Circular RNAs and their interactions with RNA-binding proteins to modulate AD-related neuropathology

摘要 新的变异，特别是在非编码区，预计将通过正在进行的AD发现 测序项目(ADSP)。该提案将研究环状RNA(CircRNAs)和RNA结合蛋白 调节这些CircRNAs或受其调节的限制性商业惯例。最近的基因组研究发现了数千种 由蛋白质编码基因和基因组的非编码区通过一个过程产生的CircRNA 这就是所谓的反向拼接。CircRNAs在神经元组织中更加丰富，通常来自基因 专用于神经元和突触功能。这些环状RNA的发现需要协调一致的 研究与CircRNA相互作用的限制性商业惯例。已知限制性商业惯例的突变和功能障碍是 额颞叶痴呆、肌萎缩侧索硬化症和阿尔茨海默病的主要病理生理机制。然而， CircRNA：RBP网络在这些疾病机制中的作用在很大程度上尚不清楚。这部小说 CircRNAs的生物学为研究ADRD的神经退化机制打开了一扇全新的窗口。CircRNAs 可通过作为海绵将miRNA/RBPs与正常隔离开来，从而导致神经变性 MRNA靶标，改变剪接或表达。限制性商业惯例还通过结合 含有许多剪接因子/限制性商业惯例的保守结合位点的环状RNA的侧翼内含子序列。 因此，限制性商业惯例在蛋白质聚集体中的隔离可能会导致CircRNAs的功能失调。这个 基因组学的历史表明，每一个新的核苷酸物种的发现都扩展了我们对 疾病机制。CircRNA的发现提供了一条尚未探索的RNA新陈代谢的主要途径 要求调查。我们假设CircRNAs水平的变化有助于 ADRD的病理生理学以及衰老过程中关键CircRNA或CircRNA-RBP相互作用的发现 人类大脑可以发现新的生物标记物、疾病机制或治疗靶点。在这 提议，通过利用大量的公共和我们自己的RNA序列数据(rRNA耗尽)，我们将应用几个 从人脑多个区域检测和鉴定AD相关CircRNA的方法，并整合 有ADSP基因发现的患者(目标1)。在目标2中，除了从人脑中发现与AD相关的限制性商业惯例 RNA-SEQ、蛋白质组学和ADSP WES/WGS数据，我们将利用ENCODE CLIP-SEQ数据进行RBP 结合，以确定推测的RBP-CircRNA与AD的相互作用，即与AD相关的功能RNA元件。最后， 在目标3中，我们将选择前10%的CircRNAs(~200)和RBP(~150)用于进一步的高吞吐量 用一种新的、功能强大的ADRD三维人体器官模型进行功能评估，名为Astad，展示了 全面的tau病理和神经退行性变。我们预计，我们对ADSP的综合分析 遗传学、CircRNA、mRNA、RBP和高通量ASAD功能屏幕读数可以帮助生成 未来分子机制实验设计的可验证性假说。

SUMMARY New variants, especially in non-coding regions, are expected to be discovered through the ongoing AD Sequencing Project (ADSP). This proposal will investigate circular RNAs (circRNAs) and RNA binding proteins (RBPs) that regulate or are regulated by these circRNAs. Recent genomic studies have discovered thousands of circRNAs produced from both protein-coding genes and non-coding regions of the genome via a process known as back-splicing. CircRNAs are more enriched in neuronal tissues and are often derived from genes specific for neuronal and synaptic function. The discovery of these circRNAs demands a coordinated investigation of RBPs that interact with the circRNAs. Mutations in and dysfunction of RBPs are known to be major mechanisms contributing to the pathophysiology in frontotemporal dementia, ALS and AD. However, the contributions of the circRNA:RBP network to these disease mechanisms are largely unknown. The novel biology of circRNAs opens an entirely new window into mechanisms of neurodegeneration in ADRD. CircRNAs could contribute to neurodegeneration by acting as sponges that sequester miRNA/RBPs away from normal mRNA targets, altering splicing or expression. RBPs also regulate circRNA production by binding to the flanking intronic sequences of circRNAs which contain many conserved binding sites of splicing factors/RBPs. Thus, sequestration of RBPs in protein aggregates could cause dysfunctional regulation of circRNAs. The history of genomics indicate that discovery of each new nucleotide species expands our understanding of disease mechanisms. The discovery of circRNA presents a major unexplored avenue of RNA metabolism that demands investigation. We hypothesize that changes in the levels of circRNAs contributes to the pathophysiology of ADRD, and that discovery of key circRNAs or circRNA-RBP interactions in aging human brains could uncover novel biomarkers, disease mechanisms or therapeutic targets. In this proposal, by leveraging large public and our own RNA-seq data (rRNA-depleted), we will apply several methods to detect and characterize AD-related circRNAs from multiple human brain regions, and integrate them with ADSP genetic findings (Aim 1). In Aim 2, aside from discovering AD-related RBPs from human brain RNA-seq, proteomics and ADSP WES/WGS data, we will leverage the ENCODE CLIP-seq data for RBP binding to identify putative RBP-circRNA interactions with AD, i.e. AD-related functional RNA elements. Finally, in Aim 3, we will select the top 10% of the circRNAs (~200) and RBPs (~150) for further high-throughput functional evaluation with a novel, powerful 3D human organoid model of ADRD, termed AstAD that exhibits the full range of tau pathology and neurodegeneration. We anticipate that our integrative analyses of ADSP genetics, circRNA, mRNA, RBP and the high-throughput AstAD functional screen readouts can help generate testable hypothesis for future molecular mechanisms experimental design.