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专用酶的特异性作用形成的。虽然我们的知识主要限于高度丰富和稳定的RNA种类，如tRNA，rRNA和snRNA，但只有零碎的数据可用于其他细胞RNA中RNA修饰的存在和精确定位，如snoRNA，小调控RNA和mRNA。最近的发展带来了一个新的层面在理解RNA修饰的作用和功能在细胞中，调用RNA修饰的表观遗传特征。这些问题的一个稳定的，持久的，和终身的RNA修饰特性的既定概念。证据支持RNA修饰是一种普遍的调节现象，因此是一种短暂的现象，其重要性可能与选择性剪接或A到I编辑相等。最近的顶级论文清楚地确定了受调控的RNA修饰以及调控RNA修饰。除了mRNA之外，这也适用于假定的混凝土铸造tRNA修饰，并且很容易预期到调节RNA的延伸。尽管这些修饰在调节细胞代谢中至关重要，但对它们在不同细胞RNA中的存在及其确切定位知之甚少。最近的突破仅限于某些修饰，并且已经通过使用RNA Seq或质谱方法实现。专家们一致认为，该领域目前的瓶颈是双重的，即：（i）检测给定RNA中修饰的化学结构和（ii）绘制其定位。目前，单个RNA分子的分析是一项困难且费力的任务，这受到对大量起始材料的要求的限制。因此，该提案旨在开发允许RNA修饰的高通量分析的新技术。作为中心策略，我们将联合收割机结合两个当前可用于高通量检测修饰的原理，即选择性化学转化和逆转录（RT）停滞。具有已知修饰的RNA将用已知或怀疑化学转化修饰的各种试剂处理，例如改变其在RT期间的行为。这种改变的行为将用于从已知修饰的RNA Seq数据中定义RT签名，然后将识别转录组范围的RNA-seq数据中的候选位点。对于验证，将通过机器人辅助技术分离承载候选位点的RNA物质，其数量足以进行LC-MS/MS分析。后者将用于确认候选位点是否存在预测的修饰。该项目的目标是为RNA修饰的全转录组分析提供工具，并将这种分析进一步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.