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Fragile X mental retardation protein (FMRP) is a multifunctional RNA-binding protein with crucial roles in neuronal development and function. Efforts aimed at elucidating how FMRP target mRNAs are selected have produced divergent sets of target mRNA and putative FMRP-bound motifs, and a clear understanding of FMRP's binding determinants has been lacking. To clarify FMRP's binding to its target mRNAs, we produced a shared dataset of FMRP consensus binding sequences (FCBS), which were reproducibly identified in two published FMRP CLIP sequencing datasets. This comparative dataset revealed that of the various sequence and structural motifs that have been proposed to specify FMRP binding, the short sequence motifs TGGA and GAC were corroborated, and a novel TAY motif was identified. In addition, the distribution of the FCBS set demonstrates that FMRP preferentially binds to the coding region of its targets but also revealed binding along 3′ UTRs in a subset of target mRNAs. Beyond probing these putative motifs, the FCBS dataset of reproducibly identified FMRP binding sites is a valuable tool for investigating FMRP targets and function.

Fragile X syndrome (FXS) results in intellectual disability (ID) most often caused by silencing of the fragile X mental retardation 1 (FMR1) gene. The resulting absence of fragile X mental retardation protein 1 (FMRP) leads to both pre- and postsynaptic defects, yet whether the pre- and postsynaptic functions of FMRP are independent and have distinct roles in FXS neuropathology remain poorly understood. Here, we demonstrate an independent presynaptic function for FMRP through the study of an ID patient with an FMR1 missense mutation. This mutation, c.413G > A (R138Q), preserves FMRP’s canonical functions in RNA binding and translational regulation, which are traditionally associated with postsynaptic compartments. However, neuronally driven expression of the mutant FMRP is unable to rescue structural defects at the neuromuscular junction in fragile x mental retardation 1 (dfmr1)-deficient Drosophila, suggesting a presynaptic-specific impairment. Furthermore, mutant FMRP loses the ability to rescue presynaptic action potential (AP) broadening in Fmr1 KO mice. The R138Q mutation also disrupts FMRP’s interaction with the large-conductance calcium-activated potassium (BK) channels that modulate AP width. These results reveal a presynaptic- and translation-independent function of FMRP that is linked to a specific subset of FXS phenotypes.

Fragile X mental retardation protein (FMRP), the protein that is absent or dysfunctional in fragile X syndrome (FXS), is a multifunctional binding protein that associates with mRNAs and proteins in the brain. The loss of FMRP results in a number of molecular problems, including dysregulated protein synthesis of a subset of genes in the postsynapse, ion channel malfunction in the presynapse, and impaired response to DNA damage in the nucleus. Identification of FMRP-binding partners would help pinpoint the pathways and processes that are perturbed by the loss of the protein and provide therapeutic targets for the treatment of FXS. This chapter will discuss the latest approaches and discoveries regarding the RNA- and protein-binding targets of FMRP.

Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle, we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies. The reactivated cells produced FMRP and exhibited a decline in DNA methylation at the FMR1 locus. These data demonstrate the excision of the expanded CGG-repeat from the fragile X chromosome can result in FMR1 reactivation.

Fragile X syndrome (FXS), the most common inherited form of developmental delay, is typically caused by CGG-repeat expansion in FMR1. However, little attention has been paid to sequence variants in FMR1. Through the use of pooled-template massively parallel sequencing, we identified 130 novel FMR1 sequence variants in a population of 963 developmentally delayed males without CGG-repeat expansion mutations. Among these, we identified a novel missense change, p.R138Q, which alters a conserved residue in the nuclear localization signal of FMRP. We have also identified three promoter mutations in this population, all of which significantly reduce in vitro levels of FMR1 transcription. Additionally, we identified 10 noncoding variants of possible functional significance in the introns and 3’-untranslated region of FMR1, including two predicted splice site mutations. These findings greatly expand the catalogue of known FMR1 sequence variants and suggest that FMR1 sequence variants may represent an important cause of developmental delay.

It has been proposed that cancer prevention results from multiple dietary agents acting together as "action packages." Here we obtain evidence that butyrate, which is generated from dietary fiber, enhances the responsiveness of colon cancer cells to all-trans retinoic acid (ATRA). Evidence was obtained that this interaction depends on histone deactylase one (HDAC1) inhibition by butyrate and retinoic acid receptor alpha (RARalpha) activation by ATRA. The enhancement of RAR beta 2 (RARbeta2) activation was accompanied by a rapid demethylation of the RARbeta2 promoter. This demethylation could be achieved by butyrate alone, and it differed from that triggered by the DNA methyltransferase inhibitor 5-Aza-2' deoxycytidine in that it was 1) sporadic on the RARbeta2 promoter, 2) not genome wide, and 3) independent of extensive DNA replication. An analysis of inter-methylated sites assay indicated that only a few percent of loci analyzed showed reduced methylation. In colon cancer cells that were particularly resistant to RARbeta2 reactivation, the actions of butyrate could be further enhanced by the soy isoflavone genistein, which has also been reported to work through an epigenetic mechanism. These data suggest that dietary compounds that modulate epigenetic programming are likely to function best in the presence of retinoids and other cancer-preventing compounds that are sensitive to a cell's epigenetic state.

The 22q11.2 deletion syndrome is the most common microdeletion disorder, with wide phenotypic variability. To investigate variation within the non-deleted allele we performed targeted resequencing of the 22q11.2 region for 127 patients, identifying multiple deletion sizes, including two deletions with atypical breakpoints. We cataloged ~12,000 hemizygous variant positions, of which 84% were previously annotated. Within the coding regions 95 non-synonymous variants, three stop gains, and two frameshift insertions were identified, some of which we speculate could contribute to atypical phenotypes. We also catalog tolerability of 22q11 gene mutations based on related autosomal recessive disorders in man, embryonic lethality in mice, cross-species conservation and observations that some genes harbor more or less variants than expected. This extensive catalog of hemizygous variants will serve as a blueprint for future experiments to correlate 22q11DS variation with phenotype. Mapping the intact chromosomes of patients who have a partial chromosomal deletion may reveal gene function. Individuals with similar deletions of a small piece of chromosome 22, known as 22q11.2 deletion syndrome (22q11.2DS), can show remarkably variable symptoms, which include heart defects, facial differences, and learning difficulties. The differences might result from unmasking of various mutations in the intact segment. Joris Vermeesch at KU Leuven in Belgium and an international team of colleagues mapped the variation in the intact arm of chromosome 22 in 127 patients with 22q11.2DS. They identified almost 12,000 variant positions, including variants of 11 of the 18 genes in the region that have unknown functions. The catalogue of variation will help in matching mutations with symptoms to determine gene function.

Fragile X syndrome is a common cause of intellectual disability and autism spectrum disorder. The gene underlying the disorder, fragile X mental retardation 1 (FMR1), is silenced in most cases by a CGG-repeat expansion mutation in the 5' untranslated region (UTR). Recently, we identified a variant located in the 3'UTR of FMR1 enriched among developmentally delayed males with normal repeat lengths. A patient-derived cell line revealed reduced levels of endogenous fragile X mental retardation protein (FMRP), and a reporter containing a patient 3'UTR caused a decrease in expression. A control reporter expressed in cultured mouse cortical neurons showed an expected increase following synaptic stimulation that was absent when expressing the patient reporter, suggesting an impaired response to neuronal activity. Mobility-shift assays using a control RNA detected an RNA-protein interaction that is lost with the patient RNA, and HuR was subsequently identified as an associated protein. Cross-linking immunoprecipitation experiments identified the locus as an in vivo target of HuR, supporting our in vitro findings. These data suggest that the disrupted interaction of HuR impairs activity-dependent translation of FMRP, which may hinder synaptic plasticity in a clinically significant fashion.

Fragile X syndrome, a common cause of intellectual disability and a well-known cause of autism spectrum disorder, is the result of loss or dysfunction of fragile X mental retardation protein (FMRP), a highly selective RNA-binding protein and translation regulator. A major research priority has been the identification of the mRNA targets of FMRP, particularly as recent studies suggest an excess of FMRP targets among genes implicated in idiopathic autism and schizophrenia. Several large-scale studies have attempted to identify mRNAs bound by FMRP through several methods, each generating a list of putative target genes, leading to distinct hypotheses by which FMRP recognizes its targets; namely, by RNA structure or sequence. However, no in depth analyses have been performed to identify the level of consensus among the studies. Here, we analyze four large FMRP target datasets to generate high-confidence consensus lists, and examine all datasets for sequence elements within the target RNAs to validate reported FMRP binding motifs (GACR, ACUK and WGGA). We found GACR to be highly enriched in FMRP datasets, while ACUK was not. The WGGA pattern was modestly enriched in several, but not all datasets. The previous association between FMRP and G-quadruplexes prompted the analysis of the distribution of WGGA in the target genes. Consistent with the requirements for G-quadruplex formation, we observed highly clustered WGGA motifs in FMRP targets compared with other genes, implicating both RNA structure and sequence in the recognition motif of FMRP. In addition, we generate a list of the top 40 FMRP targets associated with FXS-related phenotypes.

Background 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder, affecting an estimated 1 : 2000–4000 live births. Patients with 22q11.2DS have a broad spectrum of phenotypic abnormalities which generally includes congenital cardiac abnormalities, palatal anomalies, and immunodeficiency. Additional findings, such as skeletal anomalies and autoimmune disorders, can confer significant morbidity in a subset of patients. 22q11.2DS is a contiguous gene DS and over 40 genes are deleted in patients; thus deletion of several genes within this region contributes to the clinical features. Mutations outside or on the remaining 22q11.2 allele are also known to modify the phenotype. Methods We utilised whole exome, targeted exome and/or Sanger sequencing to examine the genome of 17 patients with 22q11.2 deletions and phenotypic features found in