Unbiased discovery of mechanisms regulating circRNA

 描述(由申请人提供) RNA是一种古老的生物信息载体，其许多功能成为细胞生命进化所必需的，而RNA的转录和转录后调控对健康和人类疾病的发展至关重要。值得注意的是，我们已经发现，数千个人类基因产生环状RNA(CircRNA)亚型，并且在数百个基因中，CircRNA比线性异构体更丰富。我们最近的工作表明，CircRNA的表达在人类发育过程中受到特别的调控。我们还证明了环状RNA是在相隔数十亿年进化的有机体中产生的，这表明CircRNA的机制及其功能是真核基因表达程序的核心：要么保存了数十亿年，要么是一种多次重新进化的特征，这意味着CircRNA在细胞中具有功能作用。综上所述，我们的工作提出了一个基本假设，即替代剪接除了蛋白质生产外，还具有功能后果，包括CircRNA异构体的生产。然而，该领域缺乏顺式序列和反式作用因子的预测机制模型，这些反式作用因子专门调节CircRNA，这意味着a)细胞用来产生CircRNA的生化信号通路尚不清楚；b)我们缺乏分子工具来在没有非靶标RNA背景转录的情况下特异性地表达CircRNA。这种工具是发现CircRNA功能和进行严格的实验测试所必需的。这一提议旨在发现控制CircRNA产生和调控的机制，并有望揭示生化信号如何转化为选择性剪接的RNA分子的新生物学，并为发现CircRNA的功能提供关键工具。具体地说，我们的目标是：1)设计检测和量化CircRNA变体的统计算法，以及整合不同数据集表达的统计方法；2)发现调节CircRNA生产、输出和衰退的反式作用因子；3)系统地发现CircRNA丰度的顺式序列控制。这项工作将建立在我们对人类发育中环状蛋白表达调控的发现的基础上，描述它们在正常情况下的调控，以及调控失调可能如何导致神经变性和心肌病等疾病。

 DESCRIPTION (provided by applicant) RNA is an ancient carrier of biological information, whose many functions became necessary for the evolution of cellular life, and transcriptional and post-transcriptional regulation of RNA is central to health and the progression of human disease. Remarkably, we have discovered that thousands of human genes produce circular RNA (circRNA) isoforms and in hundreds of genes, the circRNA is more abundant than the linear isoform. Our recent work has shown that circRNA expression is particularly regulated during human development. We have also demonstrated that circular RNAs are produced in organisms separated by billions of years of evolution, which suggests that the machinery, and by implication, function, of circRNA is central to eukaryotic gene expression programs: either conserved over billions of years, or a feature that has re-evolved multiple times which implies a functional role for circRNAs in the cell. Together, our work suggests a fundamental hypothesis that alternative splicing has functional consequences apart from protein production, including the production of circRNA isoforms. Yet, the field lacks a predictive mechanistic model of the cis sequences and trans-acting factors that specifically regulate circRNA, meaning a) the biochemical signaling pathways used by the cell to produce circRNA are unknown; b) we lack molecular tools to specifically express circRNA without background transcription of off-target RNAs. Such tools are required for discovery and rigorous experimental tests of circRNA function. This proposal aims to discover the mechanisms controlling circRNA production and regulation and promises to reveal novel biology regarding how biochemical signals are transduced into alternatively spliced RNA molecules and provide crucial tools for discovering the function of circRNA. Specifically, we aim to 1) engineer statistical algorithms for detecting and quantifying circRNA variants, and statistical methods for integrating expression across datasets; 2) discover trans-acting factors regulating circRNA production, export and decay; 3) systematically discover cis sequence control of circRNA abundance. The work will build on our discoveries of regulated expression of circular in human development, to delineate their regulation under normal circumstances, and how dysregulation may contribute to diseases such as neurodegeneration and cardiomyopathy.