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FMRP deficiency leads to multifactorial dysregulation of splicing and mislocalization of MBNL1 to the cytoplasm

ABSTRACT: Fragile X syndrome (FXS) is a neurodevelopmental disorder that is often modeled in Fmr1 knockout mice where the RNA-binding protein FMRP is absent. Here, we show that in Fmr1-deficient mice, RNA mis-splicing occurs in several brain regions and peripheral tissues. To assess molecular mechanisms of splicing mis-regulation, we employed N2A cells depleted of Fmr1. In the absence of FMRP, RNA-specific exon skipping events are linked to the splicing factors hnRNPF, PTBP1, and MBNL1. FMRP regulates the translation of Mbnl1 mRNA as well as Mbnl1 RNA auto-splicing. Elevated Mbnl1 auto-splicing in FMRP-deficient cells results in the loss of a nuclear localization signal (NLS)-containing exon. This in turn alters the nucleus-to-cytoplasm ratio of MBNL1. This redistribution of MBNL1 isoforms in Fmr1-deficient cells could result in downstream splicing changes in other RNAs. Indeed, further investigation revealed that splicing disruptions resulting from Fmr1 depletion could be rescued by overexpression of nuclear MBNL1. Altered Mbnl1 auto-splicing also occurs in human FXS postmortem brain. These data suggest that FMRP-controlled translation and RNA processing may cascade into a general dys-regulation of splicing in Fmr1-deficient cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE207145 | GEO | 2023/11/03

REPOSITORIES: GEO

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Antisense Oligonucleotide Rescue of CGG Expansion-Dependent FMR1 Mis-Splicing in Fragile X Syndrome Restores FMRP

Project description:Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissue of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1 expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1-217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissue. We show that in cells aberrantly expressing mis-spliced FMR1, antisense oligonucleotide (ASO) treatment reduces FMR1-217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2′-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1-217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that mis-regulated RNA processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217, ASO treatment may offer a new therapeutic approach to mitigate the disorder.

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Project description:Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Here we describe the establishment of an isogenic human pluripotent embryonic stem cell model of FXS. Using CRISPR/Cas9 to introduce indels in exon 3 of FMR1 and result in complete loss of FMRP (FMR1KO). We show that FMRP-deficient neurons exhibit a number of phenotypic abnormalities including neurite outgrowth and branching deficits and impaired electrophysiological network activity as measured by multi-electrode arrays. RNA-Seq analysis of FMRP-deficient neurons revealed transcriptional dysregulation in pathways related to neurodevelopment, neurotransmission, and the cell cycle

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Integrative analysis identifies key molecular signatures underlying neurodevelopmental deficits in fragile X syndrome

Project description:Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Here we describe the establishment of an isogenic human pluripotent embryonic stem cell model of FXS. Using CRISPR/Cas9 to introduce indels in exon 3 of FMR1 and result in complete loss of FMRP (FMR1KO). We show that FMRP-deficient neurons exhibit a number of phenotypic abnormalities including neurite outgrowth and branching deficits and impaired electrophysiological network activity as measured by multi-electrode arrays. RNA-Seq and proteome analysis of FMRP-deficient neurons revealed transcriptional dysregulation in pathways related to neurodevelopment, neurotransmission, and the cell cycle.

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Role of FMRP in neurogenesis

Project description:Fragile X syndrome (FXS) is a rare disease but is the most common form of inherited intellectual disability and a leading cause of autism. FXS is due to the absence of the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein mainly involved in translational control. Even if this molecular defect is known, no specific therapy is available for FXS. The first alteration observed in the brain of FXS patients and of Fmr1 KO mice, model for FXS, is represented by an abnormal dendritic morphology that is associated with an altered synaptic plasticity. These findings led to numerous studies focused on mature neurons. However, recently, an increasing body of evidence is pointing out the importance of FMRP in the early steps of brain development. Thus, with the purpose to decipher the earliest molecular events leading to FXS we developed a stem-cell-based disease model by knocking-down the expression of Fmr1 in mouse embryonic stem (ES) cells. To gain insights in the pathways that are affected when FMRP is repressed, we performed a gene expression profile analysis comparing total RNA from shFmr1 and shControl ES cells using whole genome mouse microarrays. Transcripts were clustered according to their Gene Ontology classification using the DAVID software. Surprisingly, the most altered functional category was Nervous system development and function, highlighting the role of FMRP also during the earliest steps of neural development. To study the precise role of FMRP in Embryonic stem (ES) cells, we used an RNAi-based loss-of-function strategy by transducing mouse ES cells with a lentivirus expressing GFP and a shRNA targeting the constitutive exon 1 of Fmr1 (shFmr1). A random shRNA (shCT) was used as a control sequence. Transduced cells were sorted by flow cytometry and established as non-clonal cell populations. RNA samples were harvested and profiling experiments were performed in â??dye-balanceâ?? as indicated for each replicate.

2016-07-04 | E-GEOD-71184 | biostudies-arrayexpress

FMR1 targets distinct mRNA sequence elements to regulate protein expression [Affymetrix]

Project description:Fragile-X Syndrome (FXS) is a multi-organ disease leading to mental retardation, macro-orchidism in males, and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASD). FXS is typically caused by the loss of FRAGILE X-MENTAL RETARDATION 1 (FMR1) expression, which encodes for the RNA-binding protein (RBP), FMR1 (or FMRP). We report the discovery of the RNA recognition elements (RREs), binding sites, and mRNA targets for wild-type and I304N mutant FMRP isoforms as well as its paralogs, FXR1 and FXR2. RRE frequency, ratio, and distribution determine target mRNA association with FMRP. Among highly-enriched targets, we identified many genes involved in ASD and demonstrate that FMRP can affect their protein levels in cell culture, mice, and human brain. Unexpectedly, we discovered that these targets are also dysregulated in Fmr1-/- mouse ovaries, showing signs of premature follicular overdevelopment. These results indicate that FMRP targets shared signaling pathways across different cellular contexts. As it is become increasingly appreciated that signaling pathways are important to FXS and ASD, our results here provide an invaluable molecular guide towards the pursuit of novel therapeutic targets for these devastating neurological disorders. The mRNA profile of RNA recovered from FLAG-antibody immunoprecipitated FMRP was compared to the mRNA profile of the starting lysate material.

2012-12-13 | E-GEOD-39504 | biostudies-arrayexpress

FMR1 targets distinct mRNA sequence elements to regulate protein expression [PAR-CLIP]

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Project description:Fragile X syndrome (FXS), the leading cause of inherited form of mental retardation and autism, is caused by the transcriptional silencing of fmr1 encoding the protein of FMRP. FMRP, acting as an RNA binding protein, FMRP is a wide expressed protein, but primarily in the brain and testis and can regulate approximately 4% of transcripts. Macroorchidism is one of the common symptoms observed both in the FXS people and mice. Thus, we analyzed the protein profiles of cerebral cortex, hippocampus and testis from both the fmr1-KO and WT mouse using a quantitative proteomics. Proteins (FMRP, RS8, RL23A and MPZ) identified by MS/MS were also verified by Western blot. Among the identified proteins, most of the significant changed proteins were downregulated in the FMRP absence. The Gene Ontology and pathway analysis revealed that the changed proteins were clustered in polyribosome and RNA binding proteins in both cerebral cortex and hippocampus, but not the same in testis. Our results provide detailed insights to the ribosome protein profiles of cerebral cortex, hippocampus and testis in the absence of FMRP. Our studies also give a better understanding of protein profile changes and underling dysregulated pathways arising from the fmr1 silencing in the FXS.

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Comparison of wildtype and Fmr1 knock-out adult mouse gene expression in the cochlear nucleus

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