Eukaryotic translation initiation factor 4AII contributes to microRNA-122 regulation of hepatitis C virus replication.

Hepatitis C virus (HCV) is a positive sense RNA virus that persistently infects human liver, leading to cirrhosis and hepatocellular carcinoma. HCV replication requires the liver-specific microRNA-122 (miR-122). In contrast to canonical miRNA-mediated repression via 3'UTR sites, miR-122 positively regulates HCV replication by a direct interaction with the 5' untranslated region (UTR) of the viral RNA. The protein factor requirements for this unusual miRNA regulation remain poorly understood. Here, we identify eIF4AII, previously implicated in miRNA-mediated repression via 3'UTR sites, as a host factor that is important for HCV replication. We demonstrate that eIF4AII interacts with HCV RNA and that this interaction is miR-122-dependent. We show that effective miR-122 binding to, and regulation of, HCV RNA are reduced following eIF4AII depletion. We find that the previously identified HCV co-factor CNOT1, which has also been implicated in miRNA-mediated repression via 3'UTR sites, contributes to regulation of HCV by eIF4AII. Finally, we show that eIF4AI knockdown alleviates the inhibition of HCV replication mediated by depletion of either eIF4AII or CNOT1. Our results suggest a competition effect between the eIF4A proteins to influence HCV replication by modulation of miR-122 function.

Targeting RNA helicases in cancer: The translation trap.

Cancer cells are reliant on the cellular translational machinery for both global elevation of protein synthesis and the translation of specific mRNAs that promote tumor cell survival. Targeting translational control in cancer is therefore increasingly recognized as a promising therapeutic strategy. In this regard, DEAD/H box RNA helicases are a very interesting group of proteins, with several family members regulating mRNA translation in cancer cells. In this review, we delineate the mechanisms by which DEAD/H box proteins modulate oncogenic translation and how inhibition of these RNA helicases can be exploited for anti-cancer therapeutics.

The RNA helicase, eIF4A-1, is required for ovule development and cell size homeostasis in Arabidopsis.

eIF4A is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of mRNA translation. The Arabidopsis genome encodes two isoforms, one of which (eIF4A-1) is required for the coordination between cell cycle progression and cell size. A T-DNA mutant eif4a1 line, with reduced eIF4A protein levels, displays slow growth, reduced lateral root formation, delayed flowering and abnormal ovule development. Loss of eIF4A-1 reduces the proportion of mitotic cells in the root meristem and perturbs the relationship between cell size and cell cycle progression. Several cell cycle reporter proteins, particularly those expressed at G2/M, have reduced expression in eif4a1 mutant meristems. Single eif4a1 mutants are semisterile and show aberrant ovule growth, whereas double eif4a1 eif4a2 homozygous mutants could not be recovered, indicating that eIF4A function is essential for plant growth and development.

eIF4AII is dispensable for miRNA-mediated gene silencing.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through partial complementary base-pairing to the 3' untranslated region (UTR) of target mRNAs. Inhibition of translation initiation has been identified as an early event of miRNA-mediated gene repression, but the underlying mechanistic details of this process are not well understood. Recently, eukaryotic initiation factor (eIF) 4AII was identified as a critical modulator of miRNA activity with depletion of this factor alleviating miRNA-mediated gene repression. Using the CRISPR/Cas9-editing system, we generated a novel cell line in which expression of eIF4AII was eliminated. The absence of eIF4AII does not affect cell viability, proliferation, or global mRNA translation. Importantly, we show that eIF4AII is dispensable for miRNA-mediated gene silencing.

Transcriptome-wide characterization of the eIF4A signature highlights plasticity in translation regulation.

BACKGROUND: Protein synthesis is tightly regulated and alterations to translation are characteristic of many cancers.Translation regulation is largely exerted at initiation through the eukaryotic translation initiation factor 4 F (eIF4F). eIF4F is pivotal for oncogenic signaling as it integrates mitogenic signals to amplify production of pro-growth and pro-survival factors. Convergence of these signals on eIF4F positions this factor as a gatekeeper of malignant fate. While the oncogenic properties of eIF4F have been characterized, genome-wide evaluation of eIF4F translational output is incomplete yet critical for developing novel translation-targeted therapies. RESULTS: To understand the impact of eIF4F on malignancy, we utilized a genome-wide ribosome profiling approach to identify eIF4F-driven mRNAs in MDA-MB-231 breast cancer cells. Using Silvestrol, a selective eIF4A inhibitor, we identify 284 genes that rely on eIF4A for efficient translation. Our screen confirmed several known eIF4F-dependent genes and identified many unrecognized targets of translation regulation. We show that 5'UTR complexity determines Silvestrol-sensitivity and altering 5'UTR structure modifies translational output. We highlight physiological implications of eIF4A inhibition, providing mechanistic insight into eIF4F pro-oncogenic activity. CONCLUSIONS: Here we describe the transcriptome-wide consequence of eIF4A inhibition in malignant cells, define mRNA features that confer eIF4A dependence, and provide genetic support for Silvestrol's anti-oncogenic properties. Importantly, our results show that eIF4A inhibition alters translation of an mRNA subset distinct from those affected by mTOR-mediated eIF4E inhibition. These results have significant implications for therapeutically targeting translation and underscore a dynamic role for eIF4F in remodeling the proteome toward malignancy.

The diverse roles of the eIF4A family: you are the company you keep.

The eIF4A (eukaryotic initiation factor 4A) proteins belong to the extensive DEAD-box RNA helicase family, the members of which are involved in many aspects of RNA metabolism by virtue of their RNA-binding capacity and ATPase activity. Three eIF4A proteins have been characterized in vertebrates: eIF4A1 and eIF4A2 are cytoplasmic, whereas eIF4A3 is nuclear-localized. Although highly similar, they have been shown to possess rather diverse roles in the mRNA lifecycle. Their specific and diverse functions are often regulated and dictated by interacting partner proteins. The key differences between eIF4A family members are discussed in the present review.

Targeting the eIF4A RNA helicase blocks translation of the MUC1-C oncoprotein.

The oncogenic MUC1 C-terminal subunit (MUC1-C) subunit is aberrantly overexpressed in most human breast cancers by mechanisms that are not well understood. The present studies demonstrate that stimulation of non-malignant MCF-10A cells with epidermal growth factor (EGF) or heregulin (HRG) results in marked upregulation of MUC1-C translation. Growth factor-induced MUC1-C translation was found to be mediated by PI3KAKT, and not by MEKERK1/2, signaling. We also show that activation of the mammalian target of rapamycin complex 1 (mTORC1)ribosomal protein S6 kinase 1 (S6K1) pathway decreases tumor suppressor programmed cell death protein 4 (PDCD4), an inhibitor of the eIF4A RNA helicase, and contributes to the induction of MUC1-C translation. In concert with these results, treatment of growth factor-stimulated MCF-10A cells with the eIF4A RNA helicase inhibitors, silvestrol and CR-1-31-B, blocked increases in MUC1-C abundance. The functional significance of the increase in MUC1-C translation is supported by the demonstration that MUC1-C, in turn, forms complexes with EGF receptor (EGFR) and promotes EGFR-mediated activation of the PI3KAKT pathway and the induction of growth. Compared with MCF-10A cells, constitutive overexpression of MUC1-C in breast cancer cells was unaffected by EGF stimulation, but was blocked by inhibiting PI3KAKT signaling. The overexpression of MUC1-C in breast cancer cells was also inhibited by blocking eIF4A RNA helicase activity with silvestrol and CR-1-31-B. These findings indicate that EGF-induced MUC1-C expression is mediated by the PI3KAKT pathway and the eIF4A RNA helicase, and that this response promotes EGFR signaling in an autoinductive loop. The findings also indicate that targeting the eIF4A RNA helicase is a novel approach for blocking MUC1-C overexpression in breast cancer cells.

Regulation of vertebrate embryogenesis by the exon junction complex core component Eif4a3.

The establishment and maintenance of cellular identity are ultimately dependent upon the accurate regulation of gene expression, the process by which genetic information is used to synthesize functional gene products. The post-transcriptional, pre-translational regulation of RNA constitutes RNA processing, which plays a prominent role in the modulation of gene expression in differentiated animal cells. The multi-protein Exon Junction Complex (EJC) serves as a critical signaling hub within the network that underlies many RNA processing events. Here, we identify a requirement for the EJC during early vertebrate embryogenesis. Knockdown of the EJC component Eukaryotic initiation factor 4a3 (Eif4a3) in embryos of the frog Xenopus laevis results in full-body paralysis, with defects in sensory neuron, pigment cell, and cardiac development; similar phenotypes are seen following knockdown of other "core" EJC protein constituents. Our studies point to an essential role for the EJC in the development of neural plate border derivatives.

A proteomic comparison of immature and mature mouse gonadotrophs reveals novel differentially expressed nuclear proteins that regulate gonadotropin gene transcription and RNA splicing.

The alphaT3-1 and LbetaT2 gonadotroph cell lines contain all the known factors required for expression of gonadotropin genes, yet only the LbetaT2 cells express the beta subunits. We hypothesized that comparison of their nuclear proteomes would reveal novel proteins and/or modifications that regulate expression of these genes. We identified nine proteins with different expression profiles in the two cell lines, of which several were chosen for further functional studies. Of those found at higher levels in alphaT3-1 nuclei, 1110005A23RIK was found associated with the Fshb gene promoter and repressed its expression. Transgelin 3 overexpression reduced transcript levels of Fshb, and its knockdown elevated Lhb and Cga transcript levels, indicating an ongoing repressive effect on these more highly expressed genes, possibly through altering levels of phosphorylated mitogen-activated protein kinase. Heterogeneous nuclear ribonucleoprotein A2/B1 repressed splicing of the Fshb primary transcript, which it binds in the first intron. Proteins at higher levels in LbetaT2 nuclei included prohibitin, the overexpression of which reduced promoter activity of all three gonadotropin subunits, and appeared to mediate the differential effect of GnRH on proliferation of the two cell lines; its knockdown also altered cell morphology. Two other splicing factors were also found at higher levels in LbetaT2 nuclei: the knockdown of PRPF19 or EIF4A3 decreased splicing of Lhb, or of both beta subunit transcripts, respectively. The levels of Eif4a3 mRNA were increased by activin, and both factors increased Fshb splicing. This study has revealed a number of novel factors that alter gonadotropin expression and gonadotroph function, and likely mediate or moderate effects of the regulatory hormones.

The NMD mRNA surveillance pathway downregulates aberrant E-cadherin transcripts in gastric cancer cells and in CDH1 mutation carriers.

Germline mutations in the gene encoding the tumour suppressor E-cadherin (CDH1) are the underlying genetic defect responsible for hereditary diffuse gastric cancer (HDGC). A remarkably high percentage ( approximately 80%) of CDH1 mutations in HDGC patients and carriers generate premature termination codons (PTCs). Here, we examined whether CDH1 transcripts harbouring PTCs are downregulated by nonsense-mediated decay (NMD), an RNA surveillance pathway that degrades PTC-bearing transcripts. Using an allele-specific expression (ASE) assay to differentiate between mutated and wild-type CDH1 alleles, we found that PTC-bearing CDH1 mRNAs are strongly downregulated in normal gastric tissue from several CDH1 mutation carriers. We show that NMD is responsible for this robust downregulation, as CDH1 transcripts harbouring PTCs in the KATO-III gastric tumour cell line were upregulated in response to protein synthesis inhibitors or depletion of the NMD factors UPF1 and eIF4AIII. Analysis of HDGC patients harbouring CDH1 alleles with PTCs at a wide variety of different positions indicates an association of their predicted ability to induce NMD and an earlier age of onset of gastric cancer. This suggests that NMD may be detrimental for HDGC patients and therefore NMD is a potentially useful therapeutic target for CDH1 mutation carriers.

Structural basis for inhibition of translation by the tumor suppressor Pdcd4.

The tumor suppressor function of Programmed Cell Death 4 (Pdcd4) is achieved through interactions between Pdcd4 and components of the translation initiation complex, namely, the RNA helicase eIF4A and the scaffolding protein eIF4G. These interactions are mediated through two MA3 domains on the Pdcd4 molecule and result in inhibition of protein synthesis. We have solved the high-resolution crystal structure of the C-terminal MA3 (cMA3) domain of Pdcd4 in several crystal forms and demonstrated its similarity to the MA3 domain of eIF4G. As predicted by the structure, the cMA3 domain competes with eIF4Gc for binding to eIF4A and surprisingly is sufficient to inhibit translation initiation. Mutations that abolish eIF4A binding negate both functions of the cMA3. Interestingly mutations in the Akt phosphorylation site influenced neither cMA3 binding to eIF4A nor its ability to inhibit translation initiation. Finally, our structural analysis reveals MA3 domains to be a novel subfamily of VHS domains.

Leishmania infantum LeIF protein is an ATP-dependent RNA helicase and an eIF4A-like factor that inhibits translation in yeast.

LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL12-mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His-tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast.

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

The two eIF4A helicases in Trypanosoma brucei are functionally distinct.

Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5' capped mini-exon which is linked to the 3' cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 x 10(5) molecules/cell) and depletion to approximately 10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (approximately 1-2 x 10(4) molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.

Growth and cell survival are unevenly impaired in pixie mutant wing discs.

It is largely unknown how growth slows and then stops in vivo. Similar to most organs, Drosophila imaginal discs undergo a fast, near-exponential growth phase followed by a slow growth phase before final target size is reached. We have used a genetic approach to study the role of an ABC-E protein, Pixie, in wing disc growth. pixie mutants, like mutants in ribosomal proteins genes (known as Minutes), show severe developmental delay with relatively mild alterations in final body size. Intriguingly, pixie mutant wing imaginal discs show complex regional and temporal defects in growth and cell survival that are compensated to result in near-normal final size. In S2 cells, Pixie, like its yeast homolog RLI1, is required for translation. However, a comparison of the growth of eukaryotic translation initiation factor eIF4A and pixie mutant clones in wing discs suggests that only a subset of translation regulators, including pixie, mediate regional differences in growth and cell survival in wing discs. Interestingly, some of the regional effects on pixie mutant clone growth are enhanced in a Minute background. Our results suggest that the role of Pixie is not merely to allow growth, as might be expected for a translation regulator. Instead, Pixie also behaves as a target of putative constraining signals that slow disc growth during late larval life. We propose a model in which a balance of growth inhibitors and promoters determines tissue growth rates and cell survival. An alteration in this balance slows growth before final disc size is reached.

Dynamics and processivity of 40S ribosome scanning on mRNA in yeast.

The eukaryotic 40S ribosomal subunit locates the translation initiation codon on an mRNA via the so-called scanning process that follows 40S binding to the capped 5' end. This key step in translation is required for the expression of almost all eukaryotic genes, yet the mechanism and dynamics of scanning are unknown. We have performed quantitative studies in vivo and in vitro of the movement of yeast 40S ribosomes along 5' untranslated regions (UTRs) of different lengths. 40S subunits perform cap-dependent scanning with high processivity for more than 1700 nucleotides in cells of Saccharomyces cerevisiae. Moreover, the observed rates of expression indicate that scanning is performed by an untethered 40S subunit that has been released from the 5' cap complex. Unexpectedly, the capability to maintain scanning competence on a long 5' UTR is more dependent on the Ded1/Dbp1 type of helicase than on eIF4A or eIF4B. In a yeast cell-free extract, scanning shows reduced processivity, with an estimated net 5'-->3' rate of approximately 10 nucleotides per second at 26 degrees C. We have developed a biased bidirectional walking model of ribosomal scanning that provides a framework for understanding the above observations as well as other known quantitative and qualitative features of this process.