[Show abstract][Hide abstract] ABSTRACT: During the last decade the concept of cellular IRES-elements has become predominant to explain the continued expression of
specific proteins in eukaryotic cells under conditions when the cap-dependent translation initiation is inhibited. However,
many cellular IRESs regarded as cornerstones of the concept, have been compromised by several recent works using a number
of modern techniques. This review analyzes the sources of artifacts associated with identification of IRESs and describes
a set of control experiments, which should be performed before concluding that a 5’ UTR of eukaryotic mRNA does contain an
IRES. Hallmarks of true IRES-elements as exemplified by well-documented IRESs of viral origin are presented. Analysis of existing
reports allows us to conclude that there is a constant confusion of the cap-independent with the IRES-directed translation
initiation. In fact, these two modes of translation initiation are not synonymous. We discuss here not numerous reports pointing
to the existence of a cap- and IRES-independent scanning mechanism of translation initiation based on utilization of special
RNA structures called cap-independent translational enhancers (CITE). We describe this mechanism and suggest it as an alternative
to the concept of cellular IRESs.
Keywords5’ CITE-5’ UTRs-cap-dependent and cap-independent translation mechanisms-IRES elements-mammalian mRNAs-translation initiation
Full-text · Article · Oct 2010 · Molecules and Cells
[Show abstract][Hide abstract] ABSTRACT: During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here,
we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian
cells. The binding of tRNAiMet occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation
complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and
A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence
of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site
of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation
initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.
Full-text · Article · Aug 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Translation of hepatitis C virus (HCV) genomic RNA is directed by an internal ribosome entry site (IRES) in the 5'-untranslated region (5'-UTR), and the HCV 3'-UTR enhances IRES activity. Since the HCV 3'-UTR has a unique structure among 3'-UTRs, we checked possible communication between the 5'- and the 3'-UTR of HCV during translation using chimeric reporter RNAs. We show that translation directed by the HCV IRES and by the HCV-like IRES of porcine teschovirus (PTV) which belongs to a quite distinct family of viruses (picornaviruses) or by the EMCV IRES is also enhanced by the HCV 3'-UTR or by a poly(A)-tail in different cell types.
[Show abstract][Hide abstract] ABSTRACT: Ribosomal protein p40 is a structural component of the eukaryotic 40S ribosomal subunit, is partly homologous to prokaryotic
ribosomal protein S2, and has a long eukaryote-specific C-terminal region. The internal ribosome entry site (IRES) of the
hepatitis C virus (HCV) RNA was tested for the binding to 40S ribosomal subunits deficient in p40, saturated with recombinant
p40, or pretreated with monoclonal antibody (MAB) 4F6 against p40. The apparent association constant of HCV IRES binding to
40S subunits was shown to directly depend on the p40 content in the subunits. MAB 4F6 prevented HCV IRES binding to 40S subunits
and blocked translation of IRES-containing RNA in a cell-free translation system. The results implicate p40 in the binding
of the HCV IRES to the ribosome and, therefore, in translation initiation on HCV RNA.
No preview · Article · Dec 2009 · Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome
Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised.
To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have
IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested
5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position
of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their
activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall
translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap
positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual
5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES
hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.
Full-text · Article · Sep 2009 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: According to generally accepted scanning model proposed by M. Kozak, the secondary structure of 5'-untranslated regions (5'-UTR) of eukaryotic mRNAs can only cause an inhibitory effect on the translation initiation since it would counteract migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. Thus, the existence of efficiently translatable mRNAs with long and highly structured 5'-UTRs is not compatible with the cap-dependent scanning mechanism. It is expected that such mRNAs should use alternative ways of translation initiation to be efficiently translated, first of all the mechanism of the internal ribosome entry mediated by special RNA structures called IRESes (for Internal Ribosome Entry Sites), which have been proposed to reside within their 5'-UTRs. In this paper, it is shown that this point of view is not correct and most probably based on experiments of mRNA translation in rabbit reticulocyte lysate. This cell free system does not reflect correctly the ratio of translation efficiencies of various mRNAs which is observed in the living cell. Using five different mRNAs of similar design which possess either relatively short leaders of cellular mRNAs (beta-globin and beta-actin mRNAs) or long and highly structured 5'-UTRs (c-myc, LINE-1, Apaf-1 mRNAs), we show that the translation activities of all tested 5'-UTRs are comparable, both in transfected cells and in a whole cytoplasmic extract of cultivated cells. This activity is strongly dependent on the presence of the cap at their 5'-ends.
Full-text · Article · Feb 2009 · Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The 5′-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome
Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S
subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed
cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding
to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy
of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275
or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the
apex of domain II cross-link to proteins S14 and S16.
Full-text · Article · Feb 2009 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met-tRNA(i) (Met), is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial-like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions.
[Show abstract][Hide abstract] ABSTRACT: RelE/RelB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "RelE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.
[Show abstract][Hide abstract] ABSTRACT: The foot-and-mouth disease virus (FMDV) RNA contains two in-frame AUG codons separated by 84 nt that direct translation initiation of the viral polyprotein. The mechanism of initiation at the IRES-proximal AUG codon (AUG1) has been previously analyzed, whereas no data on factor requirements for AUG2 have been reported. Here, using the method of 48S translation initiation complex reconstitution, we show that eIF1 is indispensable in forming the 48S initiation complex at AUG2. In contrast, it reduces the assembly of this complex at AUG1. Stabilization of a stem-loop between the initiation triplets induces a small decrease in the toeprint intensity at AUG2, accompanied by an increase in the AUG1/AUG2 ratio as well as a moderate reduction of protein synthesis initiated at AUG2 in transfected cells. PTB and ITAF45 exerted an additive positive effect on the 48S complex at AUG2, although a substantial reconstitution on both AUGs occurs on omission of either of these proteins. Relative to the beta-globin mRNA, the 48S complex formation at AUG1 and AUG2 is slow and occurs with the same kinetics as revealed by the "kinetic" toeprint assay. Mutation of AUG1 to AUA does not abrogate protein synthesis in transfected cells, and has no effect on the rate of the 48S complex formation at AUG2. We conclude that the AUG2 initiation region is selected independently of 48S complex formation at the upstream AUG1. The kinetic toeprint assay also shows that cap-dependent assembly of the 48S complex in vitro occurs faster than the FMDV IRES-mediated complex assembly.
[Show abstract][Hide abstract] ABSTRACT: Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.
Full-text · Article · Aug 2007 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Similar features in the mechanisms of mRNA translation initiation on prokaryotic and eukaryotic ribosomes are discussed with
examples from mRNAs with nonstandard 5′-untranslated regions (5′-UTRs) and mRNAs lacking 5′-UTR (leaderless mRNAs).
Full-text · Article · Jun 2006 · Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors.
Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence
of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis.
We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway
of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the
latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such
an mRNA was supplied with a 5′ leader, irrespective of whether it was in a totally single-stranded conformation or was prone
to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial
70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents
a relic from early steps in the evolution of the translation apparatus.
Full-text · Article · May 2006 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Binding of the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA to the eIF-free 40S ribosomal subunit
is the first step of initiation of translation of the viral RNA. Hairpins IIId and IIIe comprising 253–302 nt of the IRES
are known to be essential for binding to the 40S subunit. Here we have examined the molecular environment of the HCV IRES
in its binary complex with the human 40S ribosomal subunit. For this purpose, two RNA derivatives were used that bore a photoactivatable
perfluorophenyl azide cross-linker. In one derivative the cross-linker was at the nucleotide A296 in hairpin IIIe, and in
the other at G87 in domain II. Site-specific introduction of the cross-linker was performed using alkylating derivatives of
oligodeoxyribonucleotides complementary to the target RNA sequences. No cross-links with the rRNA were detected with either
RNA derivative. The RNA with the photoactivatable group at A296 cross-linked to proteins identified as S5 and S16 (major)
and p40 and S3a (minor), while no cross-links with proteins were detected with RNA modified at G87. The results obtained indicate
that hairpin IIIe is located on the solvent side of the 40S subunit head on a site opposite the beak.
Full-text · Article · Feb 2006 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.
Full-text · Article · Oct 2005 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES). Typically, picornavirus
IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it
is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5′ untranslated region direct the efficient
internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes
from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNAiMet. Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are
entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison
between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for
the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural
similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.
Full-text · Article · Jun 2004 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5' leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5' UTR of beta-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5' UTRs (with the cumulative stability of the secondary structure within the entire 5' UTR < -13 kcal/mol) and not essential for beta-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5' UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of cap-dependent cellular mRNAs.
Full-text · Article · Dec 2003 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: An approach based on complementary addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5-phosphate and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect on the binding, whereas that of hairpin IIIe substantially reduced it. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play the critical role in the binding of HCV IRES with the 40S subunit.
No preview · Article · Oct 2003 · Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus type 1 (HIV) gag/pol and env mRNAs contain cis-acting regulatory elements (INS) that impair stability, nucleocytoplasmic transport, and translation by unknown mechanisms.
This downregulation can be counteracted by the viral Rev protein, resulting in efficient export and expression of these mRNAs.
Here, we show that the INS region in HIV-1 gag mRNA is a high-affinity ligand of p54nrb/PSF, a heterodimeric transcription/splicing factor. Both subunits bound INS RNA
in vitro with similar affinity and specificity. Using an INS-containing subgenomic gag mRNA, we show that it specifically associated with p54nrb in vivo and that PSF inhibited its expression, acting via INS.
Studying the authentic HIV-1 mRNAs produced from an infectious molecular clone, we found that PSF affected specifically the
INS-containing, Rev-dependent transcripts encoding Gag-Pol and Env. Both subunits contained nuclear export and nuclear retention
signals, whereas p54nrb was continuously exported from the nucleus and associated with INS-containing mRNA in the cytoplasm,
suggesting its additional role at late steps of mRNA metabolism. Thus, p54nrb and PSF have properties of key factors mediating
INS function and likely define a novel mRNA regulatory pathway that is hijacked by HIV-1.
Full-text · Article · Oct 2003 · Molecular and Cellular Biology