[Show abstract][Hide abstract] ABSTRACT: The Cytoplasmic FMRP Interacting Protein 1 (CYFIP1), also known as Specifically RAC1 Activated Protein 1 (Sra1), plays a dual role: together with FMRP and eIF4E it forms a complex that inhibits mRNA translation; together with WAVE1, NCKAP1, ABI2, and HSPC300 it forms the WAVE-regulatory complex (WRC) that initiates, upon RAC1 activation, actin polymerization. Here, we performed Molecular Dynamics (MD) simulation on CYFIP1 extracted from the known WRC structure and show that, in the absence of its WRC partners, a butterfly-like motion brings the two ends of CYFIP1 closer together, enabling the interaction with eIF4E. Our MD simulation is supported by available data showing that CYFIP1 binding to eIF4E and to WRC is mutually exclusive and that there is a FRET between the N- and C-termini of CYFIP1. Differential interaction of RAC1-GTP with the two CYFIP1 structures predicts that RAC1 is directly responsible for the switch between these conformations.
Journal of Chemical Theory and Computation 04/2015; 11(7). DOI:10.1021/ct500431h · 5.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic by sequestering RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of Amyotrophic Lateral Sclerosis, is still poorly defined. In this work, we set out to characterise these mechanisms by identifying C9orf72 RNA binding proteins. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner FMRP, which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated to a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.
[Show abstract][Hide abstract] ABSTRACT: Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.
[Show abstract][Hide abstract] ABSTRACT: miR-29 is expressed strongly in the brain and alterations in expression have been linked to several neurological disorders. To further explore the function of this miRNA in the brain, we generated miR-29a/b-1 knockout animals. Knockout mice develop a progressive disorder characterized by locomotor impairment and ataxia. The different members of the miR-29 family are strongly expressed in neurons of the olfactory bulb, the hippocampus and in the Purkinje cells of the cerebellum. Morphological analysis showed that Purkinje cells are smaller and display less dendritic arborisation compared to their wildtype littermates. In addition, a decreased number of parallel fibers form synapses on the Purkinje cells. We identified several mRNAs significantly upregulated in the absence of the miR-29a/b-1 cluster. At the protein level, however, the voltage-gated potassium channel Kcnc3 (Kv3.3) was significantly upregulated in the cerebella of the miR-29a/b knockout mice. Dysregulation of KCNC3 expression may contribute to the ataxic phenotype.
Neurobiology of Disease 10/2014; 73. DOI:10.1016/j.nbd.2014.10.006 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In mammalian cells in general and in neurons in particular, mRNA maturation, translation, and degradation are highly complex and dynamic processes. RNA-binding proteins (RBPs) play crucial roles in all these events. First, they participate in the choice of pre-mRNA splice sites and in the selection of the polyadenylation sites, determining which of the possible isoforms is produced from a given precursor mRNA. Then, once in the cytoplasm, the protein composition of the RNP particles determines whether the mature mRNA is transported along the dendrites or the axon of a neuron to the synapses, how efficiently it is translated, and how stable it is. In agreement with their importance for neuronal function, mutations in genes that code for RBPs are associated with various neurological diseases. In this review, we illustrate how individual RBPs determine the fate of an mRNA, and we discuss how mutations in RBPs or perturbations of the mRNA metabolism can cause neurodegenerative disorders.For further resources related to this article, please visit the WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.
[Show abstract][Hide abstract] ABSTRACT: MNDs (motor neuron diseases) form a heterogeneous group of pathologies characterized by the progressive degeneration of motor neurons. More and more genetic factors associated with MND encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of MND. In the present paper we review recent developments showing a functional link between SMN (survival of motor neuron), the causative factor of SMA (spinal muscular atrophy), and FUS (fused in sarcoma), a genetic factor in ALS (amyotrophic lateral sclerosis). SMN is long known to have a crucial role in the biogenesis and localization of the spliceosomal snRNPs (small nuclear ribonucleoproteins), which are essential assembly modules of the splicing machinery. Now we know that FUS interacts with SMN and pathogenic FUS mutations have a significant effect on snRNP localization. Together with other recently published evidence, this finding potentially links ALS pathogenesis to disturbances in the splicing machinery, and implies that pre-mRNA splicing may be the common weak point in MND, although other steps in mRNA metabolism could also play a role. Certainly, further comparison of the RNA metabolism in different MND will greatly help our understanding of the molecular causes of these devastating diseases.
Biochemical Society Transactions 12/2013; 41(6):1593-1597. DOI:10.1042/BST20130142 · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In response to requests, we made available the raw data of the miRNA profiling obtained from the hippocampus of Alzheimer's patients. This will allow data re-analysis of the nCounter experiment shown in the EMM paper.
[Show abstract][Hide abstract] ABSTRACT: We also made available the raw data of the miRNA profiling obtained from the prefrontal cortex of Alzheimer's patients. This will allow data re-analysis of the nCounter experiment shown in the EMM paper.
[Show abstract][Hide abstract] ABSTRACT: The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression.
EMBO Molecular Medicine 10/2013; 5(10):1523-1536. DOI:10.1002/emmm.201302847 · 8.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An overview of miRNAs altered in Alzheimer's disease (AD) was established by profiling the hippocampus of a cohort of 41 late-onset AD (LOAD) patients and 23 controls, showing deregulation of 35 miRNAs. Profiling of miRNAs in the prefrontal cortex of a second independent cohort of 49 patients grouped by Braak stages revealed 41 deregulated miRNAs. We focused on miR-132-3p which is strongly altered in both brain areas. Downregulation of this miRNA occurs already at Braak stages III and IV, before loss of neuron-specific miRNAs. Next-generation sequencing confirmed a strong decrease of miR-132-3p and of three family-related miRNAs encoded by the same miRNA cluster on chromosome 17. Deregulation of miR-132-3p in AD brain appears to occur mainly in neurons displaying Tau hyper-phosphorylation. We provide evidence that miR-132-3p may contribute to disease progression through aberrant regulation of mRNA targets in the Tau network. The transcription factor (TF) FOXO1a appears to be a key target of miR-132-3p in this pathway.
EMBO Molecular Medicine 10/2013; 5(10). DOI:10.1002/emmm.201201974 · 8.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The CYFIP1/SRA1 gene is located in a chromosomal region linked to various neurological disorders, including intellectual disability, autism, and schizophrenia. CYFIP1 plays a dual role in two apparently unrelated processes, inhibiting local protein synthesis and favoring actin remodeling. Here, we show that brain-derived neurotrophic factor (BDNF)-driven synaptic signaling releases CYFIP1 from the translational inhibitory complex, triggering translation of target mRNAs and shifting CYFIP1 into the WAVE regulatory complex. Active Rac1 alters the CYFIP1 conformation, as demonstrated by intramolecular FRET, and is key in changing the equilibrium of the two complexes. CYFIP1 thus orchestrates the two molecular cascades, protein translation and actin polymerization, each of which is necessary for correct spine morphology in neurons. The CYFIP1 interactome reveals many interactors associated with brain disorders, opening new perspectives to define regulatory pathways shared by neurological disabilities characterized by spine dysmorphogenesis.
[Show abstract][Hide abstract] ABSTRACT: The CYFIP1/SRA1 gene is located in a chromosomal region linked to various neurological disorders, including intellectual disability, autism, and schizo-phrenia. CYFIP1 plays a dual role in two apparently unrelated processes, inhibiting local protein synthe-sis and favoring actin remodeling. Here, we show that brain-derived neurotrophic factor (BDNF)-driven synaptic signaling releases CYFIP1 from the transla-tional inhibitory complex, triggering translation of target mRNAs and shifting CYFIP1 into the WAVE regulatory complex. Active Rac1 alters the CYFIP1 conformation, as demonstrated by intramolecular FRET, and is key in changing the equilibrium of the two complexes. CYFIP1 thus orchestrates the two molecular cascades, protein translation and actin polymerization, each of which is necessary for cor-rect spine morphology in neurons. The CYFIP1 inter-actome reveals many interactors associated with brain disorders, opening new perspectives to define regulatory pathways shared by neurological disabil-ities characterized by spine dysmorphogenesis.
[Show abstract][Hide abstract] ABSTRACT: Mutations or deletions of FMRP, involved in the regulation of mRNA metabolism in brain, lead to the Fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. A severe manifestation of the disease has been associated with the Ile304Asn mutation, located on the KH2 domain of the protein. Several hypotheses have been proposed to explain the possible molecular mechanism responsible for the drastic effect of this mutation in humans. Here, we performed a molecular dynamics simulation and show that the Ile304Asn mutation destabilizes the hydrophobic core producing a partial unfolding of two α-helices and a displacement of a third one. The affected regions show increased residue flexibility and motion. Molecular docking analysis revealed strongly reduced binding to a model single-stranded nucleic acid in agreement with known data that the two partially unfolded helices form the RNA-binding surface. The third helix, which we show here to be also affected, is involved in the PAK1 protein interaction. These two functional binding sites on the KH2 domain do not overlap spatially, and therefore, they can simultaneously bind their targets. Since the Ile304Asn mutation affects both binding sites, this may justify the severe clinical manifestation observed in the patient in which both mRNA metabolism activity and cytoskeleton remodeling would be affected.
[Show abstract][Hide abstract] ABSTRACT: Genes encoding RNA-binding proteins have frequently been implicated in various motor neuron diseases, but the particular step in RNA metabolism that is vulnerable in motor neurons remains unknown. FUS, a nuclear protein, forms cytoplasmic aggregates in cells affected by amyotrophic lateral sclerosis (ALS), and mutations disturbing the nuclear import of FUS cause the disease. It is extremely likely that the cytoplasmic aggregates are cytotoxic because they trap important factors; the nature of these factors, however, remains to be elucidated. Here we show that FUS associates in a neuronal cell line with SMN, the causative factor in spinal muscular atrophy (SMA). The two genes work on the same pathway, as FUS binds to spliceosomal snRNPs downstream of the SMN function. Pathogenic FUS mutations do not disturb snRNP binding. Instead, cytoplasmic mislocalisation of FUS causes partial mis-localisation of snRNAs to the cytoplasm, which in turn causes a change in the behaviour of the alternative splicing machinery. FUS, and especially its mutations, thus have a similar effect as SMN1 deletion in SMA, suggesting that motor neurons could indeed be particularly sensitive to changes in alternative splicing.
Neurobiology of Disease 03/2013; 55. DOI:10.1016/j.nbd.2013.03.003 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The brain cytoplasmic RNA, BC1, is a small non-coding RNA that is found in different RNP particles, some of which are involved in translational control. One component of BC1-containing RNP complexes is the fragile X mental retardation protein (FMRP) that is implicated in translational repression. Peptide mapping and computational simulations show that the tudor domain of FMRP makes specific contacts to BC1 RNA. Endogenous BC1 RNA is 2'-O-methylated in nucleotides that contact the FMRP interface, and methylation can affect this interaction. In the cell body BC1 2'-O-methylations are present in both the nucleus and the cytoplasm, but they are virtually absent at synapses where the FMRP-BC1-mRNA complex exerts its function. These results strongly suggest that subcellular region-specific modifications of BC1 affect the binding to FMRP and the interaction with its mRNA targets. We finally show that BC1 RNA has an important role in translation of certain mRNAs associated to FMRP. All together these findings provide further insights into the translational regulation by the FMRP-BC1 complex at synapses.
Nucleic Acids Research 01/2012; 40(9):4086-96. DOI:10.1093/nar/gkr1254 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer, and Amyotrophic Lateral Sclerosis (ALS). In addition, aberrant mRNA splicing has been documented in neurodegeneration. To characterize the cellular response to mitochondrial perturbations at the level of gene expression and alternative pre-mRNA splicing we used splicing-sensitive microarrays to profile human neuroblastoma SH-SY5Y cells treated with paraquat, a neurotoxic herbicide that induces the formation of reactive oxygen species and causes mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant G93A-SOD1 protein, one of the genetic causes of ALS. In both models we identified a common set of genes whose expression and alternative splicing are deregulated. Pathway analysis of the deregulated genes revealed enrichment in genes involved in neuritogenesis, axon growth and guidance, and synaptogenesis. Alterations in transcription and pre-mRNA splicing of candidate genes were confirmed experimentally in the cell line models as well as in brain and spinal cord of transgenic mice carrying the G93A-SOD1 mutation. Our findings expand the realm of the pathways implicated in neurodegeneration and suggest that alterations of axonal function may descend directly from mitochondrial damage.
Human Mutation 02/2011; 32(2):168-82. DOI:10.1002/humu.21394 · 5.14 Impact Factor