High-throughput technology for detection of RNA modifications

成熟的RNA含有许多修饰的核苷酸，它们都是在转录后通过特定酶的特定作用形成的。虽然我们的知识大多局限于高度丰富和稳定的RNA物种，如tRNA、rRNA和SnRNA，但关于其他细胞RNA中RNA修饰的存在和精确定位的数据很少，如snoRNAs、小调控RNAs和mRNAs。最近的发展为理解RNA修饰在细胞中的作用和功能带来了一个新的维度，调用了RNA修饰的表观遗传学特征。这些都对RNA修饰的稳定、持久和终生特性的既定概念提出了质疑。有证据支持RNA修饰是一种普遍的调控现象，因此是一种短暂的现象，可能具有与替代剪接或A-to-I编辑同等的重要性。最近的顶级论文清楚地确定了受监管的和受监管的RNA修饰。除了mRNA，这也适用于所谓的混凝土浇注的tRNA修饰，很容易预料到调节性RNA的延伸。尽管这些修饰在调节细胞代谢方面非常重要，但人们对它们在不同细胞RNA中的存在和确切定位知之甚少。最近的突破仅限于某些修改，并通过使用RNA序列或质谱学方法得以实现。专家们一致认为，该领域目前的瓶颈有两个，即：(1)检测特定RNA中修饰的化学结构；(2)绘制其定位图。目前，单个RNA分子的分析是一项困难和繁重的任务，这受到对起始材料的大量要求的限制。因此，这项提议旨在开发能够高通量分析RNA修饰的新技术。作为中心策略，我们将结合目前可用于高通量检测修饰的两个原则，即选择性化学转化和逆转录(RT)抑制。已知修饰的RNA将用各种已知或怀疑能在化学上改变修饰的试剂处理，例如在RT过程中改变它们的行为。这种改变的行为将被用来从已知修改的RNA序列数据中定义RT-签名，然后该RT-签名将识别转录组范围的RNA-SEQ数据中的候选位点。为了验证，将通过机器人辅助技术分离候选位置所在的RNA物种，数量足以进行LC-MS/MS分析。后者将被用来确认候选地点是否存在预测的修改。该项目的目标是为RNA修饰的全转录组分析提供工具，并进一步将这种分析扩展到正常发育和病理中RNA修饰模式的全球变化。

Mature RNAs contain numerous modified nucleotides which are all formed post-transcriptionally, by specific action of dedicated enzymes. While our knowledge is mostly limited to highly abundant and stable RNA species like tRNA, rRNA and snRNA, only fragmentary data is available on the presence and precise localization of RNA modifications in other cellular RNAs, like snoRNAs, small regulatory RNAs and mRNAs. Recent developments brought a new dimension in the understanding of RNA modification roles and functions in the cell, invoking an epigenetic character of RNA modifications. These question the established concept of a stable, durable, and life-long character of RNA modifications. Evidence supports the idea of RNA modification as a general regulatory and therefore transient phenomenon, potentially of an importance equal to alternative splicing or A-to-I editing. Recent top-ranking papers clearly identified regulated as well as regulatory RNA modifications. In addition to mRNA, this also applies to the supposedly concrete-cast tRNA modifications, and an extension to regulatory RNA is easily anticipated. Despite the key importance of these modifications in regulation of the cellular metabolism, little is known on their presence in different cellular RNAs and their exact localization. The recent breakthroughs are limited to certain modifications and have been enabled by the use of either RNA Seq or mass-spectrometry approaches. There is consensus among specialists, that the current bottlenecks in this field are twofold, namely: (i) the detection of the chemical structure of modifications in a given RNA and (ii) the mapping of their localizations. Currently, the analysis of individual RNA molecules is a difficult and laborious task, which is limited by the requirement for high amounts of starting material. Hence, this proposal aims at the development of new technologies allowing high-throughput analysis of RNA modifications. As a central strategy, we will combine two current principles that are available for high- throughput detection of modifications, namely selective chemical transformation and reverse transcription (RT) arrest. RNA with known modifications will be treated with various agents known or suspected to chemically transform the modifications such as to alter their behaviour during RT. Such altered behaviour will be used to define an RT-signature from RNA Seq data of known modifications, which will then identify candidate sites in transcriptome-wide RNA-seq data. For validation, RNA species hosting candidate sites will be isolated by robot-assisted technology in quantities sufficient for LC-MS/MS analysis. The latter will be applied to confirm the existence of predicted modification at candidate sites. The goal of the project is to provide tools for whole-transcriptome analysis of modifications in RNAs, with the further extension of such analysis to global changes of RNA modification pattern in normal development and pathologies.