Mass Spectrometry of Modified RNAs

修饰 RNA 的质谱分析

• 批准号: 10551333
• 负责人: PATRICK A LIMBACH
• 金额: $ 34.62万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10551333
• 关键词: Address; Affect; Anticodon; Bar Codes; Biological; Biological Assay; Biological Process; Cardiovascular Diseases; Catalogs; Cell Line; Cells; Cellular Stress; Censuses; Code; Comparative Study; Complex; Complex Mixtures; DNA; Development; Digestion; Discipline; Disease; Disease Progression; Effectiveness; Exclusion; Fibroblasts; Fractionation; Funding; Gene Expression; Generations; Goals; Human; Human Pathology; Individual; Investigation; Knowledge; Laboratories; Life; Liquid Chromatography; Location; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Metabolic; Metastatic Melanoma; Methods; Modification; Nucleosides; Nucleotides; Oligonucleotides; Pattern; Physiological; Population; Positioning Attribute; Preparation; Process; Progress Reports; Protein Biosynthesis; Proteins; RNA; Regulation; Research; Research Personnel; Resolution; Ribonucleosides; Role; Sampling; Techniques; Time; Tissues; Transcript; Transfer RNA; Variant; Work; cell growth regulation; cell type; data acquisition; density; high throughput technology; human disease; human tissue; innovation; insight; melanocyte; melanoma; next generation sequencing; novel; posttranscriptional; proteostasis; stem; success; tandem mass spectrometry

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.

项目摘要 细胞内蛋白质的合成需要转移核糖核酸（TRNA）。 trnas 被发现包含最高密度和最大的转录后修饰多样性 任何类RNA的核苷。我们对这些功能作用的科学理解 TRNA中的修饰核苷在很大程度上受到了缺乏常规的方法的限制 识别并映射修饰到主tRNA序列上。这项研究的长期目标 继续是开发适当的质谱方法来实现生物学 研究改进的TRNA的功能意义。这种续约专注于特定 从人类样本中识别和理解经过修改的TRNA的目标。这项研究是 及时且相关，因为近270个预测独特的人tRNA序列的少于15％ 在这个细节上已经表征了。此外，许多最近的研究 暗示TRNA及其在各种不同的人类疾病和病理中的修饰。 我们对人类TRNA的完整修改概况的了解受到缺乏的限制 能够识别特定修饰的核糖核苷和特定的生物分析方法 单个TRNA中的序列位置。根据我们在上一个支持时期的进步， 该更新中的三个具体目标通过建立一个 将定义样本中表达的TRNA的群体的工作流程，修改后的人口普查 存在核苷，以及这些修饰的核苷上的映射到特异性表达的特异性 trnas。我们的创新将导致创建tRNA特定于区域的修改分析 可用于跟踪多个样本中这些修饰的动态变化的测定 类型。我们将通过检查变更来证明我们方法的实用性和意义 这些特征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， 我们将采用一种可以记录的方法 在组织，细胞系或疾病状况中表达的修饰tRNA。我们的进步将为 能够洞悉tRNA修饰状态在蛋白质合成调节中的作用的能力， 蛋白质失衡或疾病状态维持。此外，生物分析发展 这项研究可以应用于科学研究，以了解如何了解 细胞调节RNA修饰模式以及可变的RNA修饰模式 影响其他细胞调节过程。