Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs

Tristetraprolin (TTP) regulates the expression of AU-rich element-containing mRNAs through promoting the degradation and repressing the translation of target mRNA. While the mechanism for promoting target mRNA degradation has been extensively studied, the mechanism underlying translational repression is not well established. Here, we show that TTP recruits eukaryotic initiation factor 4E2 (eIF4E2) to repress target mRNA translation. TTP interacted with eIF4E2 but not with eIF4E. Overexpression of eIF4E2 enhanced TTP-mediated translational repression, and downregulation of endogenous eIF4E2 or overexpression of a truncation mutant of eIF4E2 impaired TTP-mediated translational repression. Overexpression of an eIF4E2 mutant that lost the cap-binding activity also impaired TTP''s activity, suggesting that the cap-binding activity of eIF4E2 is important in TTP-mediated translational repression. We further show that TTP promoted eIF4E2 binding to target mRNA. These results imply that TTP recruits eIF4E2 to compete with eIF4E to repress the translation of target mRNA. This notion is supported by the finding that downregulation of endogenous eIF4E2 increased the production of tumor necrosis factor alpha (TNF-α) protein without affecting the mRNA levels in THP-1 cells. Collectively, these results uncover a novel mechanism by which TTP represses target mRNA translation.     

Mextli Is a Novel Eukaryotic Translation Initiation Factor 4E-Binding Protein That Promotes Translation in Drosophila melanogaster

Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5′-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.     

Uncoupling Stress Granule Assembly and Translation Initiation Inhibition

Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAi^Met ternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.     

Viral Genome-Linked Protein (VPg) Is Essential for Translation Initiation of Rabbit Hemorrhagic Disease Virus (RHDV)

Rabbit hemorrhagic disease virus (RHDV), the causative agent of rabbit hemorrhagic disease, is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, limiting the study of the pathogenesis of RHDV. In addition, the mechanisms underlying RHDV translation and replication are largely unknown compared with other caliciviridae viruses. The RHDV replicon recently constructed in our laboratory provides an appropriate model to study the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon, we demonstrated that the viral genome-linked protein (VPg) is essential for RHDV translation in RK-13 cells for the first time. In addition, we showed that VPg interacts with eukaryotic initiation factor 4E (eIF4E) in vivo and in vitro and that eIF4E silencing inhibits RHDV translation, suggesting the interaction between VPg and eIF4E is involved in RHDV translation. Our results support the hypothesis that VPg serves as a novel cap substitute during the initiation of RHDV translation.     

Down-Regulation of eIF4GII by miR-520c-3p Represses Diffuse Large B Cell Lymphoma Development

Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.     

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m⁷G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m⁷G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.Cap-dependent translation initiation in eukaryotes is a complex process involving many factors and serves as the primary mechanism for eukaryotic translation (37, 44). The first step in the initiation process, recruitment of the m⁷G (7-methylguanosine)-capped mRNA to the ribosome, is widely considered the rate-limiting step. It begins with recognition of and binding to the m⁷G cap at the 5′ end of the mRNA by the eukaryotic translation initiation factor 4F (eIF4F) complex, which contains three proteins: eIF4E (a cap-binding protein), eIF4G (a scaffold protein with RNA binding sites), and eIF4A (an RNA helicase). eIF4G''s interaction with eIF3, itself a multisubunit complex that interacts with the 40S ribosome, facilitates the actual recruitment of capped RNA to the ribosome. With the help of several other initiation factors, the small ribosomal subunit scans the mRNA from 5′ to 3′ until a translation initiation codon (AUG) in appropriate context is identified and an 80S ribosomal complex is formed, after which the first peptide bond is formed, thus ending the initiation process (37, 44). The AUG context can play an important role in the efficiency of translation initiation (23, 44). The length, structure, and presence of AUGs or open reading frames in the mRNA 5′ untranslated region (UTR) can negatively affect cap-dependent translation and ribosomal scanning. In general, long and highly structured 5′ UTRs, as well as upstream AUGs leading to short open reading frames, can impede ribosome scanning and lead to reduced translation (23, 44). In addition, 5′ UTRs less than 10 nucleotides (nt) in length are thought to be too short to enable preinitiation complex assembly and scanning (24). Thus, several attributes of the mRNA 5′ UTR are known to negatively affect translation initiation, whereas only the AUG context and the absence of negative elements are known to have a positive effect on translation initiation (44).Two of the important mRNA features associated with cap-dependent translation, the cap and the 5′ UTR, are significantly altered by an RNA processing event known as spliced leader (SL) trans splicing (3, 8, 17, 26, 36, 47). This takes place in members of a diverse group of eukaryotic organisms, including some protozoa, sponges, cnidarians, chaetognaths, flatworms, nematodes, rotifers, crustaceans, and tunicates (17, 28, 39, 55, 56). In SL trans splicing, a separately transcribed small exon (16 to 51 nucleotides [nt]) with its own cap gets added to the 5′ end of pre-mRNAs. This produces mature mRNAs with a unique cap and a conserved sequence in the 5′ UTR. In metazoa, the m⁷G cap is replaced with a trimethylguanosine (TMG) cap (m^2,2,7GpppN) (27, 30, 46, 49). In nematodes, ∼70% of all mRNAs are trans spliced and therefore have a TMG cap and an SL (2). In general, eukaryotic eIF4E proteins do not effectively recognize the TMG cap (35). This raises the issues of how the translation machinery in trans-splicing metazoa effectively recognizes TMG-capped trans-spliced mRNAs, what role the SL sequence plays in translation initiation, and how the conserved translation initiation machinery has adapted to effectively translate trans-spliced mRNAs.Previous work has shown that efficient translation of TMG-capped messages in nematodes requires the SL sequence (22 nt) immediately downstream of the cap (5, 25, 29). In the current studies, we sought to understand the manner in which the SL enhanced the translation of TMG-capped mRNAs. Using a cell-free nematode in vitro translation system, we carried out mutational analyses that define the specific sequences in the SL that are required and sufficient for efficient translation of TMG-capped mRNAs. These analyses led to the discovery of a small, discrete stem-loop immediately adjacent to the TMG cap in trans-spliced messages required for efficient translation. Notably, the sequences involved in the base pairing of the stem are highly conserved in alternative SL sequences found in nematodes. We further show that the nematode eIF4E/G complex plays a major role in facilitating the SL enhancement of TMG-capped mRNA that likely occurs after the initial cap-binding step. The results demonstrate the importance of specific enhancing elements in the 5′ UTR and adaptation in the eIF4F complex necessary for optimal cap-dependent translation.     

NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI from Degradation by the Proteasome

The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.In eukaryotes, eukaryotic translation initiation factor 4G (eIF4G) plays a central role in the recruitment of ribosomes to the mRNA 5′ end and is therefore critical for the regulation of protein synthesis (14). Two homologues of eIF4G, eIF4GI and eIF4GII, have been cloned (15). Although they differ in various respects, both homologues clearly function in translation initiation. The most thoroughly studied of these is eIF4GI, which serves as a scaffolding protein for the assembly of eIF4F, a protein complex composed of eIF4E (the mRNA cap-binding factor) and eIF4A (an ATP-dependent RNA helicase). Thus, via its association with the mRNA cap-binding protein eIF4E and with another translation initiation factor (eIF3) which is bound to the 40S ribosomal subunit, eIF4GI creates a physical link between the mRNA cap structure and the ribosome, thus facilitating cap-dependent translation initiation (25). eIF4GI functions also in cap-independent, internal ribosome entry site (IRES)-mediated translation initiation. For instance, upon picornavirus infection, eIF4G is rapidly attacked by viral proteases. The resulting eIF4GI cleavage products serve to reprogram the cell''s translational machinery, as the N-terminal cleavage product inhibits cap-dependent translation of host cell mRNAs by sequestering eIF4E while the C-terminal cleavage product stimulates IRES-mediated translation of viral mRNAs (23). Similarly, apoptotic caspases cleave eIF4G into an N-terminal fragment that blocks cap-dependent translation and a C-terminal fragment that is utilized for IRES-mediated translation of mRNAs encoding proapoptotic proteins (22).The regulation of eIF4GI cleavage by viral proteases or apoptotic caspases has been extensively studied. Little is known, however, about the regulation of eIF4GI steady-state levels. Yet the eIF4GI amount that exists at a given moment results from the sum of the effects of de novo synthesis and ongoing degradation. Many cellular proteins are physiologically degraded by the proteasome. This has been shown to be true for eIF4GI, as the factor can be degraded by the proteasome in vitro (5) and in living cells (6). However, how eIF4GI targeting for or protection from destruction by the proteasome is regulated remains unknown.There are two major routes to degradation by the proteasome. In the more conventional route, polyubiquitinated proteins are targeted to the 26S proteasome. Alternatively, a few proteins can be degraded by the 20S proteasome (and sometimes by the 26S proteasome) in a ubiquitin-independent manner (16). Interestingly, it has been shown recently that a few of these proteins (1, 2, 13) can be protected from degradation by the 20S proteasome by binding to the NAD(P)H quinone-oxydoreductase 1 (NQO1). It has been proposed that NQO1 may interact with the 20S proteasome and may consequently block access of target proteins to the 20S degradation core. Because eIF4GI can be degraded in vitro by the 20S proteasome (5) and since it appears that proteasomes can degrade eIF4GI in living cells independently of ubiquitination (6), we asked whether NQO1 could protect eIF4GI from degradation by the proteasome.     

eIF4A,a target of siRNA derived from rice stripe virus,negatively regulates antiviral autophagy by interacting with ATG5 in Nicotiana benthamiana

Autophagy is induced by viral infection and has antiviral functions in plants, but the underlying mechanism is poorly understood. We previously identified a viral small interfering RNA (vsiRNA) derived from rice stripe virus (RSV) RNA4 that contributes to the leaf-twisting and stunting symptoms caused by this virus by targeting the host eukaryotic translation initiation factor 4A (eIF4A) mRNA for silencing. In addition, autophagy plays antiviral roles by degrading RSV p3 protein, a suppressor of RNA silencing. Here, we demonstrate that eIF4A acts as a negative regulator of autophagy in Nicotiana benthamiana. Silencing of NbeIF4A activated autophagy and inhibited RSV infection by facilitating autophagic degradation of p3. Further analysis showed that NbeIF4A interacts with NbATG5 and interferes with its interaction with ATG12. Overexpression of NbeIF4A suppressed NbATG5-activated autophagy. Moreover, expression of vsiRNA-4A, which targets NbeIF4A mRNA for cleavage, induced autophagy by silencing NbeIF4A. Finally, we demonstrate that eIF4A from rice, the natural host of RSV, also interacts with OsATG5 and suppresses OsATG5-activated autophagy, pointing to the conserved function of eIF4A as a negative regulator of antiviral autophagy. Taken together, these results reveal that eIF4A negatively regulates antiviral autophagy by interacting with ATG5 and that its mRNA is recognized by a virus-derived siRNA, resulting in its silencing, which induces autophagy against viral infection.     

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.     

Poly(A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation

Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3C^pro). However, PABP is cleaved by HAV 3C^pro in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5′ nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.     

High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation

The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation.     

Intrinsic RNA Binding by the Eukaryotic Initiation Factor 4F Depends on a Minimal RNA Length but Not on the m7G Cap

The eukaryotic initiation factor 4F (eIF4F) is thought to be the first factor to bind mRNA during 7-methylguanosine (m⁷G) cap-dependent translation initiation. The multipartite eIF4F contains the cap-binding protein eIF4E, and it is assumed that eIF4F binds mRNAs primarily at the 5′ m⁷G cap structure. We have analyzed equilibrium binding of rabbit eIF4F to a series of diverse RNAs and found no impact of the 5′-cap on the stability of eIF4F-RNA complexes. However, eIF4F preferentially and cooperatively binds to RNAs with a minimum length of ∼60 nucleotides in vitro. Furthermore, translation activity in rabbit reticulocyte lysate is strongly inhibited by RNAs exceeding this length, but not by shorter ones, consistent with the notion that eIF4F in its physiological environment preferentially binds longer RNAs, too. Collectively, our results indicate that intrinsic RNA binding by eIF4F depends on a minimal RNA length, rather than on cap recognition. The nonetheless essential m⁷G cap may either function at steps subsequent to eIF4F-RNA binding, or other factors facilitate preferential binding of eIF4F to the m⁷G cap.     

Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta

Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.     

eIF4B controls survival and proliferation and is regulated by proto-oncogenic signaling pathways

Messenger RNA translation or protein synthesis, is a fundamental biological process affecting cell growth, survival and proliferation. Initiation is the rate limiting and hence the most regulated step of translation. In eukaryotes, translation initiation is facilitated by multiple protein factors collectively called eIFs (for eukaryotic translation initiation factors). The complex consisting of the eIF4 group factors including the mRNA cap-binding eIF4E protein, large scaffolding protein eIF4G and RNA helicase eIF4A is assisted by the eIF4B co-factor to unwind local secondary structures and create a ribosome landing pad on mRNA. Recruitment of the ribosome and augmentation in the mRNA scanning process culminates in the positioning of the ribosome over the start codon. Deregulated translational control is believed to play an important role in oncogenic transformation. Indeed, many eIFs are bona fide proto-oncogenes. In many types of human cancers, eIFs are either overexpressed or ectopically activated by Ras-MAPK and PI3K-mTOR signaling cascades, resulting in increased survival and accelerated proliferation. In this review we will analyze the bulk of data describing eIF4B and its role in cell survival and proliferation. Recent studies have shown that eIF4B is phosphorylated and activated by Ras-MAPK and PI3K-mTOR signaling cascades. In addition, eIF4B regulates translation of proliferative and pro-survival mRNAs. Moreover, eIF4B depletion in cancer cells attenuates proliferation, sensitizes them to genotoxic stress-driven apoptosis. Taken together, these findings identify eIF4B as a potential target for development of anti-cancer therapies.Key words: eIF4B, translation, signaling, structured 5′UTR, helicase activity, survival, proliferation, apoptosis     

RNA Recognition Motif 2 of Yeast Pab1p Is Required for Its Functional Interaction with Eukaryotic Translation Initiation Factor 4G

The eukaryotic mRNA 3′ poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.     

Calicivirus translation initiation requires an interaction between VPg and eIF 4 E

Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 5' end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF 4 E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF 4 E by 4 E-BP 1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF 4 E and the cap structure or 4 E-BP 1, suggesting that VPg binds to eIF 4 E at a different site from both cap and 4 E-BP 1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA.