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: 7957711
• 负责人: STANLEY FIELDS
• 金额: $ 5.46万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957711
• 关键词: Autolysis; Biology; Catalytic RNA; Cells; Cleaved cell; Cloning; Computer Retrieval of Information on Scientific Projects Database; Event; Functional RNA; Funding; Fungal Genome; 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. 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 最近的实验产生了大量新型的非编码RNA。我们最近制定了一种克隆策略，用于鉴定具有末端2'，3'环状磷酸盐的RNA分子。循环磷酸盐在生物学上普遍存在：所有已知的天然核酶都留下环状磷酸末端，而从人类基因组文库中分离出的新型自我裂解的核酶则暗示了人类和其他基因组中其他核酶的存在。此外，tRNA和tRNA样剪接事件，自发自体和某些核糖核酸酶裂解事件导致循环磷酸末端。为了促进该RNA类别的研究，我们开发了一种鉴定具有3'末端循环磷酸盐的RNA分子的方法。该方法采用来自阿拉伯thaliana的tRNA连接酶，与循环磷酸盐有选择地将RNA与衔接子分子结合，从而可以选择性扩增。重要的是，使用T4 RNA连接酶进行cature的先前方法不会捕获循环磷酸终止的RNA，而该RNA类别未探索。我们已经完成了原理证明实验，这些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经历未折叠蛋白质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来识别人类总RNA制剂中的自我裂解的核酶。