FMRP loss of function causes Fragile X syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal ...translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here, we use high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results suggest that loss of a translational brake on the synthesis of a subset of synaptic proteins contributes to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD and suggest multiple targets for clinical intervention.
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► Identification of a robust, reproducible, novel set of in vivo FMRP targets ► FMRP targets encode key pre- and postsynaptic proteins and autism candidate genes ► FMRP stalls ribosomes along the coding region of its mRNA targets ► Acute and genetic loss of FMRP relieves stalling and increases protein synthesis
Exome sequencing of 343 families, each with a single child on the autism spectrum and at least one unaffected sibling, reveal de novo small indels and point substitutions, which come mostly from the ...paternal line in an age-dependent manner. We do not see significantly greater numbers of de novo missense mutations in affected versus unaffected children, but gene-disrupting mutations (nonsense, splice site, and frame shifts) are twice as frequent, 59 to 28. Based on this differential and the number of recurrent and total targets of gene disruption found in our and similar studies, we estimate between 350 and 400 autism susceptibility genes. Many of the disrupted genes in these studies are associated with the fragile X protein, FMRP, reinforcing links between autism and synaptic plasticity. We find FMRP-associated genes are under greater purifying selection than the remainder of genes and suggest they are especially dosage-sensitive targets of cognitive disorders.
► De novo mutations derive mainly from the paternal line in an age-dependent manner ► Mutations disrupting genes are twice as frequent in affected as unaffected siblings ► Many disrupted genes are associated with the fragile X protein, FMRP ► FMRP-associated genes are under unexpectedly strong purifying selection
Iossifov et al. use exome sequencing of 343 autistic families to identify de novo gene mutations associated with autism. Many of the mutated genes are associated with the fragile X protein FMRP, indicating new links between autism and synaptic plasticity.
Mammalian RNA complexity is regulated through interactions of RNA-binding proteins (RBPs) with their target transcripts. High-throughput sequencing together with UV-crosslinking and ...immunoprecipitation (HITS-CLIP) is able to globally map RBP-binding footprint regions at a resolution of ~30-60 nucleotides. Here we describe a systematic way to analyze HITS-CLIP data to identify exact crosslink sites, and thereby determine protein-RNA interactions at single-nucleotide resolution. We found that reverse transcriptase used in CLIP frequently skips the crosslinked amino-acid-RNA adduct, resulting in a nucleotide deletion. Genome-wide analysis of these crosslinking-induced mutation sites (CIMS) in HITS-CLIP data for Nova and Argonaute (Ago) proteins in mouse brain tissue revealed deletions in ~8-20% of mRNA tags, which mapped to Nova and Ago binding sites on mRNA or miRNA. CIMS analysis provides a general and more precise means of mapping protein-RNA interactions than currently available methods and insight into the biochemical properties of such interactions in living tissues.
Neurons have their own systems for regulating RNA. Several multigene families encode RNA binding proteins (RNABPs) that are uniquely expressed in neurons, including the well-known neuron-specific ...markers ELAV and NeuN and the disease antigen NOVA. New technologies have emerged in recent years to assess the function of these proteins in vivo, and the answers are yielding insights into how and why neurons may regulate RNA in special ways-to increase cellular complexity, to localize messenger RNA (mRNA) spatially, and to regulate their expression in response to synaptic stimuli. The functions of such restricted neuronal proteins are likely to be complemented by more widely expressed RNABPs that may themselves have developed specialized functions in neurons, including Argonaute/microRNAs (miRNAs). Here we review what is known about such RNABPs and explore the potential biologic and neurologic significance of neuronal RNA regulatory systems.
MicroRNAs (miRNAs) are essential components of gene regulation, but identification of miRNA targets remains a major challenge. Most target prediction and discovery relies on perfect complementarity ...of the miRNA seed to the 3′ untranslated region (UTR). However, it is unclear to what extent miRNAs target sites without seed matches. Here, we performed a transcriptome-wide identification of the endogenous targets of a single miRNA—miR-155—in a genetically controlled manner. We found that approximately 40% of miR-155-dependent Argonaute binding occurs at sites without perfect seed matches. The majority of these noncanonical sites feature extensive complementarity to the miRNA seed with one mismatch. These noncanonical sites confer regulation of gene expression, albeit less potently than canonical sites. Thus, noncanonical miRNA binding sites are widespread, often contain seed-like motifs, and can regulate gene expression, generating a continuum of targeting and regulation.
► Differential CLIP-Seq reveals transcriptome-wide sites of miR-155 binding ► Many miR-155 binding sites are noncanonical and lack a perfect seed match ► Most noncanonical binding sites contain a mismatch to the canonical seed motif ► Noncanonical sites mediate gene regulation, albeit weaker than canonical sites
Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome (FXS) and autism. ...FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with guanine (G)-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. Here we report the 2.8-Å crystal structure of the complex between the human FMRP RGG peptide bound to the in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K⁺-stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex–quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation–π interactions, and multiple hydrogen bonds. Many of these interactions critically depend on a type I β-turn, a secondary structure elementwhose formationwas not previously recognized in the RGG motif of FMRP. RNA mutagenesis and footprinting experiments indicate that interactions of the peptide with the duplex–quadruplex junction and the duplex of RNA are equally important for affinity and specificity of the RGG–RNA complex formation. These results suggest that specific binding of cellular RNAs by FMRP may involve hydrogen bonding with RNA duplexes and that RNA duplex recognition can be a characteristic RNA binding feature for RGG motifs in other proteins.
By using a cell fraction technique that separates chromatin-associated nascent RNA, newly completed nucleoplasmic mRNA and cytoplasmic mRNA, we have shown in a previous study that residues in exons ...are methylated (m
A) in nascent pre-mRNA and remain methylated in the same exonic residues in nucleoplasmic and cytoplasmic mRNA. Thus, there is no evidence of a substantial degree of demethylation in mRNA exons that would correspond to so-called "epigenetic" demethylation. The turnover rate of mRNA molecules is faster, depending on m
A content in HeLa cell mRNA, suggesting that specification of mRNA stability may be the major role of m
A exon modification. In mouse embryonic stem cells (mESCs) lacking Mettl3, the major mRNA methylase, the cells continue to grow, making the same mRNAs with unchanged splicing profiles in the absence (>90%) of m
A in mRNA, suggesting no common obligatory role of m
A in splicing. All these data argue strongly against a commonly used "reversible dynamic methylation/demethylation" of mRNA, calling into question the concept of "RNA epigenetics" that parallels the well-established role of dynamic DNA epigenetics.
The RNA-mediated disease model for myotonic dystrophy (DM) proposes that microsatellite C(C)TG expansions express toxic RNAs that disrupt splicing regulation by altering MBNL1 and CELF1 activities. ...While this model explains DM manifestations in muscle, less is known about the effects of C(C)UG expression on the brain. Here, we report that Mbnl2 knockout mice develop several DM-associated central nervous system (CNS) features including abnormal REM sleep propensity and deficits in spatial memory. Mbnl2 is prominently expressed in the hippocampus and Mbnl2 knockouts show a decrease in NMDA receptor (NMDAR) synaptic transmission and impaired hippocampal synaptic plasticity. While Mbnl2 loss did not significantly alter target transcript levels in the hippocampus, misregulated splicing of hundreds of exons was detected using splicing microarrays, RNA-seq, and HITS-CLIP. Importantly, the majority of the Mbnl2-regulated exons examined were similarly misregulated in DM. We propose that major pathological features of the DM brain result from disruption of the MBNL2-mediated developmental splicing program.
► Muscleblind-like 2 regulates alternative splicing in the brain ► Developmental regulation of splicing is disrupted in Mbnl2 knockout mice ► RNAs targeted by MBNL2 are misspliced in the myotonic dystrophy brain
Myotonic dystrophy, a disease caused by toxic RNAs, profoundly affects brain function. In this Article, Charizanis et al. demonstrate that the effect on brain function results from loss of muscleblind-like 2 RNA splicing regulation during brain development.
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.
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