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.

脆弱的X智力低下1（FMR1）编码RNA结合蛋白FMRP。 FMRP的丧失对于脆弱的X综合征（FXS）是病因，X综合征是智力障碍的主要遗传原因和最常见的自闭症形式。最近，我们首次定义了人类胚胎干细胞（HESC）和体外衍生兴奋性皮质神经元中FMRP的全局RNA靶标。从这些数据集中，我们在前MRNA靶标内含子上发现了大约三分之一的FMRP结合事件，并检测到从染色体21（HSA21）转录的大量FMRP RNA靶标，这是与唐氏综合征相关的染色体。这些RNA靶标中的许多也被绑定为前MRNA。虽然FMRP在细胞质中的翻译调节背景下最常研究，但新兴数据表明FMRP在核中转录和转录后过程中的其他功能。但是，FMRP和MRNA之间关联之间的分子机制仍然未知，因此是该应用的重点。鉴于它们的疾病相关性，我们将特别关注从HSA21转录的FMRP Pre-MRNA靶标子集。实际上，我们的数据支持了新的假设，即智力障碍，FXS和唐氏综合症（DS）的两个最常见的遗传原因，融合了共同的分子机制。在AIM I中，我们选择了一组密钥HSA21编码的FMRP靶标，以研究前MRNA结合事件和下游分子后果的功能相关性。现在，我们将定义介导前MRNA结合的相关FMRP结构域，直接测试前mRNA结合对观察到的蛋白质水平变化的相关性，并扩展患者细胞中下游分子的影响。在AIM II中，我们将测试新的假设，即FMRP结合MRNA以在剪接中起着直接抑制作用，部分基于RNA-SEQ数据，显示了FMRP丢失后差异转录本的使用情况。具体而言，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损失对剪接动力学的影响，并评估FMRP结合/拼接是否共转录。在AIM III中，我们将检验以下假设：FMRP与染色质结合使用FMRP CHIP-SEQ是其与前MRNA的关联的基础，该芯片seq可以阐明FMRP目标识别的新型机制。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的影响，并探测FMRP结合和已知染色质特征之间的关系。总的来说，该项目将阐明FMRP生物学在RNA处理中的新颖和基本方面，并有可能影响我们对FXS和Downdrome疾病生物学的理解。