Mass Spectrometry of Modified RNAs

项目总结 细胞内蛋白质的合成需要转移核糖核酸(TRNAs)。转录核糖核酸 发现含有最高密度和最大多样性的转录后修饰 任何一类RNA的核苷。我们对这些功能作用的科学认识 TRNAs中的修饰核苷在很大程度上受到缺乏常规方法的限制 识别修改并将其映射到初级tRNA序列上。这项研究的长期目标是 继续开发适当的质谱学方法，使生物 研究修饰tRNAs的功能意义。此次续订主要针对特定的 从人类样本中识别和理解修饰的tRNA的目标。这项研究是 在预测的近270个独特的人类tRNA序列中，只有不到15%的序列是及时和相关的 都被描述在这个细节的层面上。此外，最近的一些研究表明， 与多种不同的人类疾病和病理有关的tRNAs及其修饰。 我们对人类tRNAs的完整修饰图谱的了解受到缺乏的限制 生物分析方法能够识别特定的修饰核糖核苷和特定的 单个tRNA中的序列位置。根据我们在上一次支持期间的进展， 这次更新的三个具体目标通过建立一个 将定义样本中表达的tRNA群体的工作流，即修改的人口普查 核苷的存在，以及这些修饰的核苷到特定表达的 TRNAs。我们的创新将导致tRNA区域特定修饰图谱的创建 可用于跨多个样本跟踪这些修改的动态变化的分析 类型。我们将通过研究以下方面的变化来展示我们方法的实用性和重要性 这些资料使用黑素细胞和各种黑色素瘤。 这项研究计划将对多个领域和学科产生重大影响。完成后， 我们将有一种方法来记录质量和数量差异 修饰的tRNA在组织、细胞系或疾病状态下表达。我们的进步将提供 能够洞察tRNA修饰状态在蛋白质合成调节中的作用， 蛋白质代谢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，试图了解 该细胞调节RNA修饰模式以及如何改变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.