High-throughput structure probing of RNA based on specific ligation of cyclo-phosphate ends combined with deep sequencing

基于环磷酸末端特异性连接结合深度测序的RNA高通量结构探测

• 批准号: 431361947
• 负责人: Professor Dr. Mario Mörl
• 金额: --
• 依托单位: Arbeitsgruppe Biochemie und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/431361947?language=en
• 关键词: throughput; structure; probing; RNA; based

Similar to proteins, RNA molecules fold up into manifold and complex structures in order to fulfill their tasks in the cell. While highly defined folds in rRNAs, tRNAs, ribozymes, riboswitches and terminators are well-known for a long time, an increasing amount of defined structures are also found in mRNAs, correlating with stability and especially translatability. Hence, it is of great interest to identify the structures of all transcripts within a cell to better understand their functionality and regulation. While in vitro approaches to characterize RNA structures are straightforward and well established, the in vivo investigation is rather laborious. Currently, there are only a few robust methods available, like in cell SHAPE, DMS-Seq and their variants SHAPE-MaP und DMS-MaPseq. These approaches are based on the read-out of RT stop signals or specific error signatures generated during RT. A disadvantage is that RT stops are also generated by spontaneous termination or stable RNA secondary structures and that not all single-strand specific chemical modifications lead to RT signatures. We want to establish a novel method that reads ligation positions as single stranded regions. 2’3’-cyclo-phosphate and 5’OH ends induced by lead cleavage in single-stranded RNA are fused to adapters by specific ligases and identified by deep sequencing. RT-based cDNA terminations are not registered. A further reduction of the background signal is achieved by the analysis of ligation products of both generated RNA ends. In parallel to the experimental approach, we will develop a precise bioinformatic tool for qualitative and quantitative recognition of single-strand signals to achieve RNA structures as accurate as possible. As a direct measure of the structure at a certain position requires a high read coverage (10-15 reads per position), we will also develop methods that aggregate the recorded signal along small intervals so that reliable conclusions can be drawn even with lower coverage.Combined with ribosome profiling analyses, we will use this novel orthogonal strategy to clarify how mRNA structure correlates with translational efficiency in E. coli, as recently published investigations show highly conflictive results. In addition, we want to analyze in psychrophilic microorganisms how mRNA structures are regulated at extremely low temperatures to allow efficient translation.Taken together, we want to develop a novel high throughput analysis of RNA structures to address important questions concerning the impact of RNA folding on stability, translational efficiency and their regulation in different cellular systems. Expression plasmids for the optimized ligases will be deposited in the non-profit vector collection addgene, so that the scientific community has access to this method.

与蛋白质类似，RNA分子折叠成多种复杂的结构，以完成它们在细胞中的任务。虽然在rnas、tRNAs、核酶、核开关和终止子中高度定义的折叠已经为人所熟知，但在mrna中也发现了越来越多的定义结构，这与稳定性，特别是可翻译性有关。因此，识别细胞内所有转录本的结构以更好地了解它们的功能和调控是非常有意义的。虽然体外方法表征RNA结构是直接和完善的，但体内研究是相当费力的。目前，只有少数几个可靠的方法可用，如在细胞中SHAPE、DMS-Seq及其变体SHAPE- map和DMS-MaPseq。这些方法基于RT停止信号的读出或在RT过程中产生的特定错误签名。缺点是RT停止也由自发终止或稳定的RNA二级结构产生，并且并非所有单链特异性化学修饰都会导致RT签名。我们想建立一种新的方法，读取连接位置作为单链区域。单链RNA中由铅切割诱导的2 ' 3 ' -环磷酸和5'OH末端通过特定的连接酶融合到适配器上，并通过深度测序进行鉴定。基于rt的cDNA终止未被记录。进一步减少背景信号是通过分析两个生成的RNA末端的连接产物来实现的。与实验方法并行，我们将开发一种精确的生物信息学工具，用于单链信号的定性和定量识别，以尽可能准确地实现RNA结构。由于直接测量某个位置的结构需要高读取覆盖率（每个位置10-15个读取），我们还将开发沿小间隔汇总记录信号的方法，以便即使覆盖率较低也可以得出可靠的结论。结合核糖体分析，我们将使用这种新颖的正交策略来阐明mRNA结构如何与大肠杆菌的翻译效率相关，因为最近发表的研究显示了高度冲突的结果。此外，我们想分析在嗜冷微生物中mRNA结构是如何在极低温度下被调节以允许有效翻译的。综上所述，我们希望开发一种新的高通量RNA结构分析，以解决有关RNA折叠对稳定性，翻译效率及其在不同细胞系统中的调节的影响的重要问题。优化后的连接酶的表达质粒将存放在非营利载体收集addgene中，以便科学界能够使用该方法。