IDENTIFICATION OF FUNCTIONAL RNAS THROUGH CYCLIC-PHOSPHATE CAPTURE

通过环磷酸盐捕获鉴定功能性 RNA

• 批准号: 8171313
• 负责人: STANLEY FIELDS
• 金额: $ 4.42万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171313
• 关键词: Autolysis; Biology; Catalytic RNA; Cells; Cleaved cell; Cloning; Computer Retrieval of Information on Scientific Projects Database; Event; Functional RNA; Funding; Future; Genome; Genomic Library; Grant; Human; Institution; Left; Methods; Preparation; Proteins; RNA; RNA Ligase (ATP); RNA Splicing; Research; Research Personnel; Resources; Ribonucleases; Source; System; Transfer RNA; United States National Institutes of Health; Yeasts; inorganic phosphate; novel; research study; response; tRNA Ligase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent experiments have yielded an abundance of novel non-coding RNAs. We have recently developed a cloning strategy for the identification of RNA molecules with terminal 2???,3??? cyclic phosphates. Cyclic phosphates are pervasive in biology: all known natural ribozymes leave cyclic phosphate termini, and novel self-cleaving ribozymes isolated from human genomic libraries allude to the presence of additional ribozymes in human and other genomes. In addition, tRNA and tRNA-like splicing events, spontaneous autolysis and some ribonuclease cleavage events result in cyclic phosphate termini. In order to facilitate the study of this RNA class, we have developed a method for the identification of RNA molecules with 3??? terminal cyclic phosphates. The method employs the tRNA ligase from Arabadopsis thaliana to selectively ligate RNAs with cyclic phosphates to an adaptor molecule, enabling their selective amplification. Importantly, previous methods employing T4 RNA ligase for cature do not capture cyclic phosphate-terminated RNAs, leaving this RNA class unexplored. We have completed proof-of-principle experiments which demonstrate the selective capture of these molecules, and have also shown that the system can capture HAC1 splicing intermediates from yeast cells undergoing the unfolded protein response. In the future, we plan to use this method for identifying self-cleaving ribozymes from human total RNA preparations.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 最近的实验产生了大量新型非编码 RNA。 我们最近开发了一种克隆策略，用于鉴定末端 2???,3??? 的 RNA 分子。环状磷酸酯。 环状磷酸盐在生物学中普遍存在：所有已知的天然核酶都留下环状磷酸盐末端，从人类基因组文库中分离出的新型自切割核酶暗示人类和其他基因组中存在其他核酶。 此外，tRNA 和类 tRNA 剪接事件、自发自溶和一些核糖核酸酶切割事件导致环状磷酸末端。 为了促进该RNA类的研究，我们开发了一种用3??? 鉴定RNA分子的方法。末端环状磷酸酯。 该方法利用来自拟南芥的 tRNA 连接酶将带有环磷酸盐的 RNA 选择性地连接到接头分子上，从而实现选择性扩增。 重要的是，以前使用 T4 RNA 连接酶进行捕获的方法不能捕获环状磷酸盐封端的 RNA，从而导致此类 RNA 未被探索。 我们已经完成了原理验证实验，证明了这些分子的选择性捕获，并且还表明该系统可以从正在进行未折叠蛋白反应的酵母细胞中捕获 HAC1 剪接中间体。 将来，我们计划使用这种方法从人类总RNA制剂中鉴定自切割核酶。