Insights into Factorless Translational Initiation by the tRNA-Like Pseudoknot Domain of a Viral IRES

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA_i-independent IRES translation.     

Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp

The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.     

The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs

Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 5' viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 5' end of the CAT sequence, or the insertion of a small stable hairpin structure (deltaG = -18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 5' proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.     

Location of the internal ribosome entry site in the 5' non-coding region of the immunoglobulin heavy-chain binding protein (BiP) mRNA: evidence for specific RNA-protein interactions.

The 220 nucleotide 5'non-coding region (5'NCR) of the human immunoglobulin heavy chain binding protein (BiP) mRNA contains an internal ribosome entry site (IRES) that mediates the translation of the second cistron in a dicistronic mRNA in cultured mammalian cells. In this study, experiments are presented that locate the IRES immediately upstream of the start-site AUG codon in the BiP mRNA. Furthermore, crosslinking of thiouridine-labeled BiP IRES-containing RNA to cellular proteins identified the specific binding of two proteins, p60 and p95, to the 3'half of the BiP 5'NCR. Interestingly, both p60 and p95 bound also specifically to several viral IRES elements. This correlation suggests that p60 and p95 could have roles in internal initiation of cellular and viral IRES elements.     

Construction of a bicistronic vector for the co-expression of two genes in Caenorhabditis elegans using a newly identified IRES

The nematode Caenorhabditis elegans is an important model animal for biological research. Currently, transgenic C. elegans strains are mainly generated by injecting DNA encoding a gene of interest, in combination with a reporter gene, into the gonad. With this approach, the interpretation of negative results, such as the failure to observe reporter expression, is frequently required. Single, selectable vectors are urgently required. Internal ribosome entry site (IRES) elements are known to bind the eukaryotic ribosomal translation initiation complex and independently promote translation initiation. Bioinformatic analysis predicted an IRES motif upstream of the start codon of the C. elegans Hsp-3 gene. While this sequence has a Y-shaped double-hairpin secondary structure characteristic of IRES elements, it was unclear if it could function as an IRES. In the present study, this predicted Hsp-3 IRES was incorporated into a bicistronic vector driven by the myo-3 promoter, which allowed co-expression of RFP and GFP genes in the muscle tissue of C. elegans and thereby demonstrated that this IRES element is functional. This vector provides a novel, powerful tool for C. elegans research.     

Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function

Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5' untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES-40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES-40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.     

The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.     

Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon.

Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.     

A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons

The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5'-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5'-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5'-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.     

Translation of eukaryotic translation initiation factor 4GI (eIF4GI) proceeds from multiple mRNAs containing a novel cap-dependent internal ribosome entry site (IRES) that is active during poliovirus infection

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m7guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.     

Inhibition of hcv replication in HCV replicon by shRNAs

We show that the vector-derived long dsRNA specifically inhibits the replication of HCV RNA in HCV replicon. We designed a long dsRNA targeted to the full-length HCV IRES/core elements (1-to 377-nt). Our results revealed that the replication of HCV RNA was reduced to near background levels in a sequence-specific manner by the long dsRNAs in the HCV replicon. We also designed four shRNAs against several regions (120- to 139-nt, 260- to 279-nt, 330- to 349-nt, and 340- to 359-nt) of the HCV IRES/Core elements. The two HCV IRES/core-specific shRNAs, 330- to 349-nt and 340- to 359-nt, containing the AUG initiation codon sequence showed stronger HCV inhibitory effects than the other two shRNAs, 120- to 139-nt and 260- to 279-nt.     

Inhibition of HCV Replication in HCV Replicon by shRNAs

We show that the vector-derived long dsRNA specifically inhibits the replication of HCV RNA in HCV replicon. We designed a long dsRNA targeted to the full-length HCV IRES/core elements (1-to 377-nt). Our results revealed that the replication of HCV RNA was reduced to near background levels in a sequence-specific manner by the long dsRNAs in the HCV replicon. We also designed four shRNAs against several regions (120- to 139-nt, 260- to 279-nt, 330- to 349-nt, and 340- to 359-nt) of the HCV IRES/Core elements. The two HCV IRES/core-specific shRNAs, 330- to 349-nt and 340- to 359-nt, containing the AUG initiation codon sequence showed stronger HCV inhibitory effects than the other two shRNAs, 120- to 139-nt and 260- to 279-nt.     

Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site

Poliovirus translation is initiated at the internal ribosome entry site (IRES). Most likely involving the action of standard initiation factors, this highly structured cis element in the 5" noncoding region of the viral RNA guides the ribosome to an internal silent AUG. The actual start codon for viral protein synthesis further downstream is then reached by ribosomal scanning. In this study we show that two of the secondary structure elements of the poliovirus IRES, domain V and, to a minor extent, domain VI, are the determinants for binding of the eukaryotic initiation factor eIF4B. Several mutations in domain V which are known to greatly affect poliovirus growth also seriously impair the binding of eIF4B. The interaction of eIF4B with the IRES is not dependent on the presence of the polypyrimidine tract-binding protein, which also binds to the poliovirus IRES. In contrast to its weak interaction with cellular mRNAs, eIF4B remains tightly associated with the poliovirus IRES during the formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. These results indicate that the interaction of eIF4B with the 3" region of the poliovirus IRES may be directly involved in translation initiation.     

Conserved Element of the Dicistrovirus IGR IRES that Mimics an E-site tRNA/Ribosome Interaction Mediates Multiple Functions

The internal ribosome entry site within the intergenic region (IGR IRES) of the Dicistroviridae family mimics a tRNA to directly assemble 80 S ribosomes and initiate translation at a non-AUG codon from the ribosomal A-site. A comparison of IGR IRESs within this viral family reveals structural similarity but little sequence similarity. However, a few specific conserved elements exist, which likely have important roles in IRES function. In this study, we have generated a battery of mutations to characterize the role of a conserved loop (L1.1) region of the IGR IRES. Mutating specific nucleotides within the L1.1 region inhibited IGR IRES-mediated translation in rabbit reticulocyte lysates. By assaying different steps in IRES function, we found that the mutant L1.1 IRESs had reduced affinity for 80 S ribosomes but not 40 S subunits, indicating that the L1.1 region mediated either binding to preformed 80 S or 60 S joining. Furthermore, mutations in L1.1 altered the position of the ribosome on the mutant IRES, indicating that the tRNA-like anticodon/codon mimic within the ribosomal P-site is disrupted. Structural studies have revealed that the L1.1 region interacts with the L1 stalk of the 60 S subunit, which is similar to the interactions between the T-loop of the E-site tRNA and ribosomal protein rpL1. Our results demonstrate that the conserved L1.1 region directs multiple steps in IGR IRES-mediated translation including ribosome binding and positioning, which are functions that the E-site tRNA may normally mediate during translation.     

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit''s decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.     

Translation-competent 48S complex formation on HCV IRES requires the RNA-binding protein NSAP1

Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, we report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome-mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins).     

转录激活因子1(ATF1)内部核糖体进入位点的鉴定及活性分析

蛋白质翻译起始通常有两种机制,一是依赖帽结构的翻译,另一种是依赖5′非翻译区的内部核糖体进入位点(IRES).在后一种方式中,在某些IRES反式作用因子,如La蛋白、多聚嘧啶串结合蛋白1等的参与下,直接招募核糖体小亚基到mRNA的翻译起始位点,启始翻译.研究发现,参与细胞生长、分化、细胞周期进程、凋亡和压力调控的相关蛋白中通常含有IRES元件.基于功能,我们提出假说：转录激活因子1(ATF1)的5′-UTR可能具有IRES活性.为验证假说,首先构建了含全长ATF1 5′-UTR的双荧光素酶报告质粒|质粒转染结合报告酶活性分析显示,ATF1 5′-UTR在Bel7402、HCT-8和HEK293细胞中表现出不同的IRES活性|而此IRES活性与5′-UTR中的隐藏启动子无关.同时还发现,ATF1 5′-UTR在NIH3T3细胞中却没有IRES活性.与此结果相一致,Western印迹检测ATF1在这几种细胞系中的表达.结果显示,Bel7402、HCT 8和HEK293中ATF1蛋白质表达水平较高,而在NIH3T3中却极低. ATF1 5′-UTR的系列5′-删除突变及报告酶分析证明,ATF1 5′-UTR的完整性对其IRES活性大小发挥重要作用|其中5′端的204 bp序列对其IRES活性贡献较大. RNA-蛋白免疫共沉淀实验揭示,ATF1 5′-UTR可与La和PTBP1蛋白结合|抑制La和PTBP1蛋白质的表达,并可减低HEK293细胞中ATF1蛋白质表达水平.这些结果提示,La和PTBP1蛋白（两种ITAFs）为ATF1 5′-UTR发挥IRES活性所必需.总之,上述结果证明,ATF1 5′-UTR具有IRES活性,其活性发挥依赖与La和PTBP1蛋白的结合.上述发现为进一步研究La和PTBP1表达及亚细胞定位对ATF1 IRES调控机制的影响奠定了基础.     

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.