The Viral Protein K7 Inhibits Biochemical Activities and Condensate Formation by the DEAD-box Helicase DDX3X

The DEAD-box RNA helicase DDX3X promotes translation initiation and associates with stress granules. A range of diverse viruses produce proteins that target DDX3X, including hepatitis C, dengue, vaccinia, and influenza A. The interaction of some of these viral proteins with DDX3X has been shown to affect antiviral intracellular signaling, but it is unknown whether and how viral proteins impact the biochemical activities of DDX3X and its physical roles in cells. Here we show that the protein K7 from vaccinia virus, which binds to an intrinsically disordered region in the N-terminus of DDX3X, inhibits RNA helicase and RNA-stimulated ATPase activities, as well as liquid–liquid phase separation of DDX3X in vitro. We demonstrate in HCT 116 cells that K7 inhibits association of DDX3X with stress granules, as well as the formation of aberrant granules induced by expression of DDX3X with a point mutation linked to medulloblastoma and DDX3X syndrome. The results show that targeting of the intrinsically disordered N-terminus is an effective viral strategy to modulate the biochemical functions and subcellular localization of DDX3X. Our findings also have potential therapeutic implications for diseases linked to aberrant DDX3X granule formation.     

The Ded1/DDX3 subfamily of DEAD-box RNA helicases

Abstract

In eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.     

Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F

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Interactions between eIF4AI and its accessory factors eIF4B and eIF4H

Ribonucleoprotein complexes (RNP) remodeling by DEAD-box proteins is required at all stages of cellular RNA metabolism. These proteins are composed of a core helicase domain lacking sequence specificity; flanking protein sequences or accessory proteins target and affect the core's activity. Here we examined the interaction of eukaryotic initiation factor 4AI (eIF4AI), the founding member of the DEAD-box family, with two accessory factors, eIF4B and eIF4H. We find that eIF4AI forms a stable complex with RNA in the presence of AMPPNP and that eIF4B or eIF4H can add to this complex, also dependent on AMPPNP. For both accessory factors, the minimal stable complex with eIF4AI appears to have 1:1 protein stoichiometry. However, because eIF4B and eIF4H share a common binding site on eIF4AI, their interactions are mutually exclusive. The eIF4AI:eIF4B and eIF4AI:eIF4H complexes have the same RNase resistant footprint as does eIF4AI alone (9–10 nucleotides [nt]). In contrast, in a selective RNA binding experiment, eIF4AI in complex with either eIF4B or eIF4H preferentially bound RNAs much longer than those bound by eIF4AI alone (30–33 versus 17 nt, respectively). The differences between the RNase resistant footprints and the preferred RNA binding site sizes are discussed, and a model is proposed in which eIF4B and eIF4H contribute to RNA affinity of the complex through weak interactions not detectable in structural assays. Our findings mirror and expand on recent biochemical and structural data regarding the interaction of eIF4AI's close relative eIF4AIII with its accessory protein MLN51.     

A pollen-specific DEAD-box protein related to translation initiation factor eIF-4A from tobacco

A pollen-specific sequence, NeIF-4A8, has been isolated from a cDNA library from mature pollen of Nicotiana tabacum cv. Samsun. NeIF-4A8 is a full-length cDNA whose deduced amino acid sequence exhibits high homology to the eucaryotic translation initiation factor eIF-4A from mouse, Drosophila and tobacco. eIF-4A is an RNA helicase which belongs to the supergene family of DEAD-box proteins. Northern blot analysis with a gene-specific probe showed strict anther-specific expression of NeIF-4A8 starting at microspore mitosis. With antibodies raised against tobacco eIF-4A the presence of abundant eIF-4A-related proteins in developing anthers and pollen grains was demonstrated. The genomic analysis shows that the coding region is split by three introns whereas a large, fourth intron is situated in the 5-untranslated region. A promoter construct with 2137 bp of upstream sequence fused to the GUS reporter gene was used to confirm that the expression is confined to the haploid cells within the anther. NeIF-4A8 is a prime candidate for mediating translational control in the developing male gametophyte.     

Suppression of translation during in vitro maturation of pig oocytes despite enhanced formation of cap-binding protein complex eIF4F and 4E-BP1 hyperphosphorylation

In this study, we document that the overall rate of protein synthesis decreases during in vitro maturation (IVM) of pig oocytes despite enhanced formation of the 5' cap structure eIF4F. Within somatic/interphase cells, formation of the eIF4F protein complex correlates very well with overall rates of protein translation, and the formation of this complex is controlled primarily by the availability of the 5' cap binding protein eIF4E. We show that the eIF4E inhibitory protein, 4E-BP1, becomes phosphorylated during IVM, which results in gradual release of eIF4E from 4E-BP1, as documented by immunoprecipitation analyses. Isoelectric focusing and Western blotting experiments show conclusively that eIF4E becomes gradually phosphorylated with a maximum at metaphase II (M II). The activity of eIF4E and its ability to bind mRNA also increases during oocyte maturation as documented in experiments with m7-methyl GTP-Sepharose, which mimics the cap structure of mRNA. Complementary analysis of flow-through fraction for 4E-BP1, and eIF4G proteins additionally provides evidence for enhanced formation of cap-binding protein complex eIF4F. Altogether, our results bring new insights to the regulation of translation initiation during meiotic division, and more specifically clarify that 4E-BP1 hyper-phosphorylation is not the cause of the observed suppression of overall translation rates.     

Sumoylation of eIF4E activates mRNA translation

Eukaryotic translation initiation factor 4E (eIF4E) is the cap‐binding protein that binds the 5′ cap structure of cellular messenger RNAs (mRNAs). Despite the obligatory role of eIF4E in cap‐dependent mRNA translation, how the translation activity of eIF4E is controlled remains largely undefined. Here, we report that mammalian eIF4E is regulated by SUMO1 (small ubiquitin‐related modifier 1) conjugation. eIF4E sumoylation promotes the formation of the active eIF4F translation initiation complex and induces the translation of a subset of proteins that are essential for cell proliferation and preventing apoptosis. Furthermore, disruption of eIF4E sumoylation inhibits eIF4E‐dependent protein translation and abrogates the oncogenic and antiapoptotic functions associated with eIF4E. These data indicate that sumoylation is a new fundamental regulatory mechanism of protein synthesis. Our findings suggest further that eIF4E sumoylation might be important in promoting human cancers.     

Modulation of eukaryotic mRNA stability via the cap-binding translation complex eIF4F

Decapping by Dcp1 in Saccharomyces cerevisiae is a key step in mRNA degradation. However, the cap also binds the eukaryotic initiation factor (eIF) complex 4F and its associated proteins. Characterisation of the relationship between decapping and interactions involving eIF4F is an essential step towards understanding polysome disassembly and mRNA decay. Three types of observation suggest how changes in the functional status of eIF4F modulate mRNA stability in vivo. First, partial disruption of the interaction between eIF4E and eIF4G, caused by mutations in eIF4E or the presence of the yeast 4E-binding protein p20, stabilised mRNAs. The interactions of eIF4G and p20 with eIF4E may therefore act to modulate the decapping process. Since we also show that the in vitro decapping rate is not directly affected by the nature of the body of the mRNA, this suggests that changes in eIF4F structure could play a role in triggering decapping during mRNA decay. Second, these effects were seen in the absence of extreme changes in global translation rates in the cell, and are therefore relevant to normal mRNA turnover. Third, a truncated form of eIF4E (Delta196) had a reduced capacity to inhibit Dcp1-mediated decapping in vitro, yet did not change cellular mRNA half-lives. Thus, the accessibility of the cap to Dcp1 in vivo is not simply controlled by competition with eIF4E, but is subject to switching between molecular states with different levels of access.     

Sequential inverse dysregulation of the RNA helicases DDX3X and DDX3Y facilitates MYC-driven lymphomagenesis

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Specific domains in yeast translation initiation factor eIF4G strongly bias RNA unwinding activity of the eIF4F complex toward duplexes with 5'-overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.     

Sexually dimorphic RNA helicases DDX3X and DDX3Y differentially regulate RNA metabolism through phase separation

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The emerging roles of translation factor eIF4E in the nucleus

The emerging field of nuclear eIF research has yielded many surprises and led to the dissolution of some dogmatic/ideological viewpoints of the place of translation in the regulation of gene expression. Eukaryotic initiation factors (eIFs) are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. For instance, in the cytoplasm, the m7G cap-binding protein eIF4E plays a distinct role in cap-dependent translation initiation. Disruption of eIF4E's regulatory function drastically effects cell growth and may lead to oncogenic transformation. A growing number of studies indicate that many eIFs, including a substantial fraction of eIF4E, are found in the nucleus. Indeed, nuclear eIF4E participates in a variety of important RNA-processing events including the nucleocytoplasmic transport of specific, growth regulatory mRNAs. Although unexpected, it is possible that some eIFs regulate protein synthesis within the nucleus. This review will focus on the novel, nuclear functions of eIF4E and how they contribute to eIF4E's growth-activating and oncogenic properties. Both the cytoplasmic and nuclear functions of eIF4E appear to be dependent on its intrinsic ability to bind to the 5' m7G cap of mRNA. For example, Promyelocytic Leukemia Protein (PML) potentially acts as a negative regulator of nuclear eIF4E function by decreasing eIF4E's affinity for the m7G cap. Therefore, eIF4E protein is flexible enough to utilize a common biochemical activity, such as m7G cap binding, to participate in divergent processes in different cellular compartments.     

The h subunit of eIF3 promotes reinitiation competence during translation of mRNAs harboring upstream open reading frames

Upstream open reading frames (uORFs) are protein coding elements in the 5′ leader of messenger RNAs. uORFs generally inhibit translation of the main ORF because ribosomes that perform translation elongation suffer either permanent or conditional loss of reinitiation competence. After conditional loss, reinitiation competence may be regained by, at the minimum, reacquisition of a fresh methionyl-tRNA. The conserved h subunit of Arabidopsis eukaryotic initiation factor 3 (eIF3) mitigates the inhibitory effects of certain uORFs. Here, we define more precisely how this occurs, by combining gene expression data from mutated 5′ leaders of Arabidopsis AtbZip11 (At4g34590) and yeast GCN4 with a computational model of translation initiation in wild-type and eif3h mutant plants. Of the four phylogenetically conserved uORFs in AtbZip11, three are inhibitory to translation, while one is anti-inhibitory. The mutation in eIF3h has no major effect on uORF start codon recognition. Instead, eIF3h supports efficient reinitiation after uORF translation. Modeling suggested that the permanent loss of reinitiation competence during uORF translation occurs at a faster rate in the mutant than in the wild type. Thus, eIF3h ensures that a fraction of uORF-translating ribosomes retain their competence to resume scanning. Experiments using the yeast GCN4 leader provided no evidence that eIF3h fosters tRNA reaquisition. Together, these results attribute a specific molecular function in translation initiation to an individual eIF3 subunit in a multicellular eukaryote.     

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3 D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3 D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1(DDX1), which interacted with FMDV 3 D. The DDX1-3 D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA) in porcine kidney 15(PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-b activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.     

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

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

Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis

There is a desire to engineer mammalian host cell lines to improve cell growth/biomass accumulation and recombinant biopharmaceutical protein production in industrially relevant cell lines such as the CHOK1 and HEK293 cell lines. The over-expression of individual subunits of the eukaryotic translation factor eIF3 in mammalian cells has previously been shown to result in oncogenic properties being imparted on cells, including increased cell proliferation and growth and enhanced global protein synthesis rates. Here we report on the engineering of CHOK1 and HEK cells to over-express the eIF3i and eIF3c subunits of the eIF3 complex and the resultant impact on cell growth and a reporter of exogenous recombinant protein production. Transient over-expression of eIF3i in HEK293 and CHOK1 cells resulted in a modest increase in total eIF3i amounts (maximum 40% increase above control) and an approximate 10% increase in global protein synthesis rates in CHOK1 cells. Stable over-expression of eIF3i in CHOK1 cells was not achievable, most likely due to the already high levels of eIF3i in CHO cells compared to HEK293 cells, but was achieved in HEK293 cells. HEK293 cells engineered to over-express eIF3i had faster growth that was associated with increased c-Myc expression, achieved higher cell biomass and gave enhanced yields of a reporter of recombinant protein production. Whilst CHOK1 cells could not be engineered to over-express eIF3i directly, they could be engineered to over-express eIF3c, which resulted in a subsequent increase in eIF3i amounts and c-Myc expression. The CHOK1 eIF3c engineered cells grew to higher cell numbers and had enhanced cap- and IRES-dependent recombinant protein synthesis. Collectively these data show that engineering of subunits of the eIF3 complex can enhance cell growth and recombinant protein synthesis in mammalian cells in a cell specific manner that has implications for the engineering or selection of fast growing or high producing cells for production of recombinant proteins.     

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G₁/S boundary) or the Cdk1 inhibitor, RO3306 (G₂/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.