IRESdb: the Internal Ribosome Entry Site database

Internal Ribosome Entry Sites (IRES) are cis-acting RNA sequences able to mediate internal entry of the 40S ribosomal subunit on some eukaryotic and viral messenger RNAs upstream of a translation initiation codon. These sequences are very diverse and are present in a growing list of mRNAs. Novel IRES sequences continue to be added to public databases every year and the list of unknown IRESes is certainly still very large. The IRES database is a comprehensive WWW resource for internal ribosome entry sites and presents currently available general information as well as detailed data for each IRES. It is a searchable, periodically updated collection of IRES RNA sequences. Sequences are presented in FASTA form and hotlinked to NCBI GenBank files. Several subsets of data are classified according to the viral taxon (for viral IRESes), to the gene product function (for cellular IRESes), to the possible cellular regulation or to the trans-acting factor that mediates IRES function. This database is accessible at http://ifr31w3.toulouse.inserm.fr/IRESdatabase/.     

Initiation Factor eIF2-independent Mode of c-Src mRNA Translation Occurs via an Internal Ribosome Entry Site

Overexpression and activation of the c-Src protein have been linked to the development of a wide variety of cancers. The molecular mechanism(s) of c-Src overexpression in cancer cells is not clear. We report here an internal ribosome entry site (IRES) in the c-Src mRNA that is constituted by both 5′-noncoding and -coding regions. The inhibition of cap-dependent translation by m⁷GDP in the cell-free translation system or induction of endoplasmic reticulum stress in hepatoma-derived cells resulted in stimulation of the c-Src IRES activities. Sucrose density gradient analyses revealed formation of a stable binary complex between the c-Src IRES and purified HeLa 40 S ribosomal subunit in the absence of initiation factors. We further demonstrate eIF2-independent assembly of 80 S initiation complex on the c-Src IRES. These features of the c-Src IRES appear to be reminiscent of that of hepatitis C virus-like IRESs and translation initiation in prokaryotes. Transfection studies and genetic analysis revealed that the c-Src IRES permitted initiation at the authentic AUG351, which is also used for conventional translation initiation of the c-Src mRNA. Our studies unveiled a novel regulatory mechanism of c-Src synthesis mediated by an IRES element, which exhibits enhanced activity during cellular stress and is likely to cause c-Src overexpression during oncogenesis and metastasis.     

Mechanism of Initiation Site Selection Promoted by the Human Rhinovirus 2 Internal Ribosome Entry Site

Translation initiation site usage on the human rhinovirus 2 internal ribosome entry site (IRES) has been examined in a mixed reticulocyte lysate/HeLa cell extract system. There are two relevant AUG triplets, both in a base-paired hairpin structure (domain VI), with one on the 5′ side at nucleotide (nt) 576, base paired with the other at nt 611, which is the initiation site for polyprotein synthesis. A single residue was inserted in the apical loop to put AUG-576 in frame with AUG-611, and in addition another in-frame AUG was introduced at nt 593. When most of the IRES was deleted to generate a monocistronic mRNA, the use of these AUGs conformed to the scanning ribosome model: improving the AUG-576 context increased initiation at this site and decreased initiation at downstream sites, whereas the converse was seen when AUG-576 was mutated to GUA; and AUG-593, when present, took complete precedence over AUG-611. Under IRES-dependent conditions, by contrast, much less initiation occurred at AUG-576 than in a monocistronic mRNA with the same AUG-576 context, mutation of AUG-576 decreased initiation at downstream sites by ∼70%, and introduction of AUG-593 did not completely abrogate initiation at AUG-611, unless the apical base pairing in domain VI was destroyed by point mutations. These results indicate that ribosomes first bind at the AUG-576 site, but instead of initiating there, most of them are transferred to AUG-611, the majority by strictly linear scanning and a substantial minority by direct transfer, which is possibly facilitated by the occasional persistence of base pairing in the apical part of the domain VI stem.Until the recent discovery of animal picornaviruses with internal ribosome entry sites (IRESs) resembling that of hepatitis C virus, most picornavirus IRESs have been classified into two groups (1, 17): type 1 (exemplified by entero- and rhinoviruses) and type 2 (cardio- and aphthoviruses). Primary sequences and especially secondary structures are strongly conserved within each group but there is very little similarity between the two groups apart from an AUG triplet at the 3′ end of the IRES (as defined by deletion analysis), which is preceded by a ∼25 nucleotide (nt) pyrimidine-rich tract (17). In type 2 IRESs, notably encephalomyocarditis virus (EMCV), this AUG triplet is the authentic initiation codon for viral polyprotein synthesis, and the totality of the evidence indicates that all ribosomes bind at, or very close to, this AUG and that all initiate translation at this site (18, 19). The foot-and-mouth disease virus (FMDV), although a type 2 IRES, is not quite so straightforward in that a minority of initiation events occur at the AUG immediately downstream of the oligopyrimidine tract, and the rest occur at the next AUG, 84 nt downstream (3, 45).In contrast, initiation on type 1 IRESs seems much more complicated and rather puzzling. The first puzzling feature is that there is very little, if any, initiation at the AUG just downstream of the oligopyrimidine tract, at nt 586 in poliovirus type 1 (PV-1) (39), and the initiation site for polyprotein synthesis is the next AUG further downstream, at a distance of ∼160 nt in enteroviruses and ∼35 nt in rhinoviruses (17). Nevertheless, AUG-586 is important for efficient initiation at the authentic polyprotein initiation site. Mutation of AUG-586 in a PV-1 infectious clone was found to be quasi-infectious (42), while mutation of the equivalent site in PV-2 conferred a small-plaque phenotype and reduced initiation at the polyprotein initiation site by ∼70% in both in vitro assays and in transfection assays (32, 33, 37).This observation has led to the idea that ribosomes first bind at AUG-586, but instead of initiating at this site, virtually all of them get transferred to the polyprotein initiation site (17). This raises questions as to the nature of the transfer process. Because insertion of an AUG codon between PV-1 nt 586 and the authentic initiation site conferred a small-plaque phenotype and because all large-plaque pseudo-revertants had lost the inserted AUG either by deletion or point mutation (25, 26), linear scanning is likely to be important. However, as the insertion resulted in a small-plaque phenotype rather than lethality, there remains the possibility that some ribosomes were transferred directly without scanning the whole distance. This has also been suggested on the grounds that insertion of AUGs or a hairpin loop between nt 586 and the authentic initiation site of PV-1 did not seem to reduce polyprotein synthesis in vitro as much as might be expected if the authentic initiation site is accessed by strictly linear scanning (8).The final puzzle is that AUG-586 is located in a stem-loop structure, domain VI (Fig. ​(Fig.1A),1A), which is conserved in all entero- and rhinoviruses apart from bovine enterovirus. If the initiating 40S subunits do inspect AUG-586 in some way, albeit an unproductive way, this stem-loop would need to open at least partly, if not completely. This need for domain VI to be opened might be considered an impediment to efficient initiation, and yet its strong conservation suggests the opposite, namely, that it might have a positive effect. Precise deletion of the spacer downstream of AUG-586 in PV-1(Mahoney), so that polyprotein synthesis now started at 586, reduced virus yield by ∼10-fold (39), and in an independent study a deletion that brought the polyprotein initiation site to nt 586 or 580 caused a very similar growth defect in PV-1(Sabin) although the defect was considerably less in a Mahoney background (13, 27). On the other hand, two smaller deletions in PV-1(Sabin) that retained just the whole base-paired domain VI or only its 5′ side, placing the polyprotein initiation site 52 or 31 nt, respectively, downstream of AUG-586, did not confer any significant negative phenotype (13, 27). Taken together, these results would seem to imply that the base pairing in domain VI is neutral to initiation efficiency, but the primary sequence of its 5′ side may confer a moderate positive effect. In this respect it is interesting that bovine enterovirus retains most of the sequence of the 5′ side of domain VI but lacks the complementary sequence of the 3′ side.Open in a separate windowFIG. 1.(A) Sequence and base pairing of IRES domain VI of HRV-2 and PV-1(Mahoney), numbered with respect to the viral genome sequence. (B) Hypothetical model for the opening of HRV-2 domain VI in two stages, showing that in the intermediate state AUG-576 and AUG-611 are both exposed.We have reexamined these issues but in the context of human rhinovirus 2 (HRV-2), mainly because the close proximity of the polyprotein initiation site (at nt 611) to the AUG (at nt 576) just downstream of the oligopyrimidine tract makes the interpretation of results less ambiguous than is the case with enteroviruses. A recent comprehensive sequence comparison of 106 different HRV strains plus 10 field isolates shows that HRV-2 domain VI is typical of the 106 serotypes and the one field isolate that differs in domain VI from its parent strain (35). In 95% of these sequences, the number of residues between the two AUG codons is in the range of 28 to 34 nt (median, 31 nt), with five outliers at 20 or 22 nt. The two AUGs are invariably base paired in a back-to-back configuration (Fig. ​(Fig.1A),1A), and the intervening residues fold into a base-paired structure, usually with a single mismatch (Fig. ​(Fig.1A)1A) or at least one G-U codon at around the mid-point and an apical loop of 3 to 6 residues (depending on the strain). The base-paired stem of enteroviruses is considerably shorter (usually without a mismatch), and the extra length in HRV domain VI generally consists of A-U and U-A pairs (often alternating) in the apical part (Fig. ​(Fig.1A).1A). In 23% of these 107 HRV domain VI sequences, the two AUGs are in the same reading frame, and in 17 (approximately two-thirds) of these there is no in-frame stop codon between them so that any initiation at the upstream AUG would result in synthesis of a VP0 protein (and, hence, also VP4) with an N-terminal extension.We first asked whether AUG-576 in HRV-2 is similar to AUG-586 in PV-1 in that there is very little initiation at this site, and yet AUG-576 is important for efficient initiation at the downstream polyprotein initiation site. We then looked for evidence that the domain VI stem-loop opens and whether all ribosomes access the authentic initiation site (AUG-611) by strictly linear scanning from some upstream site. We conclude that most ribosomes do access AUG-611 in this way, but a significant minority may take a shortcut, which could be facilitated if the apical part of this domain remains closed and base paired, with the single mismatch in the domain VI stem possibly causing the opening of this domain to occur in two stages (Fig. ​(Fig.1B1B).     

内部核糖体进入位点研究现状及应用展望

内部核糖体进入位点 (ｉｎｔｅｒｎａｌｒｉｂｏｓｏｍｅｅｎｔｒｙｓｉｔｅ ,ＩＲＥＳ)是最早发现于动物病毒基因组 5′非编码区的一段ＤＮＡ序列 ,它具有不依赖于 5′帽子结构的翻译起始功能。1 .ＩＲＥＳ的发现基因的表达分为转录和翻译两个相互独立但又紧密联系的阶段。正常情况下真核细胞的ｍＲＮＡ前体转录完成后 ,经过剪接、5′端加帽、3′端加尾等修饰过程生成成熟的ｍＲＮＡ。 5′帽子结构除了能使ｍＲＮＡ免遭核酸酶和磷酸酶的攻击 ,在随后的翻译起始中也起到十分重要的作用。核糖体小亚基通过识别ｍＲＮＡ 5′端帽子结构来寻找蛋…     

Translation Initiation at the CUU Codon Is Mediated by the Internal Ribosome Entry Site of an Insect Picorna-Like Virus In Vitro

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3′-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3′ boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5′ terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5′ part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.     

HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication

EV71 (enterovirus 71) RNA contains an internal ribosomal entry site (IRES) that directs cap-independent initiation of translation. IRES-dependent translation requires the host’s translation initiation factors and IRES-associated trans-acting factors (ITAFs). We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs) from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2) as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication.     

Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

Purpose

Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.

Methods

The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.

Results

RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.

Conclusions

We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.     

Translation of Vascular Endothelial Growth Factor mRNA by Internal Ribosome Entry: Implications for Translation under Hypoxia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5′ untranslated region (5′UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5′UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5′UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5′UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long “improved” IRES element was abrogated, however, following substitution of a few bases near the 5′ terminus as well as substitutions close to the translation start codon. Both the full-length 5′UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.     

Alternative Ways to Think about Cellular Internal Ribosome Entry

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of “cap-dependent” and “IRES-dependent” should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation.