糖体进入位点(IRES)

真核生物大多数蛋白质合成采用了依赖帽子结构的翻译起始方式.但一组缺乏帽子构的RNA病毒的蛋白质合成起始是依赖其5′端非翻译区（untranslated region,UTR）翻译调控的顺式作用元件——内部核糖体进入位点（internal ribosome entry site, IRES）.它 们能够在一些反式作用因子的辅助下,招募核糖体小亚基到病毒mRNA的翻译起始位点.前,依赖IRES元件翻译起始的RNA病毒在哺乳动物,无脊椎动物及植物中均有发现.因此,对RNA病毒IRES元件的深入研究,不仅有助于阐明相关疾病的发生机理,而且为工业应用和疾病治疗提供借鉴意义.本文对RNA病毒IRES元件发现、分类、结构与功能等作了综述.     

Mutation of the start codon to enhance Cripavirus internal ribosome entry site-mediated translation in a wheat germ extract

Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5′ terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3–4-fold higher than that of its wild type, and also 3–4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.     

Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.     

The 3' proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits

During cap-dependent translation of eukaryotic mRNAs, initiation factors interact with the 5′ cap to attract ribosomes. When animal viruses translate in a cap-independent fashion, ribosomes assemble upstream of initiation codons at internal ribosome entry sites (IRES). In contrast, many plant viral genomes do not contain 5′ ends with substantial IRES activity but instead have 3′ translational enhancers that function by an unknown mechanism. A 393-nucleotide (nt) region that includes the entire 3′ UTR of the Turnip crinkle virus (TCV) synergistically enhances translation of a reporter gene when associated with the TCV 5′ UTR. The major enhancer activity was mapped to an internal region of ~140 nt that partially overlaps with a 100-nt structural domain previously predicted to adopt a form with some resemblance to a tRNA, according to a recent study by J.C. McCormack and colleagues. The T-shaped structure binds to 80S ribosomes and 60S ribosomal subunits, and binding is more efficient in the absence of surrounding sequences and in the presence of a pseudoknot that mimics the tRNA-acceptor stem. Untranslated TCV satellite RNA satC, which contains the TCV 3′ end and 6-nt differences in the region corresponding to the T-shaped element, does not detectably bind to 80S ribosomes and is not predicted to form a comparable structure. Binding of the TCV T-shaped element by 80S ribosomes was unaffected by salt-washing, reduced in the presence of AcPhe-tRNA, which binds to the P-site, and enhanced binding of Phe-tRNA to the ribosome A site. Mutations that reduced translation in vivo had similar effects on ribosome binding in vitro. This strong correlation suggests that ribosome entry in the 3′ UTR is a key function of the 3′ translational enhancer of TCV and that the T-shaped element contains some tRNA-like properties.     

2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site

2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure–activity relationships in the target binding of substituted benzoxazole ligands were investigated.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

Translation initiation factors are not required for Dicistroviridae IRES function in vivo

The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2•GTP•initiator tRNA^met) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo.     

Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites

Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5' end of the viral RNA and is more active than the IRES located at the 5' end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5' end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.     

Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.     

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5′ untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine–Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2′-OMe oligonucleotides.     

Plaque purification of cricket paralysis virus using an agar overlay on Drosophila cells

Cricket paralysis virus, an insect picorna-like virus, grown and assayed on Drosophila malanogaster cells, gave titers in excess of 10⁹ PFU/ml. The virus was plaque purified using an agar overlay and the intracellular polypeptides induced by “crude” and plaque-purified virions were demonstrated to be very similar.     

内部核糖体进入位点及其研究进展

内部核糖体进入位点(IRES)是mRNA5'端非编码区的一段特殊序列,允许核糖体直接在此序列结合mRNA并起始翻译。对IRES的发现、分类、特征,以及细胞中是否存在该元件进行了简要综述。     

HCV内部核糖体进入位点(IRES)结构域Ⅲ中连接点的位置对其三级结构形成的影响

丙型肝炎病毒(HCV)结构含有一个内部核糖体进入位点(IRES);该位点是一段高度保守序列,坐落于mRNA的5'-非翻译区域(5'-UTR).目前,对IRES的三级结构及相关功能研究较少.本实验通过聚丙烯酰胺凝胶电泳和放射显影技术,研究在不同离子浓度、不同RNA浓度条件下,IRES结构域Ⅲ的连接点(junction)在不同位置时对结构域Ⅲ形成的影响,以及不同的结构域Ⅱ与结构域Ⅲ的相互作用关系.实验结果表明,HCV的IRES结构域Ⅲ连接点位置对结构域Ⅲ的形成至关重要,连接点位置不同,则结构域Ⅲ形成的结构不同.因此,在HCV的疫苗和相关药物设计中,可以针对结构域Ⅲ连接点进行研究,使之成为疫苗或药物作用靶点.     

A survey for pathogens of the black field cricket, Teleogryllus commodus, in the Western District of Victoria,Australia

A pathogen survey of the black field cricket, Teleogryllus commodus, in the Western District of Victoria, Australia, during 1979 revealed that cricket paralysis virus (CrPV) was present in 42.7% of the 232 sites sampled. The fungus Metarhizium anisopliae was detected in 5.2% of the sites and represents a new pathogen record for T. commodus. The distribution of both pathogens throughout the sites sampled appeared to be random. There was a positive correlation between sample size and the likelihood of detecting a pathogen, while analysis showed that approximately 30% of the sites were probably virus free. The results are discussed in terms of the potential of CrPV and M. anisopliae as biological control agents for the black field cricket.