The flip-flop configuration of the PABP-dimer leads to switching of the translation function

Poly(A)-binding protein (PABP) is a translation initiation factor that interacts with the poly(A) tail of mRNAs. PABP bound to poly(A) stimulates translation by interacting with the eukaryotic initiation factor 4G (eIF4G), which brings the 3′ end of an mRNA close to its 5′ m⁷G cap structure through consecutive interactions of the 3′-poly(A)–PABP-eIF4G-eIF4E-5′ m⁷G cap. PABP is a highly abundant translation factor present in considerably larger quantities than mRNA and eIF4G in cells. However, it has not been elucidated how eIF4G, present in limited cellular concentrations, is not sequestered by mRNA-free PABP, present at high cellular concentrations, but associates with PABP complexed with the poly(A) tail of an mRNA. Here, we report that RNA-free PABPs dimerize with a head-to-head type configuration of PABP, which interferes in the interaction between PABP and eIF4G. We identified the domains of PABP responsible for PABP–PABP interaction. Poly(A) RNA was shown to convert the PABP–PABP complex into a poly(A)–PABP complex, with a head-to-tail-type configuration of PABP that facilitates the interaction between PABP and eIF4G. Lastly, we showed that the transition from the PABP dimer to the poly(A)–PABP complex is necessary for the translational activation function.     

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.     

Eukaryotic translation initiation factor eIF4G2 opens novel paths for protein synthesis in development,apoptosis and cell differentiation

Protein translation is a critical regulatory event involved in nearly all physiological and pathological processes. Eukaryotic translation initiation factors are dedicated to translation initiation, the most highly regulated stage of protein synthesis. Eukaryotic translation initiation factor 4G2 (eIF4G2, also called p97, NAT1 and DAP5), an eIF4G family member that lacks the binding sites for 5′ cap binding protein eIF4E, is widely considered to be a key factor for internal ribosome entry sites (IRESs)‐mediated cap‐independent translation. However, recent findings demonstrate that eIF4G2 also supports many other translation initiation pathways. In this review, we summarize the role of eIF4G2 in a variety of cap‐independent and ‐dependent translation initiation events. Additionally, we also update recent findings regarding the role of eIF4G2 in apoptosis, cell survival, cell differentiation and embryonic development. These studies reveal an emerging new picture of how eIF4G2 utilizes diverse translational mechanisms to regulate gene expression.

Eukaryotic translation initiation factors 4G2 (eIF4G2) participates in diverse translation initiation, including translation driven by internal ribosome entry sites (IRESs), cap‐independent translation enhancers (CITEs), and N6‐methyladenosine (m6A), as well as in non‐canonical eIF4F‐independent cap‐dependent translation and canonical cap‐dependent scanning translation initiation. Through the translational mechanisms to regulate gene expression, eIF4G2 is significantly involved in apoptosis, cell survival, cell differentiation, and embryonic development processes.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

Intrinsically Unstructured Sequences in the mRNA 3ʹ UTR Reduce the Ability of Poly(A) Tail to Enhance Translation

The 5? cap and 3? poly(A) tail of mRNA are known to synergistically stimulate translation initiation via the formation of the cap?eIF4E?eIF4G?PABP?poly(A) complex. Most mRNA sequences have an intrinsic propensity to fold into extensive intramolecular secondary structures that result in short end-to-end distances. The inherent compactness of mRNAs might stabilize the cap?eIF4E?eIF4G?PABP?poly(A) complex and enhance cap-poly(A) translational synergy. Here, we test this hypothesis by introducing intrinsically unstructured sequences into the 5? or 3? UTRs of model mRNAs. We found that the introduction of unstructured sequences into the 3? UTR, but not the 5? UTR, decreases mRNA translation in cell-free wheat germ and yeast extracts without affecting mRNA stability. The observed reduction in protein synthesis results from the diminished ability of the poly(A) tail to stimulate translation. These results suggest that base pair formation by the 3? UTR enhances the cap-poly(A) synergy in translation initiation.     

The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA

Here, we show a novel molecular mechanism promoted by the DEAD-box RNA helicase DDX3 for translation of the HIV-1 genomic RNA. This occurs through the adenosine triphosphate-dependent formation of a translation initiation complex that is assembled at the 5′ m⁷GTP cap of the HIV-1 mRNA. This is due to the property of DDX3 to substitute for the initiation factor eIF4E in the binding of the HIV-1 m⁷GTP 5′ cap structure where it nucleates the formation of a core DDX3/PABP/eIF4G trimeric complex on the HIV-1 genomic RNA. By using RNA fluorescence in situ hybridization coupled to indirect immunofluorescence, we further show that this viral ribonucleoprotein complex is addressed to compartmentalized cytoplasmic foci where the translation initiation complex is assembled.     

The domains of yeast eIF4G,eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

Translation initiation in eukaryotes starts with the recognition of the mRNA 5′-cap by eIF4F, a hetero-trimeric complex of eIF4E, the cap-binding protein, eIF4A, a DEAD-box helicase, and eIF4G, a scaffold protein. eIF4G comprises eIF4E- and eIF4A-binding domains (4E-BD, 4A-BD) and three RNA-binding regions (RNA1–RNA3), and interacts with eIF4A, eIF4E, and with the mRNA. Within the eIF4F complex, the helicase activity of eIF4A is increased. We showed previously that RNA3 of eIF4G is important for the stimulation of the eIF4A conformational cycle and its ATPase and helicase activities. Here, we dissect the interplay between the eIF4G domains and the role of the eIF4E/cap interaction in eIF4A activation. We show that RNA2 leads to an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E binding to the 4E-BD then further inhibits the helicase activity and closing of eIF4A, but does not affect the RNA-stimulated ATPase activity of eIF4A. The 5′-cap renders the functional interaction of mRNA with eIF4A less efficient. Overall, the activity of eIF4A at the 5′-cap is thus fine-tuned by a delicately balanced network of stimulatory and inhibitory interactions.     

Wheat germ poly(A)-binding protein increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F

Recent studies demonstrated that wheat germ poly(A)-binding protein (PABP) interacted with translation eukaryotic initiation factor (eIF)-iso4G and eIF4B, and these interactions increased the poly(A) binding activity of PABP (Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255) and the cap binding activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J., and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a substantial effect on the ATPase activity and RNA helicase activity of (eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the presence of poly(U), PABP can increase the parameter (k(cat)/K(m)) by 3.5-fold with a 2-fold decrease of K(m) for the (eIF4A + eIF-iso4F) complex. In the presence of globin messenger RNA, the ATPase activity of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence of PABP. RNA helicase assays demonstrated that the presence of PABP enhanced the RNA duplex unwinding activity of the initiation factor complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of the complex formed by eIF4A, eIF4B, and eIF4F or eIF-(iso)4F and increase the rate of translation.     

Pumilio 2 controls translation by competing with eIF4E for 7-methyl guanosine cap recognition

Pumilio 2 (Pum2) interacts with the 3′ UTR-containing pumilio binding element (PBE) of RINGO/SPY mRNA to repress translation in Xenopus oocytes. Here, we show that Pum2 also binds directly to the 5′ 7mG cap structure; in so doing, it precludes eIF4E from binding the cap. Using deletion analysis, we have mapped the cap interaction domain of Pum2 to the amino terminus of the protein and identified a conserved tryptophan residue that mediates this specific interaction. Reporter mRNA-based assays demonstrate that Pum2 requires the conserved tryptophan to repress translation in injected Xenopus oocytes. Thus, in addition to its suggested role in regulating poly(A) tail length and mRNA stability, our results suggest that vertebrate Pumilio can repress translation by blocking the assembly of the essential initiation complex on the cap.     

Poly(A) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mRNA with translation initiation factors eIF4F,eIF4B and PABP

We have investigated the effects of poly(A)-tail on binding of eIF4F, eIF4B and PABP with tobacco etch virus (TEV) IRES RNA. The fluorescence anisotropy data showed that the addition of poly(A)₂₀ increases the binding affinity of eIF4F·4B and eIF4F·PABP complexes to IRES RNA ~ 2- and 4-fold, respectively. However, the binding affinity of eIF4F with PK1 was enhanced ~ 11-fold with the addition of PABP, eIF4B, and poly(A)₂₀ together. Whereas, poly(A)₂₀ alone increases the binding affinity of eIF4F·4B·PABP with PK1 RNA about 3-fold, showing an additive effect rather than the large increase in affinity as shown for cap binding. Thermodynamic data showed that PK1 RNA binding to protein complexes in the presence of poly(A)₂₀ was enthalpy-driven and entropy-favorable. Poly(A)₂₀ decreased the entropic contribution 75% for binding of PK1 RNA to eIF4F·4B·PABP as compared to eIF4F alone, suggesting reduced hydrophobic interactions for complex formation and an overall conformational change. Overall, these results demonstrate the first direct effect of poly(A) on the equilibrium and thermodynamics of eIF4F and eIF4F·4B·PABP with IRES-RNA.     

A tale of two termini:: A functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation

A quarter of century following the prediction that mRNAs are translated in a circular form, recent biochemical and genetic evidence has accumulated to support the idea that communication between the termini of an mRNA is necessary to promote translation initiation. The poly(A)-binding protein (PABP) interacts with the cap-associated eukaryotic initiation factor (eIF) 4G (in yeast and plants) and eIF4B (in plants), a functional consequence of which is to increase the affinity of PABP for poly(A) and to increase the affinity of the cap-binding complex, eIF4F (of which eIF4G is a subunit) for the 5′ cap structure. In mammals, PABP interacts with a novel PABP-interacting protein that also binds eIF4A. The interaction between PABP and those initiation factors associated with the 5′ terminus of an mRNA may also explain the role of PABP during mRNA turnover, as it protects the 5′ cap from attack by Dcp1p, the decapping enzyme. Several of those mRNAs that have evolved functional equivalents to a cap or a poly(A) tail nevertheless require a functional interaction between terminal regulatory elements similar to that observed between the 5′ cap and poly(A) tail, suggesting that efficient translation is predicated on communication between largely-separated regulatory elements within an mRNA.     

Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E–eIF4G interactions

Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m⁷GTP cap structure at the 5′-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m⁷GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X₄)Lϕ], where X is any amino acid and Φ is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Φ) is dispensable. The LeishIF4E–LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E–eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.     

Structure of a Viral Cap-independent Translation Element That Functions via High Affinity Binding to the eIF4E Subunit of eIF4F

RNAs of many positive strand RNA viruses lack a 5′ cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3′-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the ∼100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m⁷G cap.Regulation of translation occurs primarily at the initiation step. This involves recognition of the 5′ m⁷G(5′)ppp(5′)N cap structure on the mRNA by initiation factors, which recruit the ribosome to the 5′-end of the mRNA (1–5). The 5′ cap structure and the poly(A) tail are necessary for efficient recruitment of initiation factors on eukaryotic mRNAs (3, 6–8). The cap is recognized by the eIF4E subunit of eukaryotic translation initiation factor complex eIF4F (or the eIFiso4E subunit of eIFiso4F in higher plants). The poly(A) tail is recognized by poly(A)-binding protein. In plants, eIF4F is a heterodimer consisting of eIF4E and eIF4G, the core scaffolding protein to which the other factors bind. eIF4A, an ATPase/RNA helicase, interacts with eIF4F but is not part of the eIF4F heterodimer (9, 10). For translation initiation, the purpose of eIF4E is to bring eIF4G to the capped mRNA. eIF4G then recruits the 43 S ternary ribosomal complex via interaction with eIF3.The RNAs of many positive sense RNA viruses contain a cap-independent translation element (CITE)³ that allows efficient translation in the absence of a 5′ cap structure (11–13). In animal viruses and some plant viruses, the CITE is an internal ribosome entry site (IRES) located upstream of the initiation codon. Most viral IRESes neither interact with nor require eIF4E, because they lack the m⁷GpppN structure, which, until this report, was thought to be necessary for mRNA to bind eIF4E with high affinity (3, 14). Translation initiation efficiency of mRNA is also influenced by the length of, and the degree of secondary structure in the 5′ leader (15–17).Many uncapped plant viral RNAs harbor a CITE in the 3′-UTR that confers highly efficient translation initiation at the 5′-end of the mRNA (18–22). These 3′ CITEs facilitate ribosome entry and apparently conventional scanning at the 5′-end of the mRNA (17, 23, 24). A variety of unrelated structures has been found to function as 3′ CITEs, suggesting that they recruit the ribosome by different interactions with initiation factors (13).The factors with which a plant CITE interacts to recruit the ribosome have been identified for only a potyvirus, a luteovirus, and a satellite RNA. The 143-nt 5′-UTR CITE of the potyvirus, tobacco etch virus is an IRES that functions by binding of its AU-rich pseudoknot structure with eIF4G (25). It binds eIF4G with up to 30-fold greater affinity than eIFiso4G and does not require eIF4E for IRES activity. In addition to RNA elements, the genome-linked viral protein (VPg) of potyviruses may participate in cap-independent translation initiation by interacting with the eIF4E and eIFiso4E subunits of eIF4F and eIFiso4F, respectively (26–31). In contrast, the 130-nt cap-independent translation enhancer domain (TED) in the 3′-UTR of satellite tobacco necrosis virus (STNV) RNA forms a long bulged stem-loop, which interacts strongly with both eIF4F and eIFiso4F and weakly with their eIF4E and eIFiso4E subunits (32), suggesting that the TED requires the full eIF4F or eIFiso4F for a biologically relevant interaction. Barley yellow dwarf luteovirus (BYDV) and several other viruses, have a different structure, called a BYDV-like CITE (BTE), in the 3′-UTR. The BTE is characterized by a 17-nt conserved sequence incorporated in a structure with a variable number of stem-loops radiating from a central junction (20, 33, 34). It requires and binds the eIF4G subunit of eIF4F and does not bind free eIF4E, eIFiso4E, or eIFiso4G, although eIF4E slightly enhances the BTE-eIF4G interaction (35). Other 3′ CITEs have been identified, but the host factors with which they interact are unknown.Here we describe unprecedented factor interactions of a CITE found in an umbravirus and a panicovirus. Umbraviruses show strong similarity to the Luteovirus and Dianthovirus genera in (i) the sequence of the replication genes encoded by ORFs 1 and 2, (ii) the predicted structure of the frameshift signals required for translation of the RNA-dependent RNA polymerase from ORF 2 (36, 37), (iii) the absence of a poly(A) tail, and (iv) the lack of a 5′ cap structure (37, 38). Umbraviruses are unique in that they encode no coat protein. For the umbravirus pea enation mosaic virus 2 (PEMV-2), the coat protein is provided by PEMV-1, an enamovirus (39). Uncapped PEMV-2 RNA (PEMV RNA 2), transcribed in vitro, is infectious in pea (Pisum sativa),⁴ indicating it must be translated cap-independently. The 3′-UTRs of some umbraviruses such as Tobacco bushy top virus and Groundnut rosette virus harbor sequences resembling BYDV-like CITEs (BTE).⁵ However, no BTE is apparent in the 3′-UTR of PEMV RNA 2. In this report we identify a different class of CITE in the 705-nt long 3′-UTR of PEMV RNA 2, determine its secondary structure, which may include an unusual pseudoknot, and we show that, unlike any other natural uncapped RNA, it has a high affinity for eIF4E, which is necessary to facilitate cap-independent translation.     

Translation driven by an eIF4G core domain in vivo.

Most eukaryotic mRNAs possess a 5' cap structure (m(7)GpppN) and a 3' poly(A) tail which promote translation initiation by binding the eukaryotic translation initiation factor (eIF)4E and the poly(A) binding protein (PABP), respectively. eIF4G can bridge between eIF4E and PABP, and-through eIF3-is thought to establish a link to the small ribosomal subunit. We fused the C-terminal region of human eIF4GI lacking both the eIF4E- and PABP-binding sites, to the IRE binding protein IRP-1. This chimeric protein suffices to direct the translation of the downstream cistron of bicistronic mRNAs bearing IREs in their intercistronic space in vivo. This function is preserved even when translation via the 5' end is inhibited. Deletion analysis defined the conserved central domain (amino acids 642-1091) of eIF4G as an autonomous 'ribosome recruitment core' and implicated eIF4A as a critical binding partner. Our data reveal the sufficiency of the conserved eIF4G ribosome recruitment core to drive productive mRNA translation in living cells. The C-terminal third of eIF4G is dispensable, and may serve as a regulatory domain.     

eIF4E3 forms an active eIF4F complex during stresses (eIF4FS) targeting mTOR and re-programs the translatome

The eIF4E are a family of initiation factors that bind the mRNA 5′ cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4F^S) is translationally active during stress and that it selects specific mRNA populations based on 5′ TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have ‘moon-lighting’ functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote ‘stress resistance’ and adaptation.     

The role of 5'-leader length, secondary structure and PABP concentration on cap and poly(A) tail function during translation in Xenopus oocytes

The 5′-cap structure and poly(A) tail of eukaryotic mRNAs function synergistically to promote translation initiation through a physical interaction between the proteins that bind to these regulatory elements. In this study, we have examined the effect of leader length and the presence of secondary structure on the translational competence and the function of the cap and poly(A) tail for mRNAs microinjected into Xenopus oocytes. Increasing the length of the 5′-leader from 17 to 144 nt resulted in a 2- to 4-fold increase in expression from an mRNA containing an unstructured leader but increased expression up to 20-fold for an mRNA containing 5′-proximal structure. Consequently, the presence of secondary structure was less inhibitory for those mRNAs with a longer 5′-leader. Co-injection of poly(A)-binding protein (PABP) mRNA increased the function of the cap and poly(A) tail in promoting translation from poly(A)⁺ but not poly(A)^– mRNAs, particularly for mRNAs containing secondary structure. In the absence of an internal ribosome entry site, expression from the distal cistron of a dicistronic mRNA increased as a function of the length of the intercistronic region and the concentration of PABP. The inhibitory effect of intercistronic located secondary structure on translation was position-dependent. Indeed, the effect of secondary structure was abolished if positioned 134 nt upstream of the distal cistron. These data suggest that the length of a leader, the presence of secondary structure and the concentration of PABP determine the extent to which the cap and poly(A) tail regulate translation.     

Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5′ppp- mRNAs

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K_1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K_1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K_1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.     

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA: EFFECTS OF eIF4B AND POLY(A)-BINDING PROTEIN*

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k_on) and dissociation rate constant (k_off) for eIF4F binding to IRES were 59 ± 2.1 μm⁻¹ s⁻¹ and 12.9 ± 0.3 s⁻¹, respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F ∼3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.