The Fragile X Syndrome (FXS) is an inherited neurological disorder on the autism spectrum that is caused by expansion of CGG triplets in the 5' untranslated region (UTR) of FMR1, leading to its epigenetic silencing. In the absence of the FMR1 gene product FMRP, protein synthesis in the brain is excessive, which is correlated with several manifestations of the disorder including cognitive impairment, developmental delays, social deficits, seizures, etc. Elevated protein synthesis likely drives several of these pathophysiologies, thereby underscoring the importance of understanding FMRP-regulated translation. FMRP is an RNA binding protein that represses translation and does so, at least in part, by impeding ribosome translocation on specific mRNAs. How FMRP could stall ribosomes is unclear, although reconstitution experiments suggest that it could bind the ribosome and block interactions with essential translation factors. Our studies focused initially on identifying the mRNAs that are bound with FMRP-stalled ribosomes. By modifying transcriptome-wide ribosome profiling to determine rates of ribosome transit in the mouse hippocampus, we find that thousands of mRNAs are bound by slow moving or nearly completely stalled ribosomes. FMRP in particular is necessary to stall ribosomes on a number of specific mRNAs including several that, surprisingly, code for epigenetic and transcription factors. One of these mRNAs bound by FMRP-stalled ribosomes encodes SETD2, which catalyzes the chromatin mark H3K36me3 and which is elevated ~2.5 fold in Fmr1-deficient hippocampus. ChIP-seq demonstrates that in the absence of FMRP, H3K36me3 is rearranged on chromatin including in gene bodies where it modulates pre-mRNA processing. We find substantial mis-regulation of RNA slicing, particularly exon skipping events, which strongly link FXS to autism. Based on these and other data, we propose three multi-part specific aims: 1) determine whether FMRP stalls ribosomes at specific sites on mRNA, occurs in excitatory neurons, is alleviated by synaptic activity, and takes place in cell bodies and/or dendrites; 2) investigate whether depletion of SETD2 ameliorates FXS pathophysiology in model mice; 3) determine whether exon skipping occurs in excitatory neurons, microexon skipping in the autism risk gene CPEB4 links FXS to autism, and alteration of factors that mediate exon skipping can rescue biochemical features of FXS and autism.
We propose to investigate the underlying molecular activity of FMRP, the protein produce of the FMR1 gene. Because silencing of FMR1 causes the Fragile X Syndrome, our work forms a molecular understanding of this autism spectrum disorder, and thus is directly related to human health.