Dissecting the role of FMRP in RNA processing using hPSC models

脆性X智力迟钝1 （FMR1）编码rna结合蛋白FMRP。FMRP的缺失会导致脆性X染色体综合征（FXS），这是智力残疾的主要遗传原因，也是最常见的单基因自闭症。我们最近首次定义了人类胚胎干细胞（hESCs）和体外衍生的兴奋性皮质神经元中FMRP的全局RNA靶点。从这些数据集中，我们确定了大约三分之一的FMRP结合事件在pre-mRNA靶点的内含子上，并检测到大量的FMRP RNA靶点转录自21号染色体（HSA21），这是与唐氏综合症相关的染色体。许多这些RNA靶标也作为前mrna结合。虽然FMRP最常在细胞质翻译调控的背景下进行研究，但新出现的数据指出FMRP在细胞核转录和转录后过程中的其他功能。然而，FMRP和pre- mrna之间关联的分子机制仍然未知，因此是本应用的重点。鉴于它们与疾病的相关性，我们将特别关注从HSA21转录的FMRP前mrna靶标子集。事实上，我们的数据支持了一种新的假设，即智力残疾的两种最常见的遗传原因，FXS和唐氏综合症（DS），在共同的分子机制上趋于一致。在Aim I中，我们选择了一组关键的hsa21编码的FMRP靶点来研究pre-mRNA结合事件和下游分子后果的功能相关性。我们现在将定义介导前mRNA结合的相关FMRP结构域，直接测试前mRNA结合与观察到的蛋白质水平变化的相关性，并扩展患者细胞中的下游分子影响。在Aim II中，我们将测试FMRP结合前mrna在剪接中发挥直接抑制作用的新假设，部分基于显示FMRP丢失后转录物使用差异的RNA-seq数据。具体来说，我们将研究单个FMRP前mrna结合位点如何调节剪接产物，评估FMRP丢失对剪接动力学的影响，并评估FMRP结合/剪接是否共转录发生。在第三部分中，我们将使用FMRP ChIP-seq测试FMRP与染色质结合的假设，这将阐明FMRP靶标识别的新机制。我们将进一步评估FMRP染色质结合是否对转录输出有独立的影响，并探讨FMRP结合与已知染色质特征之间的关系。总的来说，该项目将阐明RNA加工中FMRP生物学的新颖和基本方面，有可能影响我们对FXS和唐氏综合症疾病生物学的理解。

Fragile X mental retardation 1 (FMR1) encodes the RNA-binding protein, FMRP. Loss of FMRP is causative for Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and most common monogenic form of autism. We recently defined for the first time, the global RNA targets of FMRP in human embryonic stem cells (hESCs) and in vitro derived excitatory cortical neurons. From these datasets, we identified roughly one-third of FMRP binding events on introns of pre-mRNA targets and detected a significant number of FMRP RNA targets transcribed from chromosome 21 (HSA21), the chromosome associated with Down syndrome. Many of these RNA targets were also bound as pre-mRNAs. While FMRP is most frequently studied in the context of translational regulation in the cytoplasm, emerging data point to additional functions of FMRP in transcriptional and post-transcriptional processes in the nucleus. However, the molecular mechanisms underlying the association between FMRP and pre-mRNAs remain unknown and are therefore the focus of this application. Given their disease relevance, we will specifically focus on the subset of FMRP pre-mRNA targets transcribed from HSA21. Indeed, our data support the novel hypothesis that the two most common genetic causes of intellectual disability, FXS and Down Syndrome (DS), converge on common molecular mechanisms. In Aim I, we selected a set of key HSA21-encoded FMRP targets to investigate the functional relevance of pre-mRNA binding events and downstream molecular consequences. We will now define the relevant FMRP domain mediating pre-mRNA binding, directly test the relevance of pre- mRNA binding for observed protein-level changes, and expand on the downstream molecular impact in patient cells. In Aim II, we will test the novel hypothesis that FMRP binds pre-mRNAs to play a direct inhibitory role in splicing, based in part on RNA-seq data showing differential transcript usage following FMRP loss. Specifically, we will examine how individual FMRP pre-mRNA binding sites regulate splicing products, assess the impact of FMRP loss on splicing kinetics and assess whether FMRP binding/splicing occurs co-transcriptionally. In Aim III, we will test the hypothesis that FMRP binding to chromatin underlies its association with pre-mRNAs using FMRP ChIP-seq, which would illuminate a novel mechanism of FMRP target recognition. We will further assess whether FMRP chromatin binding has independent effects on transcriptional output and probe the relationship between FMRP binding and known chromatin features. Collectively, this project will illuminate novel and fundamental aspects of FMRP biology in RNA processing, with the potential to impact our understanding of FXS and Down syndrome disease biology.