IDENTIFICATION OF FUNCTIONAL RNAS THROUGH CYCLIC-PHOSPHATE CAPTURE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 最近的实验已经产生了丰富的新型非编码RNA。我们最近开发了一种克隆策略来鉴定末端为2‘，3’环磷酸的RNA分子。环状磷酸盐广泛存在于生物学中：所有已知的天然核酶都离开环状磷酸末端，从人类基因组文库中分离出的新的自切割核酶暗示在人类和其他基因组中存在额外的核酶。此外，tRNA和tRNA样剪接事件、自发自溶和一些核糖核酸酶切割事件导致环状磷酸末端。为了便于对这类RNA的研究，我们开发了一种识别具有3‘端环状磷酸盐的RNA分子的方法。该方法使用来自Arabadsis thaliana的tRNA连接酶来选择性地将带有环磷酸盐的RNA连接到接头分子上，使其能够选择性扩增。重要的是，以前使用T4 RNA连接酶进行合成的方法不能捕获环磷酸终止的RNA，这使得这一RNA类别尚未被探索。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经历未折叠蛋白质反应的酵母细胞中捕获HAC1剪接中间体。在未来，我们计划使用这种方法从人的总RNA制剂中鉴定自裂解核酶。

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.