Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation

Translation of most eukaryotic mRNAs involves the synergistic action between the 5′ cap structure and the 3′ poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.     

IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.     

Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.     

Riboproteomic analysis of polypeptides interacting with the internal ribosome-entry site element of foot-and-mouth disease viral RNA

Initiation of translation driven by internal ribosome entry site (IRES) elements depends upon the structural organization of this mRNA region. Besides translation initiation factors (eIFs), auxiliary proteins can also affect IRES activity. With the aim to identify proteins interacting with two unrelated IRESs present in the genome of foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) we have used a proteomic approach. This procedure allowed the identification of 21 RNA-binding proteins interacting with discrete regions of the FMDV IRES, domains 3 and 5, and 16 interacting with domain III of the HCV IRES. In support of the binding specificity, the factors interacting with domain 3 differed from those interacting with domain 5, and included three poly(rC)-binding protein (PCBP) members, besides proliferation-associated 2G4 (PA2G4) and deleted-azoospermia 1 (DAZ1) protein. Around 71% of the identified factors associated with the FMDV IRES differ from those interacting with the HCV IRES. The group of proteins interacting with the FMDV or the HCV IRES includes eIF4B and 5 subunits of eIF3, respectively, known to interact with each of these RNAs, validating the results of this approach. According to the function of the identified proteins, 55% are involved in translation control, whereas 35% play a role in different aspects of RNA lifespan. Compilation of factors preferentially associated with FMDV or HCV IRES provides a basis for examining the strategies used by IRESs to recruit the translation machinery.     

Herpes simplex virus virion host shutoff protein is stimulated by translation initiation factors eIF4B and eIF4H

The virion host shutoff protein (vhs) of herpes simplex virus triggers accelerated degradation of cellular and viral mRNAs while sparing other cytoplasmic RNA species. Previous work has shown that vhs forms a complex with translation initiation factor eIF4H, which displays detectable RNase activity in the absence of other viral or host proteins. However, the contributions of eIF4H and other host factors to the activity and mRNA targeting properties of vhs have not yet been directly examined. An earlier report from our laboratory demonstrated that rabbit reticulocyte lysate (RRL) contains one or more factors that strongly stimulate the RNase activity of vhs produced in Saccharomyces cerevisiae. We report here that such yeast extracts display significant vhs-dependent RNase activity in the absence of mammalian factors. This activity differs from that displayed by vhs generated in RRL in that it is not targeted to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Activity was strongly enhanced by the addition of RRL, eIF4H, or the related translation factor eIF4B. RRL also reconstituted strong targeting to the EMCV IRES, resulting in a major change in the RNA cleavage pattern. In contrast, eIF4H and eIF4B did not reconstitute IRES-directed targeting. These data indicate that eIF4B and 4H stimulate the nuclease activity of vhs, and they provide evidence that additional mammalian factors are required for targeting to the EMCV IRES.     

Interaction of Eukaryotic Initiation Factor eIF4B with the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus Is Independent of the Polypyrimidine Tract-Binding Protein

Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.     

Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors

Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.     

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry

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.     

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A^pro or L^pro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A^pro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L^pro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A^pro and L^pro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L^pro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.     

Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.

Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.     

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during 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. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

Activity of the hepatitis A virus IRES requires association between the cap-binding translation initiation factor (eIF4E) and eIF4G

The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m(7)GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.     

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.     

Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3' region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB-IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.     

Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site

Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3' border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.     

L protease from foot and mouth disease virus confers eIF2-independent translation for mRNAs bearing picornavirus IRES

The leader protease (L^pro) from foot-and-mouth disease virus (FMDV) has the ability to cleave eIF4G, leading to a blockade of cellular protein synthesis. In contrast to previous reports, our present findings demonstrate that FMDV L^pro is able to increase translation driven by FMDV IRES. Additionally, inactivation of eIF2 subsequent to phosphorylation induced by arsenite or thapsigargin in BHK cells blocks protein synthesis directed by FMDV IRES, whereas in the presence of L^pro, significant translation is found under these conditions. This phenomenon was also observed in cell-free systems after induction of eIF2 phosphorylation by addition of poly(I:C).     

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.     

A novel role for Gemin5 in mRNA translation

In eukaryotic cells translation initiation occurs through two alternative mechanisms, a cap-dependent operating in the majority of mRNAs, and a 5′-end-independent driven by internal ribosome entry site (IRES) elements, specific for a subset of mRNAs. IRES elements recruit the translation machinery to an internal position in the mRNA through a mechanism involving the IRES structure and several trans-acting factors. Here, we identified Gemin5 protein bound to the foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) IRES using two independent approaches, riboproteomic analysis and immunoprecipitation of photocroslinked factors. Functional analysis performed in Gemin5 shRNA-depleted cells, or in in vitro translation reactions, revealed an unanticipated role of Gemin5 in translation control as a down-regulator of cap-dependent and IRES-driven translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also functions as a modulator of translation activity.     

In Vitro Molecular Characterization of RNA–Proteins Interactions During Initiation of Translation of a Wild-Type and a Mutant Coxsackievirus B3 RNAs

Translation initiation of Coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5′ untranslated region. Host cell factors involved in this process include some canonical translation factors and additional RNA-binding proteins. We have, previously, described that the Sabin3-like mutation (U475 → C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. With the aim to identify proteins interacting with CVB3 wild-type and Sabin3-like IRESes and to study interactions between HeLa cell or BHK-21 protein extracts and CVB3 RNAs, UV-cross-linking assays were performed. We have observed a number of proteins that specifically interact with both RNAs. In particular, molecular weights of five of these proteins resemble to those of the eukaryotic translation initiation factors 4G, 3b, 4B, and PTB. According to cross-linking patterns obtained, we have demonstrated a better affinity of CVB3 RNA binding to BHK-21 proteins and a reduced interaction of the mutant RNA with almost cellular polypeptides compared to the wild-type IRES. On the basis of phylogeny of some initiation factors and on the knowledge of the initiation of translation process, we focused on the interaction of both IRESes with eIF3, p100 (eIF4G), and 40S ribosomal subunit by filter-binding assays. We have demonstrated a better affinity of binding to the wild-type CVB3 IRES. Thus, the reduction efficiency of the mutant RNA to bind to cellular proteins involved in the translation initiation could be the reason behind inefficient IRES function.