Dissecting the role of FMRP in RNA processing using hPSC models

使用 hPSC 模型剖析 FMRP 在 RNA 加工中的作用

• 批准号: 10121018
• 负责人: Lindy Elise Barrett
• 金额: $ 383.78万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-17 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10121018
• 关键词: 5' Untranslated Regions; Acute; Antibodies; Automobile Driving; Binding; Binding Sites; Biology; Brain; Categories; Cell Fraction; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromosome 21; Chromosomes; Cytoplasm; Data; Data Set; Disease; Down Syndrome; Epigenetic Process; Event; FMR1; FMRP; Fragile X Syndrome; Funding; Gene Targeting; Genetic; Genetic Transcription; Goals; Grant; Human; Human Development; In Vitro; Individual; Inherited; Intellectual functioning disability; Introns; Investigation; Kinetics; Longevity; Mediating; Modeling; Molecular; Mus; Neuroglia; Neurons; Nuclear; Output; Patients; Play; Prevalence; Proteins; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Reporting; Role; Series; Site; Testing; Time; Transcript; Transcription Process; Translational Regulation; Trinucleotide Repeat Expansion; Twin Multiple Birth; Up-Regulation; autism spectrum disorder; base; cell type; crosslinking and immunoprecipitation sequencing; experimental study; human disease; human embryonic stem cell; mRNA Precursor; novel; protein expression; transcriptome sequencing

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.

脆性X智力低下1（FMR 1）编码RNA结合蛋白FMRP。FMRP的缺失是脆性X综合征（FXS）的病因，FXS是智力残疾的主要遗传原因和最常见的单基因形式的自闭症。我们最近首次定义了人胚胎干细胞（hESC）和体外衍生的兴奋性皮层神经元中FMRP的全球RNA靶点。从这些数据集中，我们识别出了前mRNA靶点内含子上大约三分之一的FMRP结合事件，并检测到大量从21号染色体（HSA 21）转录的FMRP RNA靶点，该染色体与唐氏综合症相关。这些RNA靶中的许多也作为前mRNA结合。虽然FMRP最常在细胞质中的翻译调控的背景下进行研究，但新出现的数据表明FMRP在细胞核中的转录和转录后过程中具有额外的功能。然而，FMRP和前mRNA之间关联的分子机制仍然未知，因此是本申请的焦点。鉴于其疾病相关性，我们将特别关注从HSA 21转录的FMRP前mRNA靶点的子集。事实上，我们的数据支持一个新的假设，即智力残疾的两个最常见的遗传原因，FXS和唐氏综合征（DS），汇聚在共同的分子机制。在目的I中，我们选择了一组关键的HSA 21编码的FMRP靶点，以研究前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。在目标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RNA-seq数据显示FMRP丢失后的差异转录本使用。具体来说，我们将研究如何个别FMRP前mRNA结合位点调节剪接产物，评估剪接动力学的FMRP损失的影响，并评估是否FMRP结合/剪接共转录发生。在目的III中，我们将使用FMRP ChIP-seq测试FMRP与染色质结合是其与前体mRNA结合的基础的假设，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具有独立影响，并探讨FMRP结合与已知染色质特征之间的关系。总的来说，这个项目将阐明FMRP生物学在RNA加工中的新的和基本的方面，有可能影响我们对FXS和唐氏综合征疾病生物学的理解。