A cyclic PNA-based compound targeting domain IV of HCV IRES RNA inhibits in vitro IRES-dependent translation

A cyclic molecule 1 constituted by a hepta-peptide nucleic acid sequence complementary to the apical loop of domain IV of hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA has been prepared via a 'mixed' liquid-phase strategy, which relies on easily available protected PNA and poly(2-aminoethylglycinamide) building blocks. This compound 1 has been elaborated to mimic 'loop-loop' interactions. For comparison, its linear analog has also been investigated. Although preliminary biological assays have revealed the ability of 1 to inhibit in vitro the HCV IRES-dependent translation in a dose-dependent manner, the linear analog has shown a slightly higher activity.     

RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae

We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo. Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1-82 (N terminus), 492-539 (middle), and 883-952 (C terminus). The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich. The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K(d) of 5 x 10(-8) M). Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity. Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive. These data suggest that RNA-binding activity is required for eIF4G1 function.     

PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes

Background  

PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN.     

Human DDX3 functions in translation and interacts with the translation initiation factor eIF3

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.     

Neural RNA-binding protein Musashi1 inhibits translation initiation by competing with eIF4G for PABP

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural stem cells. We previously reported that Msi1 contributes to the maintenance of the immature state and self-renewal activity of neural stem cells through translational repression of m-Numb. However, its translation repression mechanism has remained unclear. Here, we identify poly(A) binding protein (PABP) as an Msi1-binding protein, and find Msi1 competes with eIF4G for PABP binding. This competition inhibits translation initiation of Msi1's target mRNA. Indeed, deletion of the PABP-interacting domain in Msi1 abolishes its function. We demonstrate that Msi1 inhibits the assembly of the 80S, but not the 48S, ribosome complex. Consistent with these conclusions, Msi1 colocalizes with PABP and is recruited into stress granules, which contain the stalled preinitiation complex. However, Msi1 with mutations in two RNA recognition motifs fails to accumulate into stress granules. These results provide insight into the mechanism by which sequence-specific translational repression occurs in stem cells through the control of translation initiation.     

Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2

In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (epsilon) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the beta subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2beta mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bepsilon employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2beta homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre-initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2.     

Functional analysis of the interaction between HCV 5'UTR and putative subunits of eukaryotic translation initiation factor eIF3.

Translation initiation in Hepatitis C Virus is controlled by the presence of an internal ribosome entry site element (IRES) principally located in its 5' untranslated region (UTR). Mutation/deletion analyses have shown that the integrity of this structure is essential for initiation of cap-independent protein synthesis. We have developed a strategy to swap the position of the two major domains (II and III) on the 5'UTR sequence. The aim was to further characterize this mechanism by preserving domain-specific interactions but possibly losing contacts that require any interdomain geometry. The expression of dicistronic mRNAs containing these different UTRs showed that the positioning of the different domains on the 5'UTR is essential for efficient IRES functioning. We then used these mutants to identify cellular factors implicated in IRES activity. Using UV crosslinking assays we found that domain III makes direct contact with two proteins (p170/p120) which can be associated with efficient IRES activity. In particular, we have mapped the binding sites of these proteins and shown that p120 binds to the apical loop segment of domain III, whilst p170 binds in the stem portion, independently of domain III position or context. Finally, we provide evidence showing that p170 and p120 represent two subunits of eukaryotic initiation factor eIF3: p170 and p116/p110.     

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.     

Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.     

eIF3: a versatile scaffold for translation initiation complexes

Translation initiation in eukaryotes depends on many eukaryotic initiation factors (eIFs) that stimulate both recruitment of the initiator tRNA, Met-tRNA(i)(Met), and mRNA to the 40S ribosomal subunit and subsequent scanning of the mRNA for the AUG start codon. The largest of these initiation factors, the eIF3 complex, organizes a web of interactions among several eIFs that assemble on the 40S subunit and participate in the different reactions involved in translation. Structural analysis suggests that eIF3 performs this scaffolding function by binding to the 40S subunit on its solvent-exposed surface rather than on its interface with the 60S subunit, where the decoding sites exist. This location of eIF3 seems ideally suited for its other proposed regulatory functions, including reinitiating translation on polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis.     

Distribution of translation initiation factor eIF3 in neutrophils in Alzheimer disease

The distribution of initiation factor eIF3 in the cytoplasm of peripheral blood neutrophils in Alzheimer disease (AD) was investigated. Using a specially developed image analysis protocol, we found a significant increase in the heterogeneity of the eIF3 distribution in AD neutrophils as compared with control cells. We also obtained an evidence that the increase in the eIF3 heterogeneity in AD neutrophils is associated with the formation of stress granules–non-membrane structures that emerge in the cytoplasm of eukaryotic cells in response to the environmental stress.     

A conserved RNA structure within the HCV IRES eIF3-binding site

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is recognized specifically by the small ribosomal subunit and eukaryotic initiation factor 3 (eIF3) before viral translation initiation. Using extensive mutagenesis and structure probing analysis, we show that the eIF3-binding domain of the HCV IRES contains an internal loop structure (loop IIIb) and an adjacent mismatched helix that are important for IRES-dependent initiation of translation. NMR studies reveal a unique three-dimensional structure for this internal loop that is conserved between viral isolates of varying primary sequence in this region. These data indicate that internal loop IIIb may be an attractive target for structure-based design of new antiviral agents.     

Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.     

Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G. Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of approximately 42 nm was derived from an association rate constant of 3.9 x 10(4) m(-1) s(-1) and dissociation rate constant of 1.5 x 10(-3) s(-1). Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.     

Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.     

Influence of the hepatitis C virus 3′-untranslated region on IRES-dependent and cap-dependent translation initiation

Translation of hepatitis C virus (HCV) genomic RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR), and the HCV 3′-UTR enhances IRES activity. Since the HCV 3′-UTR has a unique structure among 3′-UTRs, we checked possible communication between the 5′- and the 3′-UTR of HCV during translation using chimeric reporter RNAs. We show that translation directed by the HCV IRES and by the HCV-like IRES of porcine teschovirus (PTV) which belongs to a quite distinct family of viruses (picornaviruses) or by the EMCV IRES is also enhanced by the HCV 3′-UTR or by a poly(A)-tail in different cell types.     

The 3' cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation

The 3' cap-independent translation element (BTE) of Barley yellow dwarf virus RNA confers efficient translation initiation at the 5' end via long-distance base pairing with the 5'-untranslated region (UTR). Here we provide evidence that the BTE functions by recruiting translation initiation factor eIF4F. We show that the BTE interacts specifically with the cap-binding initiation factor complexes eIF4F and eIFiso4F in a wheat germ extract (wge). In wge depleted of cap-interacting factors, addition of eIF4F (and to a lesser extent, eIFiso4F) allowed efficient translation of an uncapped reporter construct (BLucB) containing the BTE in its 3' UTR. Translation of BLucB required much lower levels of eIF4F or eIFiso4F than did a capped, nonviral mRNA. Both full-length eIF4G and the carboxy-terminal half of eIF4G lacking the eIF4E binding site stimulated translation to 70% of the level obtained with eIF4F, indicating a minor role for the cap-binding protein, eIF4E. In wge inhibited by either BTE in trans or cap analog, eIF4G alone restored translation nearly as much as eIF4F, while addition of eIF4E alone had no effect. The BTE bound eIF4G (Kd = 177 nm) and eIF4F (Kd = 37 nm) with high affinity, but very weakly to eIF4E. These interactions correlate with the ability of the factors to facilitate BTE-mediated translation. These results and previous observations are consistent with a model in which eIF4F is delivered to the 5' UTR by the BTE, and they show that eIF4G, but not eIF4E, plays a major role in this novel mechanism of cap-independent translation.     

Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories. Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3. The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity. The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence. The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3. Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3. Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.