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

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rRNA、tRNA、核酶、核糖开关和终止子中高度定义的折叠是众所周知的，但在mRNAs中也发现了越来越多的定义结构，这些结构与稳定性，特别是可译性相关。因此，确定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。虽然在体外确定RNA结构的方法简单明了，但体内的研究却相当费力。目前，只有几种健壮的方法可用，如单元形状、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期间产生的特定错误签名。一个缺点是RT停止也是由自发终止或稳定的RNA二级结构产生的，并不是所有的单链特异性化学修饰都会导致RT签名。我们想要建立一种新的方法，将连接位置读取为单链区域。在单链RNA中，铅断裂诱导的2‘3’-环磷酸和5‘OH端通过特定的连接酶与接头融合，并进行深度测序。基于RT的cDNA终止不被登记。通过分析产生的两个RNA末端的连接产物，实现了背景信号的进一步减少。在实验方法的同时，我们将开发一种精确的生物信息学工具，用于定性和定量识别单链信号，以实现尽可能准确的RNA结构。由于直接测量某个位置的结构需要高阅读覆盖率(每个位置10-15次阅读)，我们还将开发按小间隔聚集记录信号的方法，以便即使在较低覆盖率的情况下也能得出可靠的结论。结合核糖体图谱分析，我们将使用这一新的正交策略来阐明在大肠杆菌中mRNA结构与翻译效率的相关性，因为最近发表的研究表明，结果高度矛盾。此外，我们想要分析在嗜冷微生物中，mRNA结构是如何在极低的温度下被调节的，从而实现高效的翻译。综上所述，我们想要开发一种新的高通量的RNA结构分析，以解决关于RNA折叠对稳定性、翻译效率的影响及其在不同细胞系统中的调节的重要问题。用于优化连接酶的表达质粒将被存放在非营利性载体收集Addgene中，这样科学界就可以使用这种方法。

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.