FMRP Regulation of Gene Expression

脆性X综合征(FXS)是自闭症谱系中的一种遗传性神经疾病，由 FMR1基因5‘非翻译区CGG三联体的扩增导致其表观遗传沉默。在 缺少FMR1基因产物FMRP，大脑中蛋白质合成过度，这与 这种疾病的几种表现包括认知障碍、发育迟缓、社交缺陷、 癫痫等。蛋白质合成增加可能推动了这些病理生理过程中的几个，从而强调 了解FMRP调节的翻译的重要性。FMRP是一种RNA结合蛋白，它能抑制 翻译，并做到这一点，至少部分是通过阻止核糖体转位在特定的mRNAs。如何使用FMRP 能否使核糖体停滞尚不清楚，尽管重组实验表明它可以与核糖体结合并 阻止与基本翻译因素的交互。我们的研究最初集中在识别 与FMRP停滞的核糖体结合。通过修改转录组范围的核糖体图谱来确定速率 关于小鼠海马体核糖体运输的研究，我们发现数千个mRNAs通过缓慢移动或 核糖体几乎完全停滞。特别是FMRP是必要的，以拖延核糖体对一些特定的 令人惊讶的是，包括几个编码表观遗传和转录因子的mRNAs。这些mRNA中的一个 与FMRP结合的停滞核糖体编码SETD2，它催化染色质标记H3K36me3，并 在Fmr1缺陷的海马区升高约2.5倍。CHIP-SEQ证明了在没有FMRP的情况下， H3K36me3在染色质上重排，包括在基因体中，在那里它调节前mRNA的加工。我们 发现RNA片断的严重不当调节，特别是外显子跳过事件，这些事件将FXS与 自闭症。基于这些和其他数据，我们提出了三个多部分的具体目标：1)确定FMRP是否 核糖体停滞在mRNA上的特定位置，发生在兴奋性神经元中，通过突触活动减轻，并且 发生在细胞体和/或树突中；2)研究SETD2的缺失是否改善FXS 3)确定兴奋性神经元、微外显子是否发生外显子跳跃 自闭症风险基因CPEB4的跳跃将FXS与自闭症联系在一起，并改变了介导外显子跳跃的因素 可以挽救FXS和自闭症的生化特征。

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.