Mass Spectrometry of Modified RNAs

项目摘要 转运核糖核酸（Transfer ribonucleic acids，tRNA）是细胞内蛋白质合成所必需的。tRNAs 被发现含有最高密度和最大多样性的转录后修饰 任何种类的RNA的核苷。我们对这些功能作用的科学理解 tRNA中修饰的核苷在很大程度上受到缺乏常规方法的限制， 鉴定修饰并将其映射到一级tRNA序列上。这项研究的长期目标是 仍然是开发适当的质谱方法，使生物 研究修饰的tRNA的功能意义。这次更新的重点是具体的 目标是从人类样本中识别和理解修饰的tRNA。本研究是 及时且相关，因为在近270个预测的独特人类tRNA序列中，只有不到15% 都被描述成这样的细节此外，最近的一些研究 在多种不同的人类疾病和病理学中涉及tRNA及其修饰。 我们对人类tRNA完整修饰谱的了解受到缺乏 能够鉴定特定修饰的核糖核苷的生物分析方法， 单个tRNA内的序列位置。根据我们在上一个支持期的进展， 这次更新的三个具体目标通过建立一个 该工作流程将定义样品中表达的tRNA群体，修饰的tRNA的普查， 存在的核苷，以及将那些修饰的核苷映射到特异性表达的 tRNA。我们的创新将导致创建tRNA区域特异性修饰谱 可用于跟踪多个样品中这些修饰的动态变化的测定 类型我们将通过检查 这些图谱使用黑色素细胞和各种黑色素瘤。 该研究计划将对多个领域和学科产生重大影响。完成后， 我们将有一种方法，可以记录质量和数量的差异， 在组织、细胞系或疾病状况中表达的修饰的tRNA。我们的进步将提供 能够深入了解tRNA修饰状态在蛋白质合成调控中的作用， 蛋白质稳态失衡或疾病状态维持。此外，生物分析的发展 这项研究的结果可以应用于科学调查，以了解如何 细胞调节RNA修饰模式， 影响其他细胞调节过程。

PROJECT SUMMARY Transfer ribonucleic acids (tRNAs) are required for the synthesis of proteins within cells. tRNAs are found to contain the highest density and greatest diversity of post-transcriptionally modified nucleosides of any class of RNAs. Our scientific understanding of the functional role of these modified nucleosides in tRNAs is limited in large part by the lack of methods that can routinely identify and map modifications onto a primary tRNA sequence. The long-term goal of this research continues to be to develop appropriate mass spectrometric approaches that enable biological studies into the functional significance of modified tRNAs. This renewal is focused on the specific goal of identifying and understanding modified tRNAs from human samples. This research is timely and relevant as less than 15% of the nearly 270 predicted unique human tRNA sequences have been characterized at this level of detail. Moreover, a number of recent studies have implicated tRNAs and their modifications in a variety of different human diseases and pathologies. Our knowledge of the complete modification profiles of human tRNAs has been limited by the lack of bioanalytical methods capable of identifying specific modified ribonucleosides and particular sequence locations within individual tRNAs. Based on our advances in the last support period, the three specific aims in this renewal address the short-comings in the field by establishing a workflow that will define the population of tRNAs expressed in the sample, the census of modified nucleosides present, and the mapping of those modified nucleosides onto the specific expressed tRNAs. Our innovations will lead to the creation of tRNA region-specific modification profiling assays that can be used to track dynamic changes in these modifications across multiple sample types. We will demonstrate the utility and significance of our approach by examining changes in these profiles using melanocytes and various melanomas. This research plan will have significant impacts on multiple fields and disciplines. When complete, we will have an approach that can document the qualitative and quantitative differences of modified tRNA expressed in tissue, cell lines or disease condition. Our advances will provide the ability to develop insights into the role of tRNA modification status in protein synthesis regulation, proteostasis imbalances or disease state maintenance. Moreover, the bioanalytical developments to result from this research can be applied to scientific investigations that seek to understand how the cell regulates RNA modification patterns as well as how variable RNA modification patterns affect other cellular regulatory processes.