scholarly journals Dicer-Derived MicroRNAs Are Utilized by the Fragile X Mental Retardation Protein for Assembly on Target RNAs

2006 ◽  
Vol 2006 ◽  
pp. 1-12 ◽  
Author(s):  
Isabelle Plante ◽  
Laetitia Davidovic ◽  
Dominique L. Ouellet ◽  
Lise-Andrée Gobeil ◽  
Sandra Tremblay ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Translational Control ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Fragile X ◽  
Rna Sequences ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein ◽  
Rnp Complex

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.

scholarly journals Oligomerization properties of fragile-X mental-retardation protein (FMRP) and the fragile-X-related proteins FXR1P and FXR2P

1999 ◽  
Vol 343 (3) ◽  
pp. 517-523 ◽  
Author(s):  
Filippo TAMANINI ◽  
Leontine VAN UNEN ◽  
Cathy BAKKER ◽  
Nicoletta SACCHI ◽  
Hans GALJAARD ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Mammalian Cells ◽  
Fragile X ◽  
Gel Filtration ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein ◽  
Heterotypic Interactions ◽  
Molecular Masses ◽  
Related Proteins

The absence of fragile-X mental-retardation protein (FMRP) results in fragile-X syndrome. Two other fragile-X-related (FXR) proteins have been described, FXR1P and FXR2P, which are both very similar in amino acid sequence to FMRP. Interaction between the three proteins as well as with themselves has been demonstrated. The FXR proteins are believed to play a role in RNA metabolism. To characterize a possible functional role of the interacting proteins the complex formation of the FXR proteins was studied in mammalian cells. Double immunofluorescence analysis in COS cells over-expressing either FMRP ISO12/FXR1P or FMRP ISO12/FXR2P confirmed heterotypic interactions. However, Western-blotting studies on cellular homogenates containing physiological amounts of the three proteins gave different indications. Gel-filtration experiments under physiological as well as EDTA conditions showed that the FXR proteins were in complexes of > 600 kDa, as parts of messenger ribonuclear protein (mRNP) particles associated with polyribosomes. Salt treatment shifted FMRP, FXR1P and FXR2P into distinct intermediate complexes, with molecular masses between 200 and 300 kDa. Immunoprecipitations of FMRP as well as FXR1P from the dissociated complexes revealed that the vast majority of the FXR proteins do not form heteromeric complexes. Further analysis by [35S]methionine labelling in vivo followed by immunoprecipitation indicated that no proteins other than the FXR proteins were present in these complexes. These results suggest that the FXR proteins form homo-multimers preferentially under physiological conditions in mammalian cells, and might participate in mRNP particles with separate functions.

scholarly journals Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit

Development ◽  
2020 ◽  
Vol 147 (21) ◽  
pp. dev188797
Author(s):  
Xiaoyu Wang ◽  
Ayelet Kohl ◽  
Xiaoyan Yu ◽  
Diego A. R. Zorio ◽  
Avihu Klar ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
Neuronal Cell ◽  
High Specificity ◽  
Fragile X Mental Retardation ◽  
Midline Crossing ◽  
Mental Retardation Protein ◽  
Axonal Targeting

ABSTRACTFragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.

scholarly journals DrosophilaTorsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Phuong Nguyen ◽  
Jong Bok Seo ◽  
Hyo-Min Ahn ◽  
Young Ho Koh
Keyword(s):  
Mental Retardation ◽  
Protein Complexes ◽  
Fragile X ◽  
Stress Sensitivity ◽  
Altered Expression ◽  
Fragile X Mental Retardation ◽  
Evolutionarily Conserved ◽  
Mental Retardation Protein ◽  
Synaptic Boutons

We investigated unknownin vivofunctions of Torsin by usingDrosophilaas a model. Downregulation ofDrosophilaTorsin (DTor) by DTor-specific inhibitory double-stranded RNA (RNAi) induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH). Altered expression of ADH has also been reported inDrosophilaFragile-X mental retardation protein (DFMRP) mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies ofdfmrpnull mutants anddfmrpmutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved.

scholarly journals The RNA-binding fragile-X mental retardation protein and its role beyond the brain

2020 ◽  
Vol 12 (4) ◽  
pp. 903-916 ◽  
Author(s):  
Cassandra Malecki ◽  
Brett D. Hambly ◽  
Richmond W. Jeremy ◽  
Elizabeth N. Robertson
Keyword(s):  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein ◽  
The Brain

scholarly journals Kinase pathway inhibition restores PSD95 induction in neurons lacking fragile X mental retardation protein

2019 ◽  
pp. 201812056
Author(s):  
Ying Yang ◽  
Yang Geng ◽  
Dongyun Jiang ◽  
Lin Ning ◽  
Hyung Joon Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
Alternative Pathways ◽  
Fragile X Mental Retardation ◽  
Mrna Targets ◽  
Mental Retardation Protein ◽  
Pathway Inhibition

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates translation of numerous mRNA targets, some of which are present at synapses. While protein synthesis deficits have long been postulated as an etiology of FXS, how FMRP loss affects distributions of newly synthesized proteins is unknown. Here we investigated the role of FMRP in regulating expression of new copies of the synaptic protein PSD95 in an in vitro model of synaptic plasticity. We find that local BDNF application promotes persistent accumulation of new PSD95 at stimulated synapses and dendrites of cultured neurons, and that this accumulation is absent in FMRP-deficient mouse neurons. New PSD95 accumulation at sites of BDNF stimulation does not require known mechanisms regulating FMRP–mRNA interactions but instead requires the PI3K-mTORC1-S6K1 pathway. Surprisingly, in FMRP-deficient neurons, BDNF induction of new PSD95 accumulation can be restored by mTORC1-S6K1 blockade, suggesting that constitutively high mTORC1-S6K1 activity occludes PSD95 regulation by BDNF and that alternative pathways exist to mediate induction when mTORC1-S6K1 is inhibited. This study provides direct evidence for deficits in local protein synthesis and accumulation of newly synthesized protein in response to local stimulation in FXS, and supports mTORC1-S6K1 pathway inhibition as a potential therapeutic approach for FXS.

scholarly journals Fragile X Mental Retardation Protein FMRP Binds mRNAs in the Nucleus

2008 ◽  
Vol 29 (1) ◽  
pp. 214-228 ◽  
Author(s):  
Miri Kim ◽  
Michel Bellini ◽  
Stephanie Ceman
Keyword(s):  
Mental Retardation ◽  
Nuclear Export ◽  
Rna Binding ◽  
Fragile X ◽  
Simian Virus 40 ◽  
Nuclear Localization Sequence ◽  
Dependent Manner ◽  
Nuclear Export Sequence ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein

ABSTRACT The fragile X mental retardation protein FMRP is an RNA binding protein that associates with a large collection of mRNAs. Since FMRP was previously shown to be a nucleocytoplasmic shuttling protein, we examined the hypothesis that FMRP binds its cargo mRNAs in the nucleus. The enhanced green fluorescent protein-tagged FMRP construct (EGFP-FMRP) expressed in Cos-7 cells was efficiently exported from the nucleus in the absence of its nuclear export sequence and in the presence of a strong nuclear localization sequence (the simian virus 40 [SV40] NLS), suggesting an efficient mechanism for nuclear export. We hypothesized that nuclear FMRP exits the nucleus through its bound mRNAs. Using silencing RNAs to the bulk mRNA exporter Tap/NXF1, we observed a significantly increased number of cells containing EGFP-FMRP in the nucleus, which was further augmented by removal of FMRP's nuclear export sequence. Nuclear-retained SV40-FMRP could be released upon treatment with RNase. Further, Tap/NXF1 coimmunoprecipitated with EGFP-FMRP in an RNA-dependent manner and contained the FMR1 mRNA. To determine whether FMRP binds pre-mRNAs cotranscriptionally, we expressed hemagglutinin-SV40 FMRP in amphibian oocytes and found it, as well as endogenous Xenopus FMRP, on the active transcription units of lampbrush chromosomes. Collectively, our data provide the first lines of evidence that FMRP binds mRNA in the nucleus.

scholarly journals Conformational-Dependent and Independent RNA Binding to the Fragile X Mental Retardation Protein

2011 ◽  
Vol 2011 ◽  
pp. 1-14 ◽  
Author(s):  
Xin Yan ◽  
Robert B. Denman
Keyword(s):  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
In Vitro Transcription ◽  
Rna Transcripts ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein ◽  
Arginine Residues ◽  
Rnase Digestion

The interaction between the fragile X mental retardation protein (FMRP) and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP.

scholarly journals Cells Lacking the Fragile X Mental Retardation Protein (FMRP) have Normal RISC Activity but Exhibit Altered Stress Granule Assembly

2009 ◽  
Vol 20 (1) ◽  
pp. 428-437 ◽  
Author(s):  
Marie-Cécile Didiot ◽  
Murugan Subramanian ◽  
Eric Flatter ◽  
Jean-Louis Mandel ◽  
Hervé Moine
Keyword(s):  
Mental Retardation ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Fragile X ◽  
Stress Granule ◽  
Translation Regulation ◽  
Positive Role ◽  
Granule Formation ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in the mRNA metabolism. The absence of FMRP in neurons leads to alterations of the synaptic plasticity, probably as a result of translation regulation defects. The exact molecular mechanisms by which FMRP plays a role in translation regulation have remained elusive. The finding of an interaction between FMRP and the RNA interference silencing complex (RISC), a master of translation regulation, has suggested that both regulators could be functionally linked. We investigated here this link, and we show that FMRP exhibits little overlap both physically and functionally with the RISC machinery, excluding a direct impact of FMRP on RISC function. Our data indicate that FMRP and RISC are associated to distinct pools of mRNAs. FMRP, unlike RISC machinery, associates with the pool of mRNAs that eventually goes into stress granules upon cellular stress. Furthermore, we show that FMRP plays a positive role in this process as the lack of FMRP or a point mutant causing a severe fragile X alter stress granule formation. Our data support the proposal that FMRP plays a role in controlling the fate of mRNAs after translation arrest.

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