RNA aptamers to mammalian initiation factor 4G inhibit cap-dependent translation by blocking the formation of initiation factor complexes

Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial multimodulatory role in mRNA translation and decay by interacting with other translation factors and mRNA-associated proteins. In this study, we isolated eight different RNA aptamers with high affinity to mammalian eIF4G by in vitro RNA selection amplification. Of these, three aptamers (apt3, apt4, and apt5) inhibited the cap-dependent translation of two independent mRNAs in a rabbit reticulocyte lysate system. The cap-independent translation directed by an HCV internal ribosome entry site was not affected. Addition of exogenous eIF4G reversed the aptamer-mediated inhibition of translation. Even though apt3 and apt4 were selected independently, they differ only by two nucleotides. The use of truncated eIF4G variants in binding experiments indicated that apt4 (and probably apt3) bind to both the middle and C-terminal domains of eIF4G, while apt5 binds only to the middle domain of eIF4G. Corresponding to the difference in the binding sites in eIF4G, apt4, but not apt5, hindered eIF4G from binding to eIF4A and eIF3, in a purified protein solution system as well as in a crude lysate system. Therefore, the inhibition of translation by apt4 (and apt3) is due to the inhibition of formation of initiation factor complexes involving eIF4A and eIF3. On the other hand, apt5 had a much weaker affinity to eIF4G than apt4, but inhibited translation much more efficiently by an unknown mechanism. The five additional aptamers have sequences and predicted secondary structures that are largely different from each other and from apt3 through apt5. Therefore, we speculate that these seven sets of aptamers may bind to different regions in eIF4G in different fashions.     

The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis.

eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.     

A Saccharomyces cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity.

The TIF3 gene of Saccharomyces cerevisiae was cloned and sequenced. The deduced amino acid sequence shows 26% identity with the sequence of mammalian translation initiation factor eIF-4B. The TIF3 gene is not essential for growth; however, its disruption results in a slow growth and cold-sensitive phenotype. In vitro translation of total yeast RNA in an extract from a TIF3 gene-disrupted strain is reduced compared with a wild-type extract. The translational defect is more pronounced at lower temperatures and can be corrected by the addition of wild-type extract or mammalian eIF-4B, but not by addition of mutant extract. In vivo translation of beta-galactosidase reporter mRNA with varying degree of RNA secondary structure in the 5' leader region in a TIF3 gene-disrupted strain shows preferential inhibition of translation of mRNA with more stable secondary structure. This indicates that Tif3 protein is an RNA helicase or contributes to RNA helicase activity in vivo.     

A new yeast translation initiation factor suppresses a mutation in the eIF-4A RNA helicase.

We have isolated a gene, STM1, which encodes a new translation initiation factor from Saccharomyces cerevisiae. The gene acts, if present on a multicopy plasmid, as a suppressor of a temperature-sensitive mutation in eIF-4A. The single copy STM1 gene is not essential, but disruption causes a slow growth phenotype. Analysis of polysomes from a strain carrying a disrupted stm1 allele shows a clear defect in translation initiation as shown by a strong reduction in polysomes and an increase in the monosomes. Sequence analysis revealed interesting features of the putative Stm1 protein. Comparison of the entire protein sequence with databanks showed some similarity with the human eIF-4B protein. The Stm1 protein has potential RNP1 and RNP2 motifs characteristic for RNA-binding proteins. The protein also contains six highly conserved direct repeats of 21-26 amino acids and one partial repeat.     

Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F.

The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.     

Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A.

eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.     

Wheat germ translation initiation factor eIF4B affects eIF4A and eIFiso4F helicase activity by increasing the ATP binding affinity of eIF4A

It has been proposed that, during translational initiation, structures in the 5' untranslated region of mRNA are unwound. eIF4A, a member of the DEAD box family of proteins (those that contain a DEAD amino acid sequence), separately or in conjunction with other eukaryotic initiation factors, utilizes the energy from ATP hydrolysis to unwind these structures. As a step in defining the mechanism of helicase activity in the wheat germ protein synthesis system, we have utilized direct fluorescence measurements, ATPase assays, and helicase assays. The RNA duplex unwinding activity of wheat germ eIF4A is similar to other mammalian systems; however, eIF4F or eIFiso4F is required, probably because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding measurements showed that eIF4A had a higher binding affinity for ADP than ATP, resulting in a limited hydrolysis and procession along the RNA in the helicase assay. The addition of eIF4B resulted in a change in binding affinity for ATP, increasing it almost 10-fold while the ADP binding affinity was approximately the same. The data presented in this paper suggest that eIF4F or eIFiso4F acts to position the eIF4A and stabilize the interaction with mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA duplex. The binding of eIF4B either prior to or after hydrolysis allows for increased affinity for ATP and for the cycle of conformational changes to proceed, resulting in further unwinding and processive movement along the mRNA.     

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.     

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.     

Translational control by a small RNA: dendritic BC1 RNA targets the eukaryotic initiation factor 4A helicase mechanism

Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5' secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.     

Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A

Eukaryotic initiation factor (eIF) 4A is the prototypic member of the DEAD box family of proteins and has been proposed to act as an RNA helicase to unwind secondary structure in the 5'-untranslated region of eukaryotic mRNAs. Previous studies have shown that the RNA helicase activity of eIF4A is dependent on the presence of a second initiation factor, eIF4B. In this report, eIF4A has been demonstrated to function independently of eIF4B as an ATP-dependent RNA helicase. The biochemical and kinetic properties of this activity were examined. By using a family of RNA duplexes with an unstructured single-stranded region followed by a duplex region of increasing length and stability, it was observed that the initial rate of duplex unwinding decreased with increasing stability of the duplex. Furthermore, the maximum amount of duplex unwound also decreased with increasing stability. Results suggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were shown to stimulate the helicase activity of eIF4A, allowing eIF4A to unwind longer, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain the mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependent manner.     

Cap-binding protein (eukaryotic initiation factor 4E) and 4E-inactivating protein BP-1 independently regulate cap-dependent translation.

Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.     

A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities. The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.     

Unwinding initiation by the viral RNA helicase NPH-II

Viral RNA helicases of the NS3/NPH-II group unwind RNA duplexes by processive, directional translocation on one of the duplex strands. The translocation is preceded by a poorly understood unwinding initiation phase. For NPH-II from vaccinia virus, unwinding initiation is rate limiting for the overall unwinding reaction. To develop a mechanistic understanding of the unwinding initiation, we studied kinetic and thermodynamic aspects of this reaction phase for NPH-II in vitro, using biochemical and single molecule fluorescence approaches. Our data show that NPH-II functions as a monomer and that different stages of the ATP hydrolysis cycle dictate distinct binding preferences of NPH-II for duplex versus single-stranded RNA. We further find that the NPH-II-RNA complex does not adopt a single conformation but rather at least two distinct conformations in each of the analyzed stages of ATP hydrolysis. These conformations interconvert with rate constants that depend on the stage of the ATP hydrolysis cycle. Our data establish a basic mechanistic framework for unwinding initiation by NPH-II and suggest that the various stages of the ATP hydrolysis cycle do not induce single, stage-specific conformations in the NPH-II-RNA complex but primarily control transitions between multiple states.     

RNA chaperone activity of translation initiation factor IF1

Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.     

Eukaryotic translation initiation factor 4F architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells

Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.     

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2'-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.     

Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation.

The immunosuppressant drug rapamycin blocks progression of the cell cycle at the G1 phase in mammalian cells and yeast. Here we show that rapamycin inhibits cap-dependent, but not cap-independent, translation in NIH 3T3 cells. Cap-dependent translation is also specifically reduced in extracts from rapamycin-treated cells, as determined by in vitro translation experiments. This inhibition is causally related to the dephosphorylation and consequent activation of 4E-BP1, a protein recently identified as a repressor of the cap-binding protein, eIF-4E, function. These effects of rapamycin are specific as FK506, a structural analogue of rapamycin, had no effect on either cap-dependent translation or 4E-BP1 phosphorylation. The rapamycin-FK506 binding protein complex is the effector of the inhibition of 4E-BP1 phosphorylation as excess of FK506 over rapamycin reversed the rapamycin-mediated inhibition of 4E-BP1 phosphorylation. Thus, inactivation of eIF-4E is, at least in part, responsible for inhibition of cap-dependent translation in rapamycin-treated cells. Furthermore, these results suggest that 4E-BP1 phosphorylation is mediated by the FRAP/TOR signalling pathway.