Genome-wide Mapping of Ribosome Occupancies with Inhibitor-Induced mRNA Covalent Labeling

标题：抑制剂诱导的mRNA共价结合的核糖体全基因组定位 标签 核糖体分析是一种通过基因组范围的定位来研究翻译的有力方法。 翻译核糖体。这项突破性的技术已经阐明了蛋白质合成的许多方面， 例如核糖体暂停、替代起始位点和蛋白质合成速率， 蜂窝系统虽然最近的创新已经将核糖体谱减少到单细胞，但核糖体谱仍然是一个复杂的过程。 分析通常需要大量的细胞来进行，因此通常不用于研究 罕见的细胞类型和临床样本。此外，核糖体分析片段mRNA，因此 不能沿着单个mRNA分子绘制多个核糖体沿着， mRNA测序数据集以说明转录物的总数。新的有效方法， 通过保持mRNA完整性同时测量核糖体占有率和mRNA丰度 这将进一步促进对翻译调控的全球研究，并提供新的基本见解。 翻译动力学为了弥合这些技术差距，我们建议开发新的小型 基于天然产物核糖体抑制剂的化学足迹核糖体分子工具 通过共价标记定位在mRNA转录物的顶部。我们的核心假设是， mRNA化学足迹可以通过易错逆转录和随后的 mRNA测序以定位核糖体占位，同时保持mRNA的完整性。这 核糖体突变分析（Ribo-MaP）。为了开发Ribo-MaP， 提出了基于结构的天然产物衍生物的设计， 伸长，同时保持多核糖体稳定性。关键的是，这些衍生品是基于经验， 核糖体抑制剂，其接触mRNA模板以阻断翻译。此外，光亲和性 标记基团将连接到这些雌激素衍生物上， 会得到考验此外，提出了mRNA共价标记的替代方法，其中 将用反应性酰化基团官能化雌激素衍生物以标记近端mRNA 2 ′- 羟基。在这两种方法中，mRNA标记效率和读出共价修饰的能力都被提高。 因为将测量易错逆转录中的突变。在第二个目标中， 将对核糖体占据基因组范围内的Ribo-MaP进行评估和基准测试， 常规核糖体分析。我们预计Ribo-MaP将绕过核糖体中的低效步骤， 分析和下降到两个强大的mRNA测序方法和新的精简工作流程 单细胞和长读mRNA测序的新兴技术。总之，Ribo-MaP将 为广泛研究细胞内的蛋白质合成和核糖体动力学提供了一种高度可行的方法。

Title: Genome-wide Mapping of Ribosome Occupancies with Inhibitor-Induced mRNA Covalent Labeling Ribosome profiling is a powerful method to study translation through genome-wide mapping of translating ribosomes. This breakthrough technology has illuminated many facets of protein synthesis, such as ribosomal pausing, alternative initiation sites, and protein synthesis rates across numerous cellular systems. Although recent innovations have reduced ribosome profiling to single cells, ribosome profiling typically requires large numbers of cells to conduct and therefore is not routinely used to study rare cell types and clinical samples. Furthermore, ribosome profiling fragments mRNA and hence cannot map multiple ribosomes along a single mRNA molecule and requires integration of separate mRNA sequencing datasets to account for the total number of transcripts. New efficient methods to simultaneously measure ribosome occupancies and mRNA abundance by preserving mRNA integrity would further enable the global study of translational regulation and provide new fundamental insight into translation dynamics. To bridge these technological gaps, here we propose to develop new small molecule tools based off the natural product ribosome inhibitors to chemically footprint ribosome positioning atop mRNA transcripts through covalent labeling. Our central hypothesis is that these mRNA chemical footprints can be detected through error prone reverse transcription and subsequent mRNA sequencing to map ribosome occupancies while maintaining the integrity of the mRNA. This approach is termed ribosome mutational profiling (Ribo-MaP). To develop Ribo-MaP, the first aim proposes the structure-based design of natural product derivatives that potently inhibit ribosome elongation while maintaining polysome stability. Critically, these derivatives are based off emetine, a ribosome inhibitor which contacts the mRNA template to block translation. Furthermore, photoaffinity labeling groups will be attached to these emetine derivatives and the ability to covalently label mRNA will be tested. Additionally, an alternative approach for mRNA covalent labeling is proposed, in which emetine derivatives will be functionalized with reactive acylating groups to label the proximal mRNA 2'- hydroxyl. In both approaches, mRNA labeling efficiency and the ability to readout covalent modifications as mutations in error prone reverse transcription will be measured. In the second aim, the ability of Ribo-MaP to map ribosome occupancies genome-wide will be evaluated and benchmarked against conventional ribosome profiling. We expect that Ribo-MaP will bypass inefficient steps in ribosome profiling and drop into the streamlined workflow of both robust mRNA sequencing methods and new emerging technologies for single-cell and long-read mRNA sequencing. Altogether, Ribo-MaP would provide a highly enabling method to broadly study protein synthesis and ribosome dynamics in cells.