CAREER: Uncovering the regulation of low fidelity mRNA splicing

职业：揭示低保真 mRNA 剪接的调控

• 批准号: 2237568
• 负责人: Athma Pai
• 金额: $ 100万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2237568&HistoricalAwards=false
• 关键词: CAREER; Uncovering; regulation; low; fidelity

The central dogma of molecular biology involves the synthesis of ribonucleic acid (RNA) from the genes defined in deoxyribonucleic acid (DNA) molecules. Some of these RNA molecules then act as messengers that shuttle instructional blueprints to enable proper cellular function. The informational content encoded by RNA is amplified by the process of splicing, which enables the creation of dozens of different RNA molecules from a single gene sequence. The process of splicing is very complex, with one of the largest molecular complexes and many auxiliary factors working together to coordinate the precise levels and compositions of messenger RNAs. This intricacy opens the door for noise and errors in splicing that may comprise a substantial fraction of RNA output in a cell. While quality control mechanisms to check for and correct such errors have been identified over the last decade, less is known about how often these errors occur and why certain erroneous RNAs escape detection and removal. This project will use novel genomics and computational tools to identify directly and quantify erroneously spliced RNAs, gaining a better understanding of the regulation of proper RNA synthesis for cellular function. More broadly, the project aims to teach the cross-cutting skills of biological data analysis to up and coming researchers and educators by (1) hosting Worcester area undergraduates for a combined summer research and outreach experience in computational biology and (2) facilitating an enrichment program at UMass Chan Medical School for Worcester Public School science teachers.For decades, the process of mRNA splicing was thought to be highly efficient, with little opportunity for deviation outside of regulated alternative splicing decisions. This idea led to a widespread perspective that all alternative mRNA isoforms must be regulated and, potentially, have biological function. Recently, however, researchers have identified pervasive, non-canonical splice site choice, including those at low-fidelity spliceosome binding sequences. These discoveries suggest that there is widespread error and cryptic splice site usage by mRNA splicing mechanisms, with unexplored roles for stochastic, biological noise in shaping metazoan transcriptome diversity. This project will use high-throughput sequencing of nascent RNA to track splicing intermediates through cellular compartments, creating a catalog of cryptic splice sites and examining genetic and biochemical features associated with these events. These studies will provide insight into how and when undesirable splicing products escape or are pruned by mRNA quality control mechanisms. Leveraging this catalog, a statistical model will be built to enable a predictive understanding of cryptic splice site usage and employ gene editing tools to test predictions from this model. The long-term research goal of this project is to define what constitutes noise in mRNA splicing and use these studies to redefine the mRNA splicing code.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

分子生物学的中心法则涉及从脱氧核糖核酸（DNA）分子中定义的基因合成核糖核酸（RNA）。然后，这些RNA分子中的一些充当信使，穿梭于指导蓝图以实现适当的细胞功能。由RNA编码的信息内容通过剪接过程放大，这使得能够从单个基因序列创建数十个不同的RNA分子。剪接的过程非常复杂，其中一个最大的分子复合物和许多辅助因子共同作用，以协调信使RNA的精确水平和组成。这种复杂性为剪接中的噪声和错误打开了大门，这些噪声和错误可能构成细胞中RNA输出的相当大一部分。虽然在过去的十年中已经确定了检查和纠正这些错误的质量控制机制，但对这些错误发生的频率以及为什么某些错误的RNA逃脱检测和去除知之甚少。该项目将使用新的基因组学和计算工具来直接识别和量化错误剪接的RNA，从而更好地了解细胞功能的正确RNA合成的调控。更广泛地说，该项目旨在通过以下方式向即将到来的研究人员和教育工作者教授生物数据分析的跨领域技能：（1）主持伍斯特地区的本科生进行计算生物学的夏季研究和推广经验，以及（2）促进马萨诸塞大学陈医学院为伍斯特公立学校科学教师提供的丰富课程。mRNA剪接过程被认为是高效的，几乎没有机会偏离受调节的选择性剪接决定。这一想法导致了一个广泛的观点，即所有替代mRNA亚型都必须受到调控，并可能具有生物学功能。然而，最近，研究人员已经确定了普遍的，非典型的剪接位点的选择，包括那些在低保真度剪接体结合序列。这些发现表明，mRNA剪接机制存在广泛的错误和隐蔽的剪接位点使用，随机生物噪声在形成后生动物转录组多样性中的作用尚未探索。该项目将使用新生RNA的高通量测序来跟踪细胞隔室中的剪接中间体，创建一个隐藏剪接位点的目录，并检查与这些事件相关的遗传和生物化学特征。这些研究将提供深入了解如何以及何时不需要的剪接产物逃脱或修剪的mRNA质量控制机制。利用这个目录，将建立一个统计模型，以实现对隐蔽剪接位点使用的预测性理解，并采用基因编辑工具来测试该模型的预测。该项目的长期研究目标是确定mRNA剪接中噪音的构成，并利用这些研究重新定义mRNA剪接代码。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。