Dual action of epidermal growth factor: extracellular signal-stimulated nuclear–cytoplasmic export and coordinated translation of selected messenger RNA

We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear–cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor–bound protein 7), which serves as an adaptor for a specific mRNA–protein export complex and a translational regulator. Using the κ–opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2–containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R–exportin-1 (CRM1) complex to form a nuclear–cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA–protein complex as well as translational activation of the exported mRNA.     

Fragile X mental retardation protein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-binding motif

Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. This region does not contain a motif known to specifically recognize RNA and thus constitutes a new RNA-binding motif. We further demonstrated that FMRP recognition involves the 5' stem loop of BC1 and that this is the region that exhibits complementarity to FMRP target mRNAs, raising the possibility that FMRP plays a direct role in BC1/mRNA annealing.     

Regulation of stress granule dynamics by Grb7 and FAK signalling pathway

Cells form stress granules (SGs) in response to environmental stresses, which constitute cytoplasmic domains where mRNAs are stored and translation is halted. Although several components are found in SGs, it is poorly understood as to how SGs are formed and dissolved. We identified growth factor receptor-bound protein 7 (Grb7), an RNA-binding, translational regulator, as an integral component of SGs, which directly interacts with Hu antigen R (HuR) and is required for cells to form SGs. When stress is terminated, Grb7 is hyperphosphorylated by focal adhesion kinase (FAK), loses its ability to directly interact with HuR and is dissociated from SG components, thereby disrupting SGs in recovering cells. Consistently, dominant-negative hypophospho mutants of FAK and Grb7 significantly attenuate SG disassembly during recovery. FAK activation followed by its phosphorylating Grb7 constitutes a cell-autonomous signalling pathway that regulates the disassembly of SGs and translational stimulation during recovery. This is the first reported pathway actively regulating the dynamics of SGs.     

Fragile X related protein 1 clusters with ribosomes and messenger RNAs at a subset of dendritic spines in the mouse hippocampus

The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.     

Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor

Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.     

Involvement of Xenopus Pumilio in the translational regulation that is specific to cyclin B1 mRNA during oocyte maturation

Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In this study, we investigated the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, in the mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound to cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain but not to its phosphorylated form in mature oocytes. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. We propose that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation.     

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

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

A nuclear protein in Schizosaccharomyces pombe with homology to the human tumour suppressor Fhit has decapping activity

A number of eukaryotic proteins are already known to orchestrate key steps of mRNA metabolism and translation via interactions with the 5' m7GpppN cap. We have characterized a new type of histidine triad (HIT) motif protein (Nhm1) that co-purifies with the cap-binding complex eIF4F of Schizosaccharomyces pombe. Nhm1 is an RNA-binding protein that binds to m7GTP-Sepharose, albeit with lower specificity and affinity for methylated GTP than is typical for the cap-binding protein known as eukaryotic initiation factor 4E. Sequence searches have revealed that proteins with strong sequence similarity over all regions of the new protein exist in a wide range of eukaryotes, yet none has been characterized up to now. However, other proteins that share specific motifs with Nhm1 include the human Fhit tumour suppressor protein and the diadenosine 5', 5"'-P1, P4-tetraphosphate asymmetrical hydrolase of S. pombe. Our experimental work also reveals that Nhm1 inhibits translation in a cell-free extract prepared from S. pombe, and that it is therefore a putative translational modulator. On the other hand, purified Nhm1 manifests mRNA decapping activity, yet is physically distinct from the Saccharomyces cerevisiae decapping enzyme Dcp1. Moreover, fluorescence and immunofluorescence microscopy show that Nhm1 is predominantly, although not exclusively, nuclear. We conclude that Nhm1 has evolved as a special branch of the HIT motif superfamily that has the potential to influence both the metabolism and the translation of mRNA, and that its presence in S. pombe suggests the utilization of a novel decapping pathway.     

Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery

Ki-1/57 is a cytoplasmic and nuclear protein of 57 kDa first identified in malignant cells from Hodgkin's lymphoma. Based on yeast-two hybrid protein interaction we found out that Ki-1/57 interacts with adaptor protein RACK1 (receptor of activated kinase 1), CIRP (cold-inducible RNA-binding protein), RPL38 (ribosomal protein L38) and FXR1 (fragile X mental retardation-related protein 1). Since these proteins are involved in the regulation of translation we suspected that Ki-1/57 may have a role in it. We show by immunoprecipitation the association of Ki-1/57 with FMRP. Confocal microscopy revealed that Ki-1/57 colocalizes with FMRP/FXR1/2 to stress granules. Furthermore Ki-1/57 cosediments with free ribosomal particles and enhances translation, when tethered to a reporter mRNA, suggesting that Ki-1/57 may be involved in translational regulation.     

Degradation of high affinity HuD targets releases Kv1.1 mRNA from miR-129 repression by mTORC1

Little is known about how a neuron undergoes site-specific changes in intrinsic excitability during neuronal activity. We provide evidence for a novel mechanism for mTORC1 kinase–dependent translational regulation of the voltage-gated potassium channel Kv1.1 messenger RNA (mRNA). We identified a microRNA, miR-129, that repressed Kv1.1 mRNA translation when mTORC1 was active. When mTORC1 was inactive, we found that the RNA-binding protein, HuD, bound to Kv1.1 mRNA and promoted its translation. Unexpectedly, inhibition of mTORC1 activity did not alter levels of miR-129 and HuD to favor binding to Kv1.1 mRNA. However, reduced mTORC1 signaling caused the degradation of high affinity HuD target mRNAs, freeing HuD to bind Kv1.1 mRNA. Hence, mTORC1 activity regulation of mRNA stability and high affinity HuD-target mRNA degradation mediates the bidirectional expression of dendritic Kv1.1 ion channels.     

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an ~16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.     

Y-box Binding Protein 1: Providing a New Angle on Translational Regulation

Current models of translational regulation are mostly focused on how translational factors engage a messenger mRNA to the ribosome to initiate translation. Since the majority of mRNAs in eukaryotes are translated in a cap-dependent manner, the mRNA 5’ cap-binding protein eIF4E was characterized as a key player responsible for the recruitment of mRNAs to the initiation complex. The availability of eIF4E is believed to be especially critical for translational activation of mRNAs with extensive secondary structures in their 5’UTRs, many of which code for labile regulatory proteins essential for cell growth or viability. Surprisingly, little attention is paid to the other side of translational control, e.g., to define mechanisms responsible for translational silencing and storage of the above messages. In this review, we discuss the possibility that eIF4E per se may not be sufficient to release mRNAs from translational block. We found that many growth- and stress-related mRNAs are associated with the translational repressor YB-1, which can compete with the eIF4E-driven translation initiation complex for binding to the capped 5’ mRNA terminus. Moreover, the cap-dependent repressor activity of YB-1 appears to be negatively regulated via Akt-mediated phosphorylation of the Ser-102 residue of YB-1. Taken together with recent evidence suggesting that translational activation of growth-related messages is a primary cellular response to activation of Ras-Erk and PI3K-Akt signaling pathways, our data suggest that differential expression of specific mRNA subsets is regulated by the PI3K-Akt pathway and achieved via coordinated activation of the components of translational machinery and inactivation of general translational repressors such as YB-1.     

FUS contributes to mTOR-dependent inhibition of translation

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.