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In brief RNA granules travel through the cumulus cell network of transzonal projections which is associated with oocyte developmental competence, and RNA packaging involves RNA-binding proteins of the Fragile X protein family. Abstract The determinants of oocyte developmental competence have puzzled scientists for decades. It is known that follicular conditions can nurture the production of a high-quality oocyte, but the underlying mechanisms remain unknown. Somatic cumulus cells most proximal to the oocyte are known to have cellular extensions that reach across the zona pellucida and contact with the oocyte plasma membrane. Herein, it was found that transzonal projections (TZPs) network quality is associated with developmental competence. Knowing that ribonucleoparticles are abundant within TZPs, the distribution of RNA-binding proteins was studied. The Fragile X-related proteins (FXR1P and FXR2P) and two partnering protein families, namely cytoplasmic FMRP-interacting protein and nuclear FMRP-interacting protein, exhibited distinctive patterns consistent with roles in regulating mRNA packaging, transport, and translation. The expression of green fluorescent protein (GFP)–FMRP fusion protein in cumulus cells showed active granule formation and their transport and transfer through filipodia connecting with neighboring cells. Near the projections’ ends was found the cytoskeletal anchoring protein Filamin A and active protein synthesis sites. This study highlights key proteins involved in delivering mRNA to the oocyte. Thus, cumulus cells appear to indeed support the development of high-quality oocytes via the transzonal network.

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.

Fragile X syndrome is the most common form of inherited mental retardation affecting approximately 1/7000 females and 1/4000 males worldwide. The syndrome is due to the silencing/dysfuntion of a single gene, the FRAGILE X MENTAL RETARDATION 1 gene, and affected patients display a variety of physical and mental abnormalities. In the last 15 years, research advances in the fragile X syndrome field have lead to a better understanding of its molecular basis as well as of the pleiotropic phenotypic effects caused by the absence of the fragile X mental retardation protein. Keywords: mental retardation; fragile X; neuron; RNA translation; RNA trafficking

Objectives The Fragile X Mental Retardation Protein (FMRP) is a widely expressed RNA-binding protein involved in translation regulation. Since the absence of FMRP leads to Fragile X Syndrome (FXS) and autism, FMRP has been extensively studied in brain. The functions of FMRP in peripheral organs and on metabolic homeostasis remain elusive; therefore, we sought to investigate the systemic consequences of its absence. Methods Using metabolomics, in vivo metabolic phenotyping of the Fmr1-KO FXS mouse model and in vitro approaches, we show that the absence of FMRP induced a metabolic shift towards enhanced glucose tolerance and insulin sensitivity, reduced adiposity, and increased β-adrenergic-driven lipolysis and lipid utilization. Results Combining proteomics and cellular assays, we highlight that FMRP loss increased hepatic protein synthesis and impacted pathways notably linked to lipid metabolism. Mapping metabolomic and proteomic phenotypes onto a signaling and metabolic network, we predicted that the coordinated metabolic response to FMRP loss was mediated by dysregulation in the abundances of specific hepatic proteins. We experimentally validated these predictions, demonstrating that the translational regulator FMRP associates with a subset of mRNAs involved in lipid metabolism. Finally, we highlight that FXS patients mirror metabolic variations observed in Fmr1-KO mice with reduced circulating glucose and insulin and increased free fatty acids. Conclusions Loss of FMRP results in a widespread coordinated systemic response that notably involves upregulation of protein translation in the liver, increased utilization of lipids, and significant changes in metabolic homeostasis. Our study unravels metabolic phenotypes in FXS and further supports the importance of translational regulation in the homeostatic control of systemic metabolism., Graphical abstract Image 1, Highlights • Loss of the translational regulator FMRP impacts glucose and lipid homeostasis in mouse and human. • FMR1-deficiency modifies blood metabolic markers. • Loss of FMRP enhances the insulin response and lipolysis. • Loss of FMRP exaggerates hepatic protein synthesis. • FMRP controls the translation of key hepatic proteins involved in lipid metabolism.

// Pauline Adjibade 1, 2, 3 , Valerie Grenier St-Sauveur 1, 2, 3 , Miguel Quevillon Huberdeau 1, 2, 3 , Marie-Josee Fournier 1, 2, 3 , Andreanne Savard 1, 2, 3 , Laetitia Coudert 1, 2, 3 , Edouard W. Khandjian 4, 5 , Rachid Mazroui 1, 2, 3 1 Centre de Recherche du toCHU de Quebec, Universite Laval, Quebec, PQ, Canada 2 Departement de Biologie Moleculaire, Biochimie Medicale et Pathologie, Faculte de Medecine, Universite Laval, Quebec, PQ, Canada 3 Centre de Recherche en Cancerologie de l’Universite Laval, Universite Laval, Quebec, PQ, Canada 4 Centre de Recherche, Institut Universitaire en Sante Mentale de Quebec, Universite Laval, Quebec, PQ, Canada 5 Departement de Psychiatrie et de Neurosciences, Faculte de Medecine, Universite Laval, Quebec, PQ, Canada Correspondence to: Rachid Mazroui, e-mail: rachid.mazroui@crsfa.ulaval.ca Keywords: stress granules, sorafenib, PERK, eIF2a, ATF4 Received: July 13, 2015 Accepted: October 04, 2015 Published: November 02, 2015 ABSTRACT Stress granules (SGs) are cytoplasmic RNA multimeric bodies that form under stress conditions known to inhibit translation initiation. In most reported stress cases, the formation of SGs was associated with the cell recovery from stress and survival. In cells derived from cancer, SGs formation was shown to promote resistance to either proteasome inhibitors or 5-Fluorouracil used as chemotherapeutic agents. Despite these studies, the induction of SGs by chemotherapeutic drugs contributing to cancer cells resistance is still understudied. Here we identified sorafenib, a tyrosine kinase inhibitor used to treat hepatocarcinoma, as a potent chemotherapeutic inducer of SGs. The formation of SGs in sorafenib-treated hepatocarcionoma cells correlates with inhibition of translation initiation; both events requiring the phosphorylation of the translation initiation factor eIF2α. Further characterisation of the mechanism of sorafenib-induced SGs revealed PERK as the main eIF2α kinase responsible for SGs formation. Depletion experiments support the implication of PERK-eIF2α-SGs pathway in hepatocarcinoma cells resistance to sorafenib. This study also suggests the existence of an unexpected complex regulatory balance between SGs and phospho-eIF2α where SGs dampen the activation of the phospho-eIF2α-downstream ATF4 cell death pathway.

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.

So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.

SMN1, the causative gene for spinal muscular atrophy (SMA), plays a housekeeping role in the biogenesis of small nuclear RNA ribonucleoproteins. SMN is also present in granular foci along axonal projections of motoneurons, which are the predominant cell type affected in the pathology. These so-called RNA granules mediate the transport of specific mRNAs along neurites and regulate mRNA localization, stability, as well as local translation. Recent work has provided evidence suggesting that SMN may participate in the assembly of RNA granules, but beyond that, the precise nature of its role within these structures remains unclear. Here, we demonstrate that SMN associates with polyribosomes and can repress translation in an in vitro translation system. We further identify the arginine methyltransferase CARM1 as an mRNA that is regulated at the translational level by SMN and find that CARM1 is abnormally up-regulated in spinal cord tissue from SMA mice and in severe type I SMA patient cells. We have previously characterized a novel regulatory pathway in motoneurons involving the SMN-interacting RNA-binding protein HuD and CARM1. Thus, our results suggest the existence of a potential negative feedback loop in this pathway. Importantly, an SMA-causing mutation in the Tudor domain of SMN completely abolished translational repression, a strong indication for the functional significance of this novel SMN activity in the pathology.

RNA-binding proteins (RBPs) are key components in RNA metabolism, regulating the temporal, spatial and functional dynamics of RNAs. Altering the expression of RBPs has profound implications for cellular physiology, affecting RNA processes from pre-mRNA splicing to protein translation. Recent genetic and proteomic data and evidence from animal models reveal that RBPs are involved in many human diseases ranging from neurologic disorders to cancer. Here we review the emerging evidence showing the involvement of RBPs in many disease networks and conclude that defects in RNA metabolism caused by aberrations in RBPs might underlie a broader spectrum of complex human disorders.

Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome., Author Summary Fragile X syndrome is the most common form of inherited mental retardation affecting approximately 1 female out of 7000 and 1 male out of 4000 worldwide. The syndrome is due to the silencing of a single gene, the Fragile Mental Retardation 1 (FMR1), that codes for the Fragile X mental retardation protein (FMRP). This protein is highly expressed in brain and controls local protein synthesis essential for neuronal development and maturation as well as the formation of neural circuits. Several studies suggest a role for FMRP in the regulation of mRNA transport along axons and dendrites to distant synaptic locations in structures called RNA granules. Here we report the isolation of a particular subpopulation of these structures and the analysis of their architecture and composition in terms of RNA and protein. Also, using time-lapse video microscopy, we monitored granule transport and fusion throughout neuronal processes. These findings open new avenues for the study of RNA transport dysfunctions in animal models of nervous system disorders.

Fragile X syndrome is caused by the absence of the fragile X mental retardation protein (FMRP). This RNA-binding protein is widely expressed in human and mouse tissues, and it is particularly abundant in the brain because of its high expression in neurons, where it localizes in the cell body and in granules throughout dendrites. Although FMRP is thought to regulate trafficking of repressed mRNA complexes and to influence local protein synthesis in synapses, it is not known whether it has additional functions in the control of translation in the cell body. Here, we have used recently developed approaches to investigate whether FMRP is associated with the translation apparatus. We demonstrate that, in the brain, FMRP is present in actively translating polyribosomes, and we show that this association is acutely sensitive to the type of detergent required to release polyribosomes from membranous structures. In addition, proteomic analyses of purified brain polyribosomes reveal the presence of several RNA-binding proteins that, similarly to FMRP, have been previously localized in neuronal granules. Our findings highlight the complex roles of FMRP both in actively translating polyribosomes and in repressed trafficking ribonucleoparticle granules.

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.

This study determined: (1) whether dopamine (DA) D2-receptor (R) and D1-R mRNA levels in the carotid body (CB), petrosal ganglion (PG) and superior cervical ganglion (SCG) are modulated by hypoxia; (2) the role of hypoxia intensity and exposure duration, and (3) whether the pattern of modulation differs between newborn (1-day-old) and adult rabbits. Rabbits were exposed to five FiO2 conditions: 21% (control); 15%/6 h; 15%/24 h; 8%/6 h and 8%/24 h. D2- and D1-R mRNA expression levels were calculated for each hypoxia condition relative to control using RT-PCR analysis. In the CB of 1-day-old rabbits, D2- and D1-R transcript levels increased and decreased after exposure to 15 and 8% O2, respectively. In the adult CB, D2- and D1-R transcript levels decreased independently of hypoxia intensities. Only changes in D1-R mRNA levels were dependent on exposure time. In the PG, both hypoxia intensities decreased the D2-R transcript levels in 1-day-old and adult rabbits; but for the D1-R mRNA levels, hypoxia decreased its level in 1-day-old rabbits and increased it in adults regardless of exposure duration. In the SCG, hypoxia had no determinant effect on the D2-R mRNA levels either in newborn or adult rabbits while it induced a decrease of D1-R transcript levels in 1-day-old rabbits and an increase in adult rabbits. It is concluded that hypoxia affects DA D2- or D1-R mRNA levels in the chemoreflex pathway in an age-dependent and site-specific manner.

Stress granules (SGs) are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies) and are bone fide SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G1 phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs.

Fragile X mental retardation 1 protein (FMRP) is the archetype of a class of cytoplasmic mRNA-binding proteins that includes the fragile X-related 1 and 2 proteins (FXR1P and FXR2P). Whereas absence of FMRP is the cause of fragile X syndrome, it is not known if FXR1P and FXR2P are associated with any pathology. It is also still elusive whether these homologous proteins can partially compensate for the absence of FMRP in the case of the fragile X syndrome. FXR1 is widely expressed in mammals and its expression pattern is complex since several mRNA variants and protein isoforms are detected. In mouse, we observed that the highest level of FXR1 is found in the adult testis. This tissue is an exception, since all known FXR1P isoforms, some of which have been considered as tissue specific, are detected in it. In young animals, changes in mRNA-spliced variants and their corresponding protein isoforms occur during spermatogenesis. Using biochemical, immunohistochemical and electron microscopic techniques, we show that FXR1P is associated with microtubule elements. Since the cytoskeletal framework is implicated in cellular plasticity as well as in mRNA transport, we propose new possibilities for the function(s) of the FXR proteins.

Transforming infection of Go/G1-arrested primary mouse kidney cell cultures with simian virus 40 (SV40) induces cells to re-enter the S-phase of the cell cycle. In Go-arrested cells, no p53 is detected, whereas in cells induced to proliferate by infection, a gradual accumulation of p53 complexed to SV40 large T-antigen is observed in the nucleus. Heat treatment of actively proliferating SV40-infected cells leads to inhibition of DNA synthesis and growth arrest. To determine the fate of p53 after heat treatment, proliferating infected cells were exposed to mild heat (42.5°C) for increasing lengths of time. The results presented here show that after ninety minutes of treatment, the arrest of DNA synthesis by heat correlates with the disruption of the p53/LT-antigen complex. Longer treatments induce, in addition, a reduction in the solubility of p53, which was recovered tightly associated with the nuclear fraction. This contrasted with large T-antigen, whose solubility remained unaffected by heat treatment. Although the total amount of p53 in the nucleus remained constant, as shown by immunoblot analyses, p53 was no longer detectable after immunoprecipitation or by immunofluorescent staining techniques. These results suggest that heat treatment had either induced conformational changes in its antigenic sites, or had sequestered the sites through aggregation or binding to insoluble nuclear components.Key words: p53, heat shock, LT-antigen/p53 complex, S-phase.

The fragile X syndrome, an X-linked disease, is the most frequent cause of inherited mental retardation. The syndrome results from the absence of expression of the FMR1 gene (fragile mental retardation 1) owing to the expansion of a CGG trinucleotide repeat located in the 5prime untranslated region of the gene and the subsequent methylation of its CpG island. The FMR1 gene product (FMRP) is a cytoplasmic protein that contains two KH domains and one RGG box, characteristics of RNA-binding proteins. FMRP is associated with mRNP complexes containing poly(A)+mRNA within actively translating polyribosomes and contains nuclear localization and export signals making it a putative transporter (chaperone) of mRNA from the nucleus to the cytoplasm. FMRP is the archetype of a novel family of cytoplasmic RNA-binding proteins that includes FXR1P and FXR2P. Both of these proteins are very similar in overall structure to FMRP and are also associated with cytoplasmic mRNPs. Members of the FMR family are widely expressed in mouse and human tissues, albeit at various levels, and seem to play a subtle choreography of expression. FMRP is most abundant in neurons and is absent in muscle. FXR1P is strongly expressed in muscle and low levels are detected in neurons. The complex expression patterns of the FMR1 gene family in different cells and tissues suggest that independent, however similar, functions for each of the three FMR-related proteins might be expected in the selection and metabolism of tissue-specific classes of mRNA. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated as well as the putative role(s) of FXR1P and FXR2P as compensatory molecules.Key words: RNA-binding proteins, polyribosomes, messenger ribonucleoprotein, messenger ribonucleoparticles, nucleocytoplasmic trafficking, mental retardation.

The fragile X syndrome results from transcriptional silencing of the FMR1 gene and the absence of its encoded FMRP protein. Two autosomal homologues of the FMR1 gene, FXR1 and FXR2, have been identified and the overall structures of the corresponding proteins are very similar to that of FMRP. Using antibodies raised against FXR1P, we observed that two major protein isoforms of relative MW of 78 and 70 kDa are expressed in different mammalian cell lines and in the majority of mouse tissues. In mammalian cells grown in culture as well as in brain extracts, both P78 and P70 isoforms are associated with mRNPs within translating polyribosomes, similarly to their closely related FMRP homologues. In muscle tissues as well as in murine myoblastic cell lines induced to differentiate into myotubes, FXR1P78 and P70 isoforms are replaced by novel unpredicted isoforms of 81‐84 kDa and a novel FXR1 exon splice variant was detected in muscle RNA. While P81‐84 isoforms expressed after fusion into myotubes in murine myoblast cell lines grown in culture are associated with polyribosomes, this is not the case when isolated from muscle tissues since they sediment with lower S values. Immunohistochemical studies showed coexpression of FMRP and FXR1P70 and P78 in the cytoplasm of brain neurons, while in muscle no FMRP was detected and FXR1P81‐84 were mainly localized to structures within the muscle contractile bands. The complex expression pattern of FXR1P suggests tissue-specific expression for the various isoforms of FXR1 and the differential expression of FMRP and FXR1Ps suggests that in certain types of cells and tissues, complementary functions may be fulfilled by the various FMRP family members.

Dopamine is a major neurotransmitter in the carotid body of several animal species and its functional role at the level of peripheral arterial chemoreflex pathway is attributed to the presence of the dopamine D2-receptors. We present evidence that the dopamine D1-receptor mRNA is also expressed in the carotid body of adult rabbits, cats and rats. A DNA fragment of 611 bp of this receptor was first isolated from rabbit. The nucleic acid sequence of this fragment was found to be 84.5% identical to that of rat. This specific 611 bp fragment was used as a probe to detect, either by Northern analysis or by the reverse transcription-polymerase chain reaction, the dopamine D1-receptor mRNA. The results revealed the presence of dopamine D1-receptor transcript in the carotid body as well as in the petrosal ganglion and the superior cervical ganglion from the three animal models studied here. The physiological significance of dopamine D1-receptor expression in the carotid body is discussed.

Fragile X mental retardation syndrome is associated with an expansion of a CGG repeat within the 5'UTR of the first exon of the FMR1 gene, abnormal methylation of the CpG island in the promoter region, and a transcriptional silencing of this gene. We studied transcriptional regulation of the FMR1 gene using protein footprint analysis of the active and inactive gene in vivo . We identified four footprints within the FMR1 promoter region which correspond to consensus binding sites of known transcription factors, alpha-PAL/NRF1, Sp1, H4TF1/Sp1-like and c-myc. These footprints were present in normal cells with a transcriptionally active FMR1 gene. The same footprints were present in different cell types: primary fibroblasts, lymphoblastoid cells and peripheral lymphocytes. However, for the 1.1 kb region analyzed, no footprints were detected in a variety of cell types derived from patients with fragile X syndrome which have a transcriptionally inactive FMR1 gene. A BLAST nucleotide search identified sequence similarities between the region of the FMR1 gene containing the footprints and an analogous region within the promoter region of the gene for the heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a member of a family of ribonucleoproteins implicated in mRNA processing and nuclear-cytoplasm transport. The nucleotide sequences identified in the hnRNP-A2 promoter region correspond to the same consensus binding sites showing DNA-protein interactions in the FMR1 gene. Our previous functional studies and the studies of others demonstrate that FMR proteins, like hnRNP-A2, are also ribonucleoproteins which appear to be involved in mRNA transport. The results from our footprint studies suggest that the expression of the FMR1 gene is regulated by the binding of specific transcription factors to sequence elements in the 5' region of the gene and that this expression may be regulated by elements in common with the hnRNP-A2 gene. Common regulation of these two genes might play an important role in the cooperative processing and transport of mRNA from the nucleus to the translation machinery.

The Fragile X Mental Retardation protein (FMRP) is an RNA-binding protein and its absence leads to the Fragile X syndrome, the most common form of inherited mental retardation. Because it has been acknowledged for a long time that FMRP is associated with polyribosomal mRNPs in all non-neuronal cellular systems studied so far, it is thought that it regulates translation in neurons also; however, its exact function remains elusive. Recently, it has been reported that, contrary to non-neuronal cells, brain FMRP is not associated with the translation machinery, but is part of repressed small RNP complexes excluded from polyribosomes.(27) To elucidate this puzzling result, Stefani et al.(17) and Khandjian et al.(32) have optimized methods to analyze brain polyribosomes and now provide definitive evidence for the association of FMRP with brain polyribosomes. In addition, the data presented in these two reports clearly indicate that FMRP's function resides at the translation control level.

The two isoforms of dopamine D2 receptor, D2 short (D2s) and D2 long, are generated by alternative splicing of premessenger RNA and differ in the length of their third cytoplasmic loop. Expression of these two isoforms has not yet been studied at the level of the peripheral arterial chemoreflex pathway. Using reverse transcriptase-polymerase chain reaction, we evaluated the relative abundance of dopamine D2 receptor mRNA by amplifying a common segment between both D2 receptor mRNA isoforms which reflects the total D2-receptor mRNA and the two isoforms in the carotid bodies, the petrosal ganglia, and the superior cervical ganglia of 1-, 10-, and 25-day-old and adult rabbits, GH4C1 cell line, which does not express D2 receptors, was used as negative control, whereas GH4C1 19 cell line, which expresses only the D2s mRNA and the striatum of each age, were used as positive controls. Both D2-receptor mRNA isoforms were found to be expressed in all organs studied of newborn and adult rabbits. The expression of total D2 receptor mRNA in 10- and 25-day-old and adult rabbits was found to be 3-, 5-, and 1.5-fold more, respectively, compared with 1-day-old rabbits for all organs studied. Our data suggest that the expression of D2-receptor mRNA is modulated with age at the level of the peripheral arterial chemoreflex pathway.

Gene expression involves multiple steps from the transcription of a mRNA in the nucleus to the production of the encoded protein in the cytoplasm. This final step occurs through a highly regulated process of mRNA translation on ribosomes that is required to maintain cell homeostasis. Alterations in the control of mRNA translation may lead to cell transformation, a hallmark of cancer development. Indeed, recent advances indicated that increased translation of mRNAs encoding tumor-promoting proteins may be a key mechanism of tumor resistance in several cancers. Moreover, it was found that proteins whose encoding mRNAs are translated at higher efficiencies may be effective biomarkers. Evaluation of global changes in translation efficiency in human tumors has thus the potential of better understanding what can be used as biomarkers and therapeutic targets. Investigating changes in translation efficiency in human cancer cells has been made possible through the development and the use of the polyribosome profiling combined with DNA microarray or deep RNA sequencing. While helpful, the use of cancer cell lines has many limitations and it is essential to define translational changes in human tumor samples in order to properly prioritize genes implicated in cancer phenotype. We present an optimized polyribosome RNA-Seq protocol suitable for quantitative analysis of mRNA translation that occurs in human tumor samples and murine xenografts. Applying this innovative approach to human tumors, which requires a complementary bioinformatics analysis, unlocks the potential to identify key mRNAs which are preferentially translated in tumor tissue compared to benign tissue as well as translational changes which occur following treatment. These technical advances will be of interest to those researching all solid tumors, opening possibilities for understanding what may be therapeutic Achilles heels’ or relevant biomarkers.

The fragile X syndrome is an X-linked inherited disease and is the result of transcriptional inactivation of the FMR1 gene and the absence of its encoded FMR protein (FMRP). Using a specific monoclonal antibody directed against human FMRP, we have studied the steady-state levels of its murine homolog in several tissues and organs of adult and young mice. In immunoblot analyses, the antibody recognizes a heterogeneous subset of proteins with apparent molecular weights ranging from 80 to 70 kDa. These proteins are detected in all the 27 tissues tested; however, the relative proportion of each polypeptide recognized varies between tissues, and a significantly higher expression is observed in young animals. Northern blot analysis of RNA extracted from selected tissues from adult mouse shows that these tissues express the major 4.8 kb mRNA, although at different levels, and contain several additional shorter transcripts, particularly in muscular tissues. We also report that expression of the FMR1 gene is modulated in proliferating and quiescent primary mouse kidney cell cultures with an inverse relationship between levels of FMR1 mRNA and of its encoded proteins. This suggests that FMRPs are highly stable in quiescent cells and that FMR1 expression is likely post-transcriptionally controlled. Our results document the widespread expression of the FMR1 gene, and suggest that it is controlled by different mechanisms implicated in cell growth and differentiation.

There was a typographical error in the title and citation. The correct title is, "Fragile X Mental Retardation Protein Interacts with the RNA-Binding Protein Caprin1 in Neuronal RiboNucleoProtein Complexes" and the correct citation is: El Fatimy R, Tremblay S, Dury AY, Solomon S, De Koninck P, et al. (2012) Fragile X Mental Retardation Protein Interacts with the RNA-Binding Protein Caprin1 in Neuronal RiboNucleoProtein Complexes. PLoS ONE 7(6): e39338. doi:10.1371/journal.pone.0039338

Fragile X syndrome is caused by the absence of the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein. FMRP is associated with messenger RiboNucleoParticles (mRNPs) present in polyribosomes and its absence in neurons leads to alteration in synaptic plasticity as a result of translation regulation defects. The molecular mechanisms by which FMRP plays a role in translation regulation remain elusive. Using immunoprecipitation approaches with monoclonal Ab7G1-1 and a new generation of chicken antibodies, we identified Caprin1 as a novel FMRP-cellular partner. In vivo and in vitro evidence show that Caprin1 interacts with FMRP at the level of the translation machinery as well as in trafficking neuronal granules. As an RNA-binding protein, Caprin1 has in common with FMRP at least two RNA targets that have been identified as CaMKIIα and Map1b mRNAs. In view of the new concept that FMRP species bind to RNA regardless of known structural motifs, we propose that protein interactors might modulate FMRP functions.

The frog is a model of choice to study gene function during early development, since a large number of eggs are easily obtained and rapidly develop external to the mother. This makes it a highly flexible model system in which direct tests of gene function can be investigated by microinjecting RNA antisense reagents. Two members of the Fragile X Related (FXR) gene family, namely xFmr1 and xFxr1 have been identified in Xenopus. While the tissue distribution of their products was found to be identical to that in mammals, the pattern of isoform expression is less complex. Translational silencing of the xFmr1 and xFxr1 mRNAs by microinjection of antisense morpholino oligonucleotides (MO) induced dramatic morphological alterations, revealing tissue-specific requirements for each protein during development and in maintaining the steady state levels of a range of transcripts in these tissues. The power and versatility of the frog model is that the MO-induced phenotypes can be rescued by microinjection of the corresponding MO-insensitive mRNAs. Most importantly, this animal model allows one rapidly to determine whether any member of the FXR family can compensate for the absence of another, an approach that cannot be performed in other animal models.

The frog is a model of choice to study gene function during early development, since a large number of eggs are easily obtained and rapidly develop external to the mother. This makes it a highly flexible model system in which direct tests of gene function can be investigated by microinjecting RNA antisense reagents. Two members of the Fragile X Related (FXR) gene family, namely xFmr1 and xFxr1 have been identified in Xenopus. While the tissue distribution of their products was found to be identical to that in mammals, the pattern of isoform expression is less complex. Translational silencing of the xFmr1 and xFxr1 mRNAs by microinjection of antisense morpholino oligonucleotides (MO) induced dramatic morphological alterations, revealing tissue-specific requirements for each protein during development and in maintaining the steady state levels of a range of transcripts in these tissues. The power and versatility of the frog model is that the MO-induced phenotypes can be rescued by microinjection of the corresponding MO-insensitive mRNAs. Most importantly, this animal model allows one rapidly to determine whether any member of the FXR family can compensate for the absence of another, an approach that cannot be performed in other animal models.

In neurons, the presence of mRNAs, together with the translation machinery at presynaptic terminals or postsynaptic dendritic spines, enables an extrasomatic activity-dependent protein synthesis. The mechanisms of transport, targeting, and release of these mRNAs are beginning to be unveiled, and involve granule-like motile macromolecular ribonucleoparticles termed RNA granules. These complexes contain repressed mRNAs packed together with RNA-binding proteins and ribosomes and travel along microtubules toward synaptic terminals. Upon stimulation, granules are unfolded, allowing local protein synthesis to take place. The presence of the translation apparatus at pre- and postsynaptic subdomains allows rapid delivery of proteins necessary for neuronal development and synaptic plasticity.

In Iacoangeli et al. (1), five independent groups report that results published by Zalfa et al. (2) are not reproducible. Bagni now suggests (3) that different reagents, antibodies, or procedures might explain this lack of reproducibility. Iacoangeli et al. replicated the experimental conditions reported by Zalfa et al. whenever possible, as indicated. In several cases, however, reagents used by Zalfa et al. were not available. For instance, Zalfa et al. generated antibody rAM1 and used it to probe BC1–FMRP interactions in supershift and other assays. The antibody did not produce a supershift in brain extracts—as one would have expected if BC1 RNA did in fact bind to FMRP in vivo—but abolished the regular mobility shift (2). Despite repeated requests by several of the undersigned, antibody rAM1 was not provided. We urge the Bagni group (2) to make antibody rAM1 available for independent examination. In contrast, Iacoangeli et al. used two established anti-FMRP antibodies that have been independently validated and are publicly available. Bagni suggests that two other groups (4, 5) have published data in support of her claims. Both articles were quoted and discussed by Iacoangeli et al. (1). One of the undersigned (E.W.K.) coauthored one of these articles (4) and has confirmed that, although BC1 RNA does bind to FMRP in vitro, this binding is entirely nonspecific because it is completely abrogated by competitor tRNA (1). Bagni does not mention that Gabus et al. (4) reported a Kd of FMRP for tRNA of 25 nM, the same as for BC1 RNA. The second article (5) reported a weak BC1 RT-PCR signal in FMRP cross-linked immunoprecipitates and a stronger signal in MAP2 cross-linked immunoprecipitates. However, considering that MAP2 is not a known RNA binding protein, mere copurification cannot be taken as evidence for physical association (1). Thus, there is no confirmation, independent of the Bagni group, of a specific physical link between FMRP and BC1 RNA, as posited by Zalfa et al. (2). Similarly, the Zalfa et al. claim that FMRP is not associated with polyribosomes in neurons could not be confirmed in subsequent work (6, 7).

Fragile X mental retardation 1 protein (FMRP) is an RNA-binding protein whose absence results in the fragile X syndrome, the most common inherited form of mental retardation. FMRP contains multiple domains with apparently differential affinity to mRNA and interacts also with protein partners present in ribonucleoprotein complexes called RNA granules. In neurons, these particles travel along dendrites and axons to translocate mRNAs to specific destinations in spines and growth cones, where local synthesis of neuro-specific proteins is taking place. However, the molecular mechanisms of how RNA granules are translocated to dendrites remained unknown. We report here the identification and characterization of the motor protein KIF3C as a novel FMRP-interacting protein. In addition, using time-lapse videomicroscopy, we studied the dynamics and kinetics of FMRP-containing RNA granules in dendrites and show that a KIF3C dominant-negative impedes their distal transport. We therefore propose that, in addition to modulate the translation of its mRNA targets, FMRP acts also as a molecular adaptor between RNA granules and the neurospecific kinesin KIF3C that powers their transport along neuronal microtubules.

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of expression of the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein with high specificity for G-quartet RNA structure. FMRP is involved in several steps of mRNA metabolism: nucleocytoplasmic trafficking, translational control and transport along dendrites in neurons. Fragile X Related Protein 1 (FXR1P), a homologue and interactor of FMRP, has been postulated to have a function similar to FMRP, leading to the hypothesis that it can compensate for the absence of FMRP in Fragile X patients. Here we analyze the ability of three isoforms of FXR1P, expressed in different tissues, to bind G-quartet RNA structure specifically. Only the longest FXR1P isoform was found to be able to bind specifically the G-quartet RNA, albeit with a lower affinity as compared to FMRP, whereas the other two isoforms negatively regulate the affinity of FMRP for G-quartet RNA. This result is important to decipher the molecular basis of fragile X syndrome, through the understanding of FMRP action in the context of its multimolecular complex in different tissues. In addition, we show that the action of FXR1P is synergistic rather than compensatory for FMRP function.

Fragile X syndrome (FXS) – the leading cause of inherited mental retardation – is an X-linked disease caused by loss of expression of the FMR1 (fragile X mental retardation 1) gene. In addition to impairment of higher-cognitive functions, FXS patients show a variety of physical and other mental abnormalities. FMRP, the protein encoded by the FMR1 gene, is thought to play a key role in translation, trafficking and targeting of mRNA in neurons. To better understand FMRP's functions, the protein partners and mRNA targets that interact with FMRP have been sought. These and functional studies have revealed links with processes such as cytoskeleton remodelling via the RhoGTPase pathway and mRNA processing via the RNA interference pathway. In this review, we focus on recent insights into the function of FMRP and speculate on how the absence of FMRP might cause the clinical phenotypes seen in FXS patients. Finally, we explore potential therapies for FXS.

The fragile X syndrome, the leading cause of inherited mental retardation, is due to the inactivation of the fragile mental retardation 1 gene (FMR1) and the subsequent absence of its gene product FMRP. This RNA-binding protein is thought to control mRNA translation and its absence in fragile X cells leads to alteration in protein synthesis. In neurons, FMRP is thought to repress specific mRNAs during their transport as silent ribonucleoparticles (mRNPs) from the cell body to the distant synapses which are the sites of local synthesis of neuro-specific proteins. The mechanism by which FMRP sorts out its different mRNAs targets might be tuned by the intervention of different proteins. Using a yeast two-hybrid system, we identified MicroSpherule Protein 58 (MSP58) as a novel FMRP-cellular partner. In cell cultures, we found that MSP58 is predominantly present in the nucleus where it interacts with the nuclear isoform of FMRP. However, in neurons but not in glial cells, MSP58 is also present in the cytoplasmic compartment, as well as in neurites, where it co-localizes with FMRP. Biochemical evidence is given that MSP58 is associated with polyribosomal poly(A)+ mRNPs. We also show that MSP58, similar to FMRP, is present on polyribosomes prepared from synaptoneurosomes and that it behaves as an RNA-binding protein with a high affinity to the G-quartet structure. We propose that this novel cellular partner for FMRP escorts FMRP-containing mRNP from the nucleus and nucleolus to the somato-dendritic compartment where it might participate in neuronal translation regulation.

In mammalian cells, fragile X mental retardation protein (FMRP) has been reported to be part of a microRNA (miRNA)-containing effector ribonucleoprotien (RNP) complex believed to mediate translational control of specific mRNAs. Here, using recombinant proteins, we demonstrate that human FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences. The miRNA assembler property of FMRP was abrogated upon deletion of its single-stranded (ss) RNA binding K-homology domains. The requirement of FMRP for efficient RNA interference (RNAi) in vivo was unveiled by reporter gene silencing assays using various small RNA inducers, which also supports its involvement in an ss small interfering RNA (siRNA)-containing RNP (siRNP) effector complex in mammalian cells. Our results define a possible role for FMRP in RNA silencing and may provide further insight into the molecular defects in patients with the fragile X syndrome.

Using the Reverse transcription-Polymerase chain reaction, we detected dopamine D2 receptor mRNA short and long isoforms in the adult carotid body of rats, cats, and rabbits. For these animals, the relative short/long ratios were 0.60, 0.65 and 0.57, respectively. Our results suggest that the variety of dopamine effects on carotid chemoreceptor activity, that has been related to species differences, may not be dependent on the expression levels of the dopamine D2 receptor mRNA isoforms in the studied species.

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).

RNA chaperones are ubiquitous proteins that play pivotal roles in cellular RNA metabolism and RNA virus replication. Here we propose that they act by organizing complex and highly dynamic networks of RNA-RNA, RNA-protein and protein-protein interactions. How this is achieved and how their malfunction may lead to disease will be discussed through the examples of human immunodeficiency virus type 1 nucleocapsid protein (NCp7), the fragile X mental retardation protein and the prion protein.

Tumor necrosis factor (TNF) is regulated post-transcriptionally by the AU-rich element (ARE) within the 3′-untranslated region of its mRNA. This regulation modulates translational efficacy and mRNA stability. By using a cRNA probe containing the TNF ARE sequence, we screened a macrophage protein expression library and identified FXR1P. Macrophages that we generated from FXR1 knock-out mice had enhanced TNF protein production compared with wild type macrophages following activation. Expression of several other proteins that are regulated by ARE sequences was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3′-untranslated region of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. Finally, we found that the ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes demonstrating the important role of FXR1P in the post-transcriptional regulation of TNF expression. Our data suggest that release of this repression by FXR1P occurs during lipopolysaccharide-induced macrophage activation. Finally, complementation of the knock-out macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation.

Fragile X Mental Retardation protein (FMRP) is an RNA-binding protein that contains multiple domains with apparently differential affinity to mRNA and to the ribonucleotide homopolymer poly(G). Attempts have been made to map the RNA-binding sites along the protein sequence with a view to determining which of the KH1, KH2 and RGG domains are required to recognize and bind to RNA. While these studies have greatly contributed to the delineation of domains that bind homopolymers or mRNA in vitro, little is known concerning their implications in FMRP function(s) in vivo. To address this question, we have prepared a series of FMRP versions, in which each known in vitro functional domain has been individually deleted, leaving the rest of the protein intact. Constructs with deletions in the protein-protein interaction and RNA-binding as well as in the phosphorylation domains were expressed in STEK-KO cells lacking FMRP and their recruitment into polyribosomal mRNPs and their intra-cellular localization were determined. Our results indicate that the KH RNA-binding domains and the Protein-Protein Interacting domain are essential for FMRP to associate with polyribosomal mRNPs, while the RGG box and the phosphorylated domains are dispensable.