Site-specific R-loops induce CGG repeat contraction and fragile X gene reactivation.

Here, we describe an approach to correct the genetic defect in fragile X syndrome (FXS) via recruitment of endogenous repair mechanisms. A leading cause of autism spectrum disorders, FXS results from epigenetic silencing of FMR1 due to a congenital trinucleotide (CGG) repeat expansion. By investigating conditions favorable to FMR1 reactivation, we find MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We trace the mechanism to DNA demethylation and site-specific R-loops, which are necessary and sufficient for repeat contraction. A positive feedback cycle comprising demethylation, de novo FMR1 transcription, and R-loop formation results in the recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction is specific to FMR1 and restores the production of FMRP protein. Our study therefore identifies a potential method of treating FXS in the future.

Excess Translation of Epigenetic Regulators Contributes to Fragile X Syndrome and Is Alleviated by Brd4 Inhibition.

Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.

BRCA1 levels and DNA-damage response are controlled by the competitive binding of circHIPK3 or FMRP to the BRCA1 mRNA.

Circular RNAs (circRNAs) are covalently closed RNA molecules widely expressed in eukaryotes and deregulated in several pathologies, including cancer. Many studies point to their activity as microRNAs (miRNAs) and protein sponges; however, we propose a function based on circRNA-mRNA interaction to regulate mRNA fate. We show that the widely tumor-associated circHIPK3 directly interacts in vivo with the BRCA1 mRNA through the back-splicing region in human cancer cells. This interaction increases BRCA1 translation by competing for the binding of the fragile-X mental retardation 1 protein (FMRP) protein, which we identified as a BRCA1 translational repressor. CircHIPK3 depletion or disruption of the circRNA-mRNA interaction decreases BRCA1 protein levels and increases DNA damage, sensitizing several cancer cells to DNA-damage-inducing agents and rendering them susceptible to synthetic lethality. Additionally, blocking FMRP interaction with BRCA1 mRNA with locked nucleic acid (LNA) restores physiological protein levels in BRCA1 hemizygous breast cancer cells, underscoring the importance of this circRNA-mRNA interaction in regulating DNA-damage response.

FMRP phosphorylation modulates neuronal translation through YTHDF1.

RNA-binding proteins (RBPs) control messenger RNA fate in neurons. Here, we report a mechanism that the stimuli-induced neuronal translation is mediated by phosphorylation of a YTHDF1-binding protein FMRP. Mechanistically, YTHDF1 can condense with ribosomal proteins to promote the translation of its mRNA targets. FMRP regulates this process by sequestering YTHDF1 away from the ribosome; upon neuronal stimulation, FMRP becomes phosphorylated and releases YTHDF1 for translation upregulation. We show that a new small molecule inhibitor of YTHDF1 can reverse fragile X syndrome (FXS) developmental defects associated with FMRP deficiency in an organoid model. Our study thus reveals that FMRP and its phosphorylation are important regulators of activity-dependent translation during neuronal development and stimulation and identifies YTHDF1 as a potential therapeutic target for FXS in which developmental defects caused by FMRP depletion could be reversed through YTHDF1 inhibition.

Integrative omics indicate FMRP sequesters mRNA from translation and deadenylation in human neuronal cells.

How fragile X syndrome protein (FMRP) binds mRNAs and regulates mRNA metabolism remains unclear. Our previous work using human neuronal cells focused on mRNAs targeted for nonsense-mediated mRNA decay (NMD), which we showed are generally bound by FMRP and destabilized upon FMRP loss. Here, we identify >400 high-confidence FMRP-bound mRNAs, only ∼35% of which are NMD targets. Integrative transcriptomics together with SILAC-LC-MS/MS reveal that FMRP loss generally results in mRNA destabilization and more protein produced per FMRP target. We use our established RIP-seq technology to show that FMRP footprints are independent of protein-coding potential, target GC-rich and structured sequences, and are densest in 5' UTRs. Regardless of where within an mRNA FMRP binds, we find that FMRP protects mRNAs from deadenylation and directly binds the cytoplasmic poly(A)-binding protein. Our results reveal how FMRP sequesters polyadenylated mRNAs into stabilized and translationally repressed complexes, whose regulation is critical for neurogenesis and synaptic plasticity.

The Genetic Control of Stoichiometry Underlying Autism.

Autism is a common and complex neurologic disorder whose scientific underpinnings have begun to be established in the past decade. The essence of this breakthrough has been a focus on families, where genetic analyses are strongest, versus large-scale, case-control studies. Autism genetics has progressed in parallel with technology, from analyses of copy number variation to whole-exome sequencing (WES) and whole-genome sequencing (WGS). Gene mutations causing complete loss of function account for perhaps one-third of cases, largely detected through WES. This limitation has increased interest in understanding the regulatory variants of genes that contribute in more subtle ways to the disorder. Strategies combining biochemical analysis of gene regulation, WGS analysis of the noncoding genome, and machine learning have begun to succeed. The emerging picture is that careful control of the amounts of transcription, mRNA, and proteins made by key brain genes-stoichiometry-plays a critical role in defining the clinical features of autism.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

Dosage sensitivity to Pumilio1 variants in the mouse brain reflects distinct molecular mechanisms.

Different mutations in the RNA-binding protein Pumilio1 (PUM1) cause divergent phenotypes whose severity tracks with dosage: a mutation that reduces PUM1 levels by 25% causes late-onset ataxia, whereas haploinsufficiency causes developmental delay and seizures. Yet PUM1 targets are derepressed to equal degrees in both cases, and the more severe mutation does not hinder PUM1's RNA-binding ability. We therefore considered the possibility that the severe mutation might disrupt PUM1 interactions, and identified PUM1 interactors in the murine brain. We find that mild PUM1 loss derepresses PUM1-specific targets, but the severe mutation disrupts interactions with several RNA-binding proteins and the regulation of their targets. In patient-derived cell lines, restoring PUM1 levels restores these interactors and their targets to normal levels. Our results demonstrate that dosage sensitivity does not always signify a linear relationship with protein abundance but can involve distinct mechanisms. We propose that to understand the functions of RNA-binding proteins in a physiological context will require studying their interactions as well as their targets.

Truncated variants of MAGEL2 are involved in the etiologies of the Schaaf-Yang and Prader-Willi syndromes.

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS.

Loss of FMRP affects ovarian development and behaviour through multiple pathways in a zebrafish model of fragile X syndrome.

Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder and the leading genetic cause of autism spectrum disorders. FXS is caused by loss of function mutations in Fragile X mental retardation protein (FMRP), an RNA binding protein that is known to regulate translation of its target mRNAs, predominantly in the brain and gonads. The molecular mechanisms connecting FMRP function to neurodevelopmental phenotypes are well understood. However, neither the full extent of reproductive phenotypes, nor the underlying molecular mechanisms have been as yet determined. Here, we developed new fmr1 knockout zebrafish lines and show that they mimic key aspects of FXS neuronal phenotypes across both larval and adult stages. Results from the fmr1 knockout females also showed that altered gene expression in the brain, via the neuroendocrine pathway contribute to distinct abnormal phenotypes during ovarian development and oocyte maturation. We identified at least three mechanisms underpinning these defects, including altered neuroendocrine signaling in sexually mature females resulting in accelerated ovarian development, altered expression of germ cell and meiosis promoting genes at various stages during oocyte maturation, and finally a strong mitochondrial impairment in late stage oocytes from knockout females. Our findings have implications beyond FXS in the study of reproductive function and female infertility. Dissection of the translation control pathways during ovarian development using models like the knockout lines reported here may reveal novel approaches and targets for fertility treatments.

Antisense oligonucleotide rescue of CGG expansion-dependent FMR1 mis-splicing in fragile X syndrome restores FMRP.

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 tissues 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 tissues. 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 misregulated 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 therapeutic approach to mitigate the disorder.

Complex interplay between FMRP and DHX9 during DNA replication stress.

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.

Fragile X Syndrome: Lessons Learned and What New Treatment Avenues Are on the Horizon.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading single-gene form of autism spectrum disorder, encompassing cognitive, behavioral, and physical forms of clinical involvement. FXS is caused by large expansions of a noncoding CGG repeat (>200 repeats) in the FMR1 gene, at which point the gene is generally silenced. Absence of FMR1 protein (FMRP), important for synaptic development and maintenance, gives rise to the neurodevelopmental disorder. There is, at present, no therapeutic approach that directly reverses the loss of FMRP; however, there is an increasing number of potential treatments that target the pathways dysregulated in FXS, including those that address the enhanced activity of the mGluR5 pathway and deficits in GABA pathways. Based on studies of targeted therapeutics to date, the prospects are good for one or more effective therapies for FXS in the near future.

RNA-binding FMRP and Staufen sequentially regulate the Coracle scaffold to control synaptic glutamate receptor and bouton development.

Both mRNA-binding Fragile X mental retardation protein (FMRP; Fmr1) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. Here, we tested whether these RNA-binding proteins act jointly in a common mechanism. We found that both dfmr1 and staufen mutants, and trans-heterozygous double mutants, displayed increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNA interference, we showed a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, we showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and we confirmed that Staufen binds to coracle mRNA. We found that FMRP and Staufen act sequentially to co-regulate postsynaptic Coracle expression, and showed that Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, we found that dfmr1, staufen and coracle mutants elevate neurotransmission strength. We also identified that FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development.

The Drosophila fragile X mental retardation protein modulates the neuronal cytoskeleton to limit dendritic arborization.

Dendritic arbor development is a complex, highly regulated process. Post-transcriptional regulation mediated by RNA-binding proteins plays an important role in neuronal dendrite morphogenesis by delivering on-site, on-demand protein synthesis. Here, we show how the Drosophila fragile X mental retardation protein (FMRP), a conserved RNA-binding protein, limits dendrite branching to ensure proper neuronal function during larval sensory neuron development. FMRP knockdown causes increased dendritic terminal branch growth and a resulting overelaboration defect due, in part, to altered microtubule stability and dynamics. FMRP also controls dendrite outgrowth by regulating the Drosophila profilin homolog chickadee (chic). FMRP colocalizes with chic mRNA in dendritic granules and regulates its dendritic localization and protein expression. Whereas RNA-binding domains KH1 and KH2 are both crucial for FMRP-mediated dendritic regulation, KH2 specifically is required for FMRP granule formation and chic mRNA association, suggesting a link between dendritic FMRP granules and FMRP function in dendrite elaboration. Our studies implicate FMRP-mediated modulation of both the neuronal microtubule and actin cytoskeletons in multidendritic neuronal architecture, and provide molecular insight into FMRP granule formation and its relevance to FMRP function in dendritic patterning.

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

The cap epitranscriptome: Early directions to a complex life as mRNA.

Animal, protist and viral messenger RNAs (mRNAs) are most prominently modified at the beginning by methylation of cap-adjacent nucleotides at the 2'-O-position of the ribose (cOMe) by dedicated cap methyltransferases (CMTrs). If the first nucleotide of an mRNA is an adenosine, PCIF1 can methylate at the N6 -position (m6 A), while internally the Mettl3/14 writer complex can methylate. These modifications are introduced co-transcriptionally to affect many aspects of gene expression including localisation to synapses and local translation. Of particular interest, transcription start sites of many genes are heterogeneous leading to sequence diversity at the beginning of mRNAs, which together with cOMe and m6 Am could constitute an extensive novel layer of gene expression control. Given the role of cOMe and m6 A in local gene expression at synapses and higher brain functions including learning and memory, such code could be implemented at the transcriptional level for lasting memories through local gene expression at synapses.

FMRP Long-Range Transport and Degradation Are Mediated by Dynlrb1 in Sensory Neurons.

The fragile X messenger ribonucleoprotein 1 (FMRP) is a multifunctional RNA-binding protein implicated in human neurodevelopmental and neurodegenerative disorders. FMRP mediates the localization and activity-dependent translation of its associated mRNAs through the formation of phase-separated condensates that are trafficked by microtubule-based motors in axons. Axonal transport and localized mRNA translation are critical processes for long-term neuronal survival and are closely linked to the pathogenesis of neurological diseases. FMRP dynein-mediated axonal trafficking is still largely unexplored but likely to constitute a key process underlying FMRP spatiotemporal translational regulation. Here, we show that dynein light chain roadblock 1 (Dynlrb1), a subunit of the dynein complex, is a critical regulator of FMRP function. In sensory axons, FMRP associates with endolysosomal organelles, likely through annexin A11, and is retrogradely trafficked by the dynein complex in a Dynlrb1-dependent manner. Moreover, Dynlrb1 silencing induced FMRP granule accumulation and repressed the translation of microtubule-associated protein 1b, one of its primary mRNA targets. Our findings suggest that Dynlrb1 regulates FMRP function through the control of its transport and targeted degradation.

FMRP promotes transcription-coupled homologous recombination via facilitating TET1-mediated m5C RNA modification demethylation.

RNA modifications regulate a variety of cellular processes including DNA repair.The RNA methyltransferase TRDMT1 generates methyl-5-cytosine (m5C) on messen-ger RNA (mRNA) at DNA double-strand breaks (DSBs) in transcribed regions, pro-moting transcription-coupled homologous recombination (HR). Here, we identifiedthat Fragile X mental retardation protein (FMRP) promotes transcription-coupled HRvia its interaction with both the m5C writer TRDMT1 and the m5C eraser ten-eleventranslocation protein 1 (TET1). TRDMT1, FMRP, and TET1 function in a temporalorder at the transcriptionally active sites of DSBs. FMRP displays a higher affinity forDNA:RNA hybrids containing m5C-modified RNA than for hybrids without modifica-tion and facilitates demethylation of m5C by TET1 in vitro. Loss of either the chroma-tin- or RNA-binding domain of FMRP compromises demethylation of damage-inducedm5C in cells. Importantly, FMRP is required for R-loop resolving in cells. Due to unre-solved R-loop and m5C preventing completion of DSB repair, FMRP depletion or lowexpression leads to delayed repair of DSBs at transcriptionally active sites and sensitizescancer cells to radiation in a BRCA-independent manner. Together, ourfindings presentan m5C reader, FMRP, which acts as a coordinator between the m5C writer and eraserto promote mRNA-dependent repair and cell survival in cancer.

Post-transcriptional regulation of mouse neurogenesis by Pumilio proteins.

Despite extensive studies on mammalian neurogenesis, its post-transcriptional regulation remains under-explored. Here we report that neural-specific inactivation of two murine post-transcriptional regulators, Pumilio 1 (Pum1) and Pum2, severely reduced the number of neural stem cells (NSCs) in the postnatal dentate gyrus (DG), drastically increased perinatal apoptosis, altered DG cell composition, and impaired learning and memory. Consistently, the mutant DG neurospheres generated fewer NSCs with defects in proliferation, survival, and differentiation, supporting a major role of Pum1 and Pum2 in hippocampal neurogenesis and function. Cross-linking immunoprecipitation revealed that Pum1 and Pum2 bind to thousands of mRNAs, with at least 694 common targets in multiple neurogenic pathways. Depleting Pum1 and/or Pum2 did not change the abundance of most target mRNAs but up-regulated their proteins, indicating that Pum1 and Pum2 regulate the translation of their target mRNAs. Moreover, Pum1 and Pum2 display RNA-dependent interaction with fragile X mental retardation protein (FMRP) and bind to one another's mRNA. This indicates that Pum proteins might form collaborative networks with FMRP and possibly other post-transcriptional regulators to regulate neurogenesis.