Combined transcriptomics and proteomics unveil the impact of vitamin C in modulating specific protein abundance in the mouse liver.

BACKGROUND: Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to the key enzyme gulonolactone oxidase (Gulo) unlike humans. In the current investigation, we used Gulo-/- mice, which cannot synthesize their own ascorbate to determine the impact of this vitamin on both the transcriptomics and proteomics profiles in the whole liver. The study included Gulo-/- mouse groups treated with either sub-optimal or optimal ascorbate concentrations in drinking water. Liver tissues of females and males were collected at the age of four months and divided for transcriptomics and proteomics analysis. Immunoblotting, quantitative RT-PCR, and polysome profiling experiments were also conducted to complement our combined omics studies. RESULTS: Principal component analyses revealed distinctive differences in the mRNA and protein profiles as a function of sex between all the mouse cohorts. Despite such sexual dimorphism, Spearman analyses of transcriptomics data from females and males revealed correlations of hepatic ascorbate levels with transcripts encoding a wide array of biological processes involved in glucose and lipid metabolisms as well as in the acute-phase immune response. Moreover, integration of the proteomics data showed that ascorbate modulates the abundance of various enzymes involved in lipid, xenobiotic, organic acid, acetyl-CoA, and steroid metabolism mainly at the transcriptional level, especially in females. However, several proteins of the mitochondrial complex III significantly correlated with ascorbate concentrations in both males and females unlike their corresponding transcripts. Finally, poly(ribo)some profiling did not reveal significant enrichment difference for these mitochondrial complex III mRNAs between Gulo-/- mice treated with sub-optimal and optimal ascorbate levels. CONCLUSIONS: Thus, the abundance of several subunits of the mitochondrial complex III are regulated by ascorbate at the post-transcriptional levels. Our extensive omics analyses provide a novel resource of altered gene expression patterns at the transcriptional and post-transcriptional levels under ascorbate deficiency.

The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans.

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3'UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.

Localized control of oxidized RNA.

The oxidation of biological molecules by reactive oxygen species (ROS) can render them inactive or toxic. This includes the oxidation of RNA, which appears to underlie the detrimental effects of oxidative stress, aging and certain neurodegenerative diseases. Here, we investigate the management of oxidized RNA in the chloroplast of the green alga Chlamydomonas reinhardtii. Our immunofluorescence microscopy results reveal that oxidized RNA (with 8-hydroxyguanine) is localized in the pyrenoid, a chloroplast microcompartment where CO2 is assimilated by the Calvin cycle enzyme Rubisco. Results of genetic analyses support a requirement for the Rubisco large subunit (RBCL), but not Rubisco, in the management of oxidized RNA. An RBCL pool that can carry out such a 'moonlighting' function is revealed by results of biochemical fractionation experiments. We also show that human (HeLa) cells localize oxidized RNA to cytoplasmic foci that are distinct from stress granules, processing bodies and mitochondria. Our results suggest that the compartmentalization of oxidized RNA management is a general phenomenon and therefore has some fundamental significance.

Control of mRNA turnover: implication of cytoplasmic RNA granules.

The control of mRNA turnover is essential for the cell to rationalize its mRNA content both under physiological conditions and upon stress. Several mechanisms involved in the control of mRNA turnover have been elucidated. These include surveillance mechanisms such as nonsense-mediated decay, non-stop mediated decay and non-go-mediated decay that eliminate aberrant mRNAs, and regulatory mechanisms including AU-mediated decay, GU-mediated decay, and CDE-mediated decay that ensure mRNA plasticity. In general, the mechanisms of RNA decay rely on interactions between specific cis-acting RNA elements and selected RNA-binding proteins that either prevent the degradation of mRNA targets or induce the recruitment of decaying effectors leading to mRNA degradation. Formation of cytoplasmic RNA granules including processing bodies, stress granules, UV granules, and exosome granules have recently emerged as an additional mechanism that control mRNA turnover of selected mRNAs. Here we will review briefly review the main mechanisms that control mRNA decay and highlight possible implication of RNA granules in such mechanisms.

[Role of HRI in apoptosis resistance]. / Rôle de l'heme regulated inhibitor (HRI) dans la résistance à l'apoptose.

When exposed to environmental stresses, cells activate defence mechanisms to adapt stress and inhibit apoptotic pathways leading to their survival. Stressed cells also reduce their general metabolism in part by inhibiting mRNA translation, thereby saving energy needed to repair stress-induced damages. Under stress conditions, the inhibition of mRNA translation occurs mainly at its initiation step through the phosphorylation of the translation initiation factor eIF2α. One of the four kinases known to phosphorylate eIF2α is heme-regulated inhibitor (HRI). The activation of HRI occurs under conditions of heme deficiency, oxidative stress and treatment with anti-cancer drugs such as proteasome inhibitors. In this article, we discuss the role of HRI in promoting cell resistance to stress-mediated apoptosis.

The RNA Demethylases ALKBH5 and FTO Regulate the Translation of ATF4 mRNA in Sorafenib-Treated Hepatocarcinoma Cells.

Translation is one of the main gene expression steps targeted by cellular stress, commonly referred to as translational stress, which includes treatment with anticancer drugs. While translational stress blocks the translation initiation of bulk mRNAs, it nonetheless activates the translation of specific mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, the ATF4 mRNA encodes a transcription factor that reprograms gene expression in cells responding to various stresses. Although the stress-induced translation of the ATF4 mRNA relies on the presence of uORFs (upstream to the main ATF4 ORF), the mechanisms mediating this effect, particularly during chemoresistance, remain elusive. Here, we report that ALKBH5 (AlkB Homolog 5) and FTO (FTO: Fat mass and obesity-associated protein), the two RNA demethylating enzymes, promote the translation of ATF4 mRNA in a transformed liver cell line (Hep3B) treated with the chemotherapeutic drug sorafenib. Using the in vitro luciferase reporter translational assay, we found that depletion of both enzymes reduced the translation of the reporter ATF4 mRNA upon drug treatment. Consistently, depletion of either protein abrogates the loading of the ATF3 mRNA into translating ribosomes as assessed by polyribosome assays coupled to RT-qPCR. Collectively, these results indicate that the ALKBH5 and FTO-mediated translation of the ATF4 mRNA is regulated at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into an ATF4 mRNA reporter renders its translation insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. In summary, our data show that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating the translation of ATF4 mRNA.

The Identification of Nuclear FMRP Isoform Iso6 Partners.

A deficiency of FMRP, a canonical RNA-binding protein, causes the development of Fragile X Syndrome (FXS), which is characterised by multiple phenotypes, including neurodevelopmental disorders, intellectual disability, and autism. Due to the alternative splicing of the encoding FMR1 gene, multiple FMRP isoforms are produced consisting of full-length predominantly cytoplasmic (i.e., iso1) isoforms involved in translation and truncated nuclear (i.e., iso6) isoforms with orphan functions. However, we recently implicated nuclear FMRP isoforms in DNA damage response, showing that they negatively regulate the accumulation of anaphase DNA genomic instability bridges. This finding provided evidence that the cytoplasmic and nuclear functions of FMRP are uncoupled played by respective cytoplasmic and nuclear isoforms, potentially involving specific interactions. While interaction partners of cytoplasmic FMRP have been reported, the identity of nuclear FMRP isoform partners remains to be established. Using affinity purification coupled with mass spectrometry, we mapped the nuclear interactome of the FMRP isoform iso6 in U2OS. In doing so, we found FMRP nuclear interaction partners to be involved in RNA processing, pre-mRNA splicing, ribosome biogenesis, DNA replication and damage response, chromatin remodeling and chromosome segregation. By comparing interactions between nuclear iso6 and cytoplasmic iso1, we report a set of partners that bind specifically to the nuclear isoforms, mainly proteins involved in DNA-associated processes and proteasomal proteins, which is consistent with our finding that proteasome targets the nuclear FMRP iso6. The specific interactions with the nuclear isoform 6 are regulated by replication stress, while those with the cytoplasmic isoform 1 are largely insensitive to such stress, further supporting a specific role of nuclear isoforms in DNA damage response induced by replicative stress, potentially regulated by the proteasome.

Prostate cancer resistance leads to a global deregulation of translation factors and unconventional translation.

Emerging evidence associates translation factors and regulators to tumorigenesis. However, our understanding of translational changes in cancer resistance is still limited. Here, we generated an enzalutamide-resistant prostate cancer (PCa) model, which recapitulated key features of clinical enzalutamide-resistant PCa. Using this model and poly(ribo)some profiling, we investigated global translation changes that occur during acquisition of PCa resistance. We found that enzalutamide-resistant cells exhibit an overall decrease in mRNA translation with a specific deregulation in the abundance of proteins involved in mitochondrial processes and in translational regulation. However, several mRNAs escape this translational downregulation and are nonetheless bound to heavy polysomes in enzalutamide-resistant cells suggesting active translation. Moreover, expressing these corresponding genes in enzalutamide-sensitive cells promotes resistance to enzalutamide treatment. We also found increased association of long non-coding RNAs (lncRNAs) with heavy polysomes in enzalutamide-resistant cells, suggesting that some lncRNAs are actively translated during enzalutamide resistance. Consistent with these findings, expressing the predicted coding sequences of known lncRNAs JPX, CRNDE and LINC00467 in enzalutamide-sensitive cells drove resistance to enzalutamide. Taken together, this suggests that aberrant translation of specific mRNAs and lncRNAs is a strong indicator of PCa enzalutamide resistance, which points towards novel therapeutic avenues that may target enzalutamide-resistant PCa.

TDRD3, a novel Tudor domain-containing protein, localizes to cytoplasmic stress granules.

Our previous work has demonstrated that the Tudor domain of the 'survival of motor neuron' protein and the Tudor domain-containing protein 3 (TDRD3) are highly similar and that they both have the ability to interact with arginine-methylated polypeptides. TDRD3 has been identified among genes whose overexpression has a strong predictive value for poor prognosis of estrogen receptor-negative breast cancers, although its precise function remains unknown. TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain. We report here that TDRD3 localizes predominantly to the cytoplasm, where it co-sediments with the fragile X mental retardation protein on actively translating polyribosomes. We also demonstrate that TDRD3 accumulates into stress granules (SGs) in response to various cellular stresses. Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process. Pull down experiments identified five novel TDRD3 interacting partners, most of which are potentially methylated RNA-binding proteins. Our findings revealed that two of these proteins, SERPINE1 mRNA-binding protein 1 and DEAD/H box-3 (a gene often deleted in Sertoli-cell-only syndrome), are also novel constituents of cytoplasmic SGs. Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.

The chemotherapeutic agent bortezomib induces the formation of stress granules.

BACKGROUND: Cytoplasmic stress granules (SGs) are specialized storage sites of untranslated mRNAs whose formation occurs under different stress conditions and is often associated with cell survival. SGs-inducing stresses include radiations, hypoxia, viral infections, and chemical inhibitors of specific translation initiation factors. The FDA-approved drug bortezomib (Velcade(R)) is a peptide boronate inhibitor of the 26S proteasome that is very efficient for the treatment of myelomas and other hematological tumors. Solid tumors are largely refractory to bortezomib. In the present study, we investigated the formation of SGs following bortezomib treatment. RESULTS: We show that bortezomib efficiently induces the formation of SGs in cancer cells. This process involves the phosphorylation of translation initiation factor eIF2alpha by heme-regulated inhibitor kinase (HRI). Depletion of HRI prevents bortezomib-induced formation of SGs and promotes apoptosis. CONCLUSIONS: This is the first study describing the formation of SGs by a chemotherapeutic compound. We speculate that the activation of HRI and the formation of SGs might constitute a mechanism by which cancer cells resist bortezomib-mediated apoptosis.

The RNA-binding protein HuR promotes cell migration and cell invasion by stabilizing the beta-actin mRNA in a U-rich-element-dependent manner.

A high expression level of the beta-actin protein is required for important biological mechanisms, such as maintaining cell shape, growth, and motility. Although the elevated cellular level of the beta-actin protein is directly linked to the long half-life of its mRNA, the molecular mechanisms responsible for this effect are unknown. Here we show that the RNA-binding protein HuR stabilizes the beta-actin mRNA by associating with a uridine-rich element within its 3' untranslated region. Using RNA interference to knock down the expression of HuR in HeLa cells, we demonstrate that HuR plays an important role in the stabilization but not in the nuclear/cytoplasmic distribution of the beta-actin mRNA. HuR depletion in HeLa cells alters key beta-actin-based cytoskeleton functions, such as cell adhesion, migration, and invasion, and these defects correlate with a loss of the actin stress fiber network. Together our data establish that the posttranscriptional event involving HuR-mediated beta-actin mRNA stabilization could be a part of the regulatory mechanisms responsible for maintaining cell integrity, which is a prerequisite for avoiding transformation and tumor formation.

Inhibition of the ubiquitin-proteasome system induces stress granule formation.

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2alpha by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2alpha phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21(cip1) mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.

Treatment of cancer cells with Lapatinib negatively regulates general translation and induces stress granules formation.

Stress granules (SG) are cytoplasmic RNA granules that form during various types of stress known to inhibit general translation, including oxidative stress, hypoxia, endoplasmic reticulum stress (ER), ionizing radiations or viral infection. Induction of these SG promotes cell survival in part through sequestration of proapoptotic molecules, resulting in the inactivation of cell death pathways. SG also form in cancer cells, but studies investigating their formation upon treatment with chemotherapeutics are very limited. Here we identified Lapatinib (Tykerb / Tyverb®), a tyrosine kinase inhibitor used for the treatment of breast cancers as a new inducer of SG in breast cancer cells. Lapatinib-induced SG formation correlates with the inhibition of general translation initiation which involves the phosphorylation of the translation initiation factor eIF2α through the kinase PERK. Disrupting PERK-SG formation by PERK depletion experiments sensitizes resistant breast cancer cells to Lapatinib. This study further supports the assumption that treatment with anticancer drugs activates the SG pathway, which may constitute an intrinsic stress response used by cancer cells to resist treatment.

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.

Combined transcriptomics and proteomics unveil the impact of vitamin C in modulating specific protein abundance in the mouse liver

Background Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to the key enzyme gulonolactone oxidase (Gulo) unlike humans. In the current investigation, we used Gulo-/- mice, which cannot synthesize their own ascorbate to determine the impact of this vitamin on both the transcriptomics and proteomics profiles in the whole liver. The study included Gulo-/- mouse groups treated with either sub-optimal or optimal ascorbate concentrations in drinking water. Liver tissues of females and males were collected at the age of four months and divided for transcriptomics and proteomics analysis. Immunoblotting, quantitative RT-PCR, and polysome profiling experiments were also conducted to complement our combined omics studies. Results Principal component analyses revealed distinctive differences in the mRNA and protein profiles as a function of sex between all the mouse cohorts. Despite such sexual dimorphism, Spearman analyses of transcriptomics data from females and males revealed correlations of hepatic ascorbate levels with transcripts encoding a wide array of biological processes involved in glucose and lipid metabolisms as well as in the acute-phase immune response. Moreover, integration of the proteomics data showed that ascorbate modulates the abundance of various enzymes involved in lipid, xenobiotic, organic acid, acetyl-CoA, and steroid metabolism mainly at the transcriptional level, especially in females. However, several proteins of the mitochondrial complex III significantly correlated with ascorbate concentrations in both males and females unlike their corresponding transcripts. Finally, poly(ribo)some profiling did not reveal significant enrichment difference for these mitochondrial complex III mRNAs between Gulo-/- mice treated with sub-optimal and optimal ascorbate levels. Conclusions Thus, the abundance of several subunits of the mitochondrial complex III are regulated by ascorbate at the post-transcriptional levels. Our extensive omics analyses provide a novel resource of altered gene expression patterns at the transcriptional and post-transcriptional levels under ascorbate deficiency.

PRMT7 methylates eukaryotic translation initiation factor 2α and regulates its role in stress granule formation.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that modify proteins by methylating the guanidino nitrogen atoms of arginine residues to regulate cellular processes such as chromatin remodeling, pre-mRNA splicing, and signal transduction. PRMT7 is the single type III PRMT solely capable of arginine monomethylation. To date, other than histone proteins, there are very few identified substrates of PRMT7. We therefore performed quantitative mass spectrometry experiments to identify PRMT7's interactome and potential substrates to better characterize the enzyme's biological function(s) in cells. These experiments revealed that PRMT7 interacts with and can methylate eukaryotic translation initiation factor 2 alpha (eIF2α), in vitro and in breast cancer cells. Furthermore, we uncovered a potential regulatory interplay between eIF2α arginine methylation by PRMT7 and stress-induced phosphorylation status of eIF2α at serine 51. Finally, we demonstrated that PRMT7 is required for eIF2α-dependent stress granule formation in the face of various cellular stresses. Altogether, our findings implicate PRMT7 as a novel mediator of eIF2α-dependent cellular stress response pathways.

NF-kappa B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release.

Muscle wasting (cachexia) is a consequence of chronic diseases, such as cancer, and is associated with degradation of muscle proteins such as MyoD. The cytokines tumor necrosis factor alpha and gamma interferon induce muscle degeneration by activating the transcription factor NF-kappaB and its target genes. Here, we show that a downstream target of NF-kappaB is the nitric oxide (NO) synthase gene (iNos) and suggest that NO production stimulates MyoD mRNA loss. In fact, although cytokine treatment of iNos(-/-) mice activated NF-kappaB, it did not trigger MyoD mRNA degeneration, demonstrating that NF-kappaB-mediated muscle wasting requires an active iNOS-NO pathway. The induced expression of iNOS by cytokines relies on both transcriptional activation via NF-kappaB and increased mRNA stability via the RNA-binding protein HuR. Moreover, we show that HuR regulates iNOS expression in an AMP-activated protein kinase (AMPK)-dependent manner. Furthermore, AMPK activation results in HuR nuclear sequestration, inhibition of iNOS synthesis, and reduction in cytokine-induced MyoD loss. These results define iNOS and HuR as critical players in cytokine-induced cachexia, establishing them as potential therapeutic targets.

The RNA-binding protein fragile X-related 1 regulates somite formation in Xenopus laevis.

Fragile X-related 1 protein (FXR1P) is a member of a small family of RNA-binding proteins that includes the Fragile X mental retardation 1 protein (FMR1P) and the Fragile X-related 2 protein (FXR2P). These proteins are thought to transport mRNA and to control their translation. While FMR1P is highly expressed in neurons, substantial levels of FXR1P are found in striated muscles and heart, which are devoid of FMRP and FXR2P. However, little is known about the functions of FXR1P. We have isolated cDNAs for Xenopus Fxr1 and found that two specific splice variants are conserved in evolution. Knockdown of xFxr1p in Xenopus had highly muscle-specific effects, normal MyoD expression being disrupted, somitic myotomal cell rotation and segmentation being inhibited, and dermatome formation being abnormal. Consistent with the absence of the long muscle-specific xFxr1p isoform during early somite formation, these effects could be rescued by both the long and short mRNA variants. Microarray analyses showed that xFxr1p depletion affected the expression of 129 known genes of which 50% were implicated in muscle and nervous system formation. These studies shed significant new light on Fxr1p function(s).

DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression.

Accumulation of unfolded and potentially toxic proteins in the endoplasmic reticulum (ER) activates a cell stress adaptive response, which involves a reprogramming of general gene expression. ATF4 is a master stress-induced transcription factor that orchestrates gene expression in cells treated with various ER stress inducers including those used to treat cancers. ER stress-induced ATF4 expression occurs mainly at the translational level involving the activity of the phosphorylated (P) translation initiation factor (eIF) eIF2α. While it is well established that under ER stress PeIF2α drives ATF4 expression through a specialised mode of translation re-initiation, factors (e.g. RNA-binding proteins and specific eIFs) involved in PeIF2α-mediated ATF4 translation remain unknown. Here we identified the RNA-binding protein named DDX3 as a promotor of ATF4 expression in cancer cells treated with sorafenib, an ER stress inducer used as a chemotherapeutic. Depletion experiments showed that DDX3 is required for PeIF2α-mediated ATF4 expression. Luciferase and polyribosomes assays showed that DDX3 drives ER stress-induced ATF4 mRNA expression at the translational level. Protein-interaction assays showed that DDX3 binds the eIF4F complex, which we found to be required for ER stress-induced ATF4 expression. This study thus showed that PeIF2α-mediated ATF4 mRNA translation requires DDX3 as a part of the eIF4F complex.

The fragile X mental retardation protein has nucleic acid chaperone properties.

The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA-RNA interactions and thus on the structural status of mRNAs.