Internal ribosome entry sites of viral and cellular RNAs

In recent years mechanism of internal initation of translation in eukaryotic cells commands the attention of molecular biologists in increasing frequency. Ten years ago, experiments with picornaviruses demonstrated the ability of 40S ribosomal subunits to bind to nucleotide sequences localized far from the 5′ ends of RNA molecules, and since then numerous viral and even cellular RNAs were shown to be capable of internal initiation of translation. In the present survey, data on the localization, structure, and functional load of these internal ribosome entry sites (IRES elements) of viral and cellular RNAs, as well as on proteins capable of strong and highly specific binding to IRES elements, are discussed. A conclusion is that a unified model of structure and fuctioning of viral and cellular IRES elements cannot be suggested.     

Identification of the internal ribosome entry sites (IRES) of prion protein gene

Many studies demonstrated that there are several type bands of prion protein in cells. However, the formation of different prion protein bands is elusive. After several low molecular weight bands of prion protein appeared in SMB-S15 cells infected with scrapie agent Chandler, we think that IRES-dependent translation mechanism induced by prion is involved in the formation of prion protein bands. Then we designed a series of pPrP-GFP fusing plasmids and bicistronic plasmids to identify the IRES sites of prion protein gene and found 3 IRES sites inside of PrP mRNA. We also demonstrated that cap-independent translation of PrP was associated with the ER stress through Tunicamycin treatment. We still found that only IRE1 and PERK pathway regulated the IRES-dependent translation of PrP in this study. Our results indicated, we found that PrP gene had an IRES-dependent translation initiation mechanism and we successfully identified the IRESs inside of the prion protein gene.     

The efficiency of different IRESs (Internal Ribosomes Entry Site) in monocistronic mRNAS

The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.     

DDX60 selectively reduces translation off viral type II internal ribosome entry sites

Co‐opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell''s own protein synthesis. Here, we describe an interferon‐stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5′ cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV‐1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5′ capped mRNA. Our study identifies a novel facet in the repertoire of interferon‐stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.     

Internal ribosome entry sites in cellular mRNAs: mystery of their existence

Although studies on viral gene expression were essential for the discovery of internal ribosome entry sites (IRESs), it is becoming increasingly clear that IRES activities are present in a significant number of cellular mRNAs. Remarkably, many of these IRES elements initiate translation of mRNAs encoding proteins that protect cells from stress (when the translation of the vast majority of cellular mRNAs is significantly impaired). The purpose of this review is to summarize the progress on the discovery and function of cellular IRESs. Recent findings on the structures of these IRESs and specifically regulation of their activity during nutritional stress, differentiation, and mitosis will be discussed.     

Identification of internal ribosome entry segment (IRES)-trans-acting factors for the Myc family of IRESs

The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.     

Translation initiation by the c-myc mRNA internal ribosome entry sequence and the poly(A) tail

Eukaryotic mRNAs possess a poly(A) tail that enhances translation via the (7)mGpppN cap structure or internal ribosome entry sequences (IRESs). Here we address the question of how cellular IRESs recruit the ribosome and how recruitment is augmented by the poly(A) tail. We show that the poly(A) tail enhances 48S complex assembly by the c-myc IRES. Remarkably, this process is independent of the poly(A) binding protein (PABP). Purification of native 48S initiation complexes assembled on c-myc IRES mRNAs and quantitative label-free analysis by liquid chromatography and mass spectrometry directly identify eIFs 2, 3, 4A, 4B, 4GI, and 5 as components of the c-myc IRES 48S initiation complex. Our results demonstrate for the first time that the poly(A) tail augments the initiation step of cellular IRES-driven translation and implicate a distinct subset of translation initiation factors in this process. The mechanistic distinctions from cap-dependent translation may allow specific translational control of the c-myc mRNA and possibly other cellular mRNAs that initiate translation via IRESs.     

Internal ribosome entry site biology and its use in expression vectors.

Internal ribosome entry sites (IRESs) are cis-acting elements that recruit the small ribosomal subunits to an internal initiator codon in the mRNA with the help of cellular trans-acting factors. The recent discovery of the IRES recognition site of the eIF4G initiation factor is beginning to shed some light into how IRES elements are recognized by the translational machinery. Additionally, the progress made in the understanding of the parameters that influence start codon selection will be instrumental in establishing the rational design of bicistronic expression vectors.     

Minor contribution of an internal ribosome entry site in the 5'-UTR of ornithine decarboxylase mRNA on its translation

The mechanism of synthesis of ornithine decarboxylase (ODC) at the level of translation was studied using cell culture and cell-free systems. Synthesis of firefly luciferase (Fluc) from the second open reading frame (ORF) in a bicistronic construct transfected into FM3A and HeLa cells was enhanced by the presence of the 5′-untranslated region (5′-UTR) of ODC mRNA between the two ORFs. However, cotransfection of the gene encoding 2A protease inhibited the synthesis of Fluc. Synthesis of Fluc from the second cistron in the bicistronic mRNA in a cell-free system was not affected significantly by the 5′-UTR of ODC mRNA. Synthesis of ODC from ODC mRNA in a cell-free system was inhibited by 2A protease and cap analogue (m⁷GpppG). Rapamycin inhibited ODC synthesis by 40-50% at both the G₁/S boundary and the G₂/M phase. These results indicate that an IRES in the 5′-UTR of ODC mRNA does not function effectively.     

Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors

Riboswitches are RNA elements in mRNA that control gene expression in cis in response to their specific ligands. Because artificial riboswitches make it possible to regulate any gene with an arbitrary molecule, they are expected to function as biosensors, in which the output is easily detectable protein expression. I report herein a fully rational design strategy for artificially constructing novel riboswitches that work in a eukaryotic cell-free translation system (wheat germ extract). In these riboswitches, translation mediated by an internal ribosome entry site (IRES) is promoted only in the presence of a specific ligand (ON), while it is inhibited in the absence of the ligand (OFF). The first rationally designed riboswitch, which is regulated by theophylline, showed a high switching efficiency and dependency on theophylline. In addition, based on the design of the theophylline-dependent riboswitch, other three kinds of riboswitches controlled by FMN, tetracycline, and sulforhodamine B, were constructed only by calculating the ΔG value of one stem-loop structure. The rational design strategy described herein is therefore useful for easily producing various ligand-dependent riboswitches, which are available as biosensors for detecting their ligands.     

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease to show that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production and also on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.     

Antimicrobial peptides (AMPs): A promising class of antimicrobial compounds

Antimicrobial peptides (AMPs) are compounds, which have inhibitory activity against microorganisms. In the last decades, AMPs have become powerful alternative agents that have met the need for novel anti-infectives to overcome increasing antibiotic resistance problems. Moreover, recent epidemics and pandemics are increasing the popularity of AMPs, due to the urgent necessity for effective antimicrobial agents in combating the new emergence of microbial diseases. AMPs inhibit a wide range of microorganisms through diverse and special mechanisms by targeting mainly cell membranes or specific intracellular components. In addition to extraction from natural sources, AMPs are produced in various hosts using recombinant methods. More recently, the synthetic analogues of AMPs, designed with some modifications, are predicted to overcome the limitations of stability, toxicity and activity associated with natural AMPs. AMPs have potential applications as antimicrobial agents in food, agriculture, environment, animal husbandry and pharmaceutical industries. In this review, we have provided an overview of the structure, classification and mechanism of action of AMPs, as well as discussed opportunities for their current and potential applications.     

A Cucumis sativus cell-free translation system: preparation, optimization and sensitivity to some antibiotics and ribosome inactivating proteins

A cell-free translation system was prepared from 3- to 5-day-old embryonic axes of gherkin ( Cucumis sativus L.). The system was optimized for Mg²⁺, K⁺, NH⁺₄, high speed supernatants, tRNA mixture from wheat germ, time and temperature. The system translates efficiently both endogenous mRNA (using a 30000 g supernatant) and polyuridylic acid (using either a 30000 g supernatant or a 100000 g supernatant supplemented with purified ribosomes). Translation by gherkin ribosomes was inhibited by several well-known eukaryotic inhibitors, antibiotics and ribosome-inactivating proteins. A translational inhibitory activity found in Cucumis sativus L. dry seeds acted on polypeptide synthesis carried out by cell-free systems from several mammals and plants, including gherkin embryonic axes. Our results indicate that the inhibitor is located in the seed bark and cotyledons, and is either blocked or absent in the embryonic axes, thus allowing the isolation of active gherkin ribosomes. The presence of the putative inhibitor appeared to be unevenly distributed in developing plants.     

Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites

Translation of the open reading frames (ORF) of the hepatitis C virus (HCV) and closely related GB virus B (GBV-B) genomes is driven by internal ribosome entry site (IRES) elements located within the 5' non-translated RNA. The functioning of these IRES elements is highly dependent on primary and higher order RNA structures. We present here the solution structures of a common, critical domain within each of these IRESs, stem-loop IIIc. These ten-nucleotide hairpins have nearly identical sequences and similar overall tertiary folds. The final refined structure of each shows a stem with three G:C base-pairs and a novel tetraloop fold. Although the bases are buckled, the first and fourth nucleotides of both tetraloops form a Watson-Crick type base-pair, while the apical nucleotides are located in the major groove where they adopt C(2)-endo sugar puckering with B-form geometry. No hydrogen bonding interactions were observed involving the two apical residues of the tetraloop. Stability of the loops appears to be derived primarily from the stacking of bases, and the hydrogen bonding between the fourth and seventh residues. Mutational analysis shows that the primary sequence of stem-loop IIIc is important for IRES function and that the stem and first and fourth nucleotides of the tetraloop contribute to the efficiency of internal ribosome entry. Base-pair formation between these two positions is essential. In contrast, the apical loop nucleotides differ between HCV and GBV-B, and substitutions in this region of the hairpin are tolerated without major loss of function.     

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.     

N-乙酰氨基葡萄糖转移酶（OGT）研究进展

O-GlcNAc是一种广泛存在于蛋白质丝/苏氨酸残基上的动态、可逆的蛋白翻译后修饰,它广泛分布在细胞浆和细胞核中,参与调节多种细胞途径。研究表明蛋白的O-GlcNAc糖基化与神经退行性疾病、糖尿病和癌症等疾病相关。在体内,O-GlcNAc动态修饰由N-乙酰氨基葡萄糖转移酶（OGT）和N-乙酰氨基葡萄糖苷酶（OGA）协同完成。近年来,OGT逐渐成为糖生物学领域的研究热点,在其结构、作用机制及晶体学方面取得了快速发展。