IDENTIFICATION OF FUNCTIONAL RNAS THROUGH CYCLIC-PHOSPHATE CAPTURE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 最近的实验已经产生了大量的新的非编码RNA。 我们最近开发了一种克隆策略，用于鉴定具有末端2？的RNA分子，三个？环状磷酸盐 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，并且从人类基因组文库中分离的新型自切割核酶暗示了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。 为了便于这类RNA的研究，我们开发了一种用3？末端环状磷酸酯。 该方法采用来自拟南芥的tRNA连接酶来选择性地将具有环状磷酸酯的RNA连接到衔接子分子，从而使其能够选择性扩增。 重要的是，以前的方法采用T4 RNA连接酶的cature不捕获环状磷酸封端的RNA，留下这种RNA类未探索。 我们已经完成了证明这些分子的选择性捕获的原理性实验，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HAC 1剪接中间体。 在未来，我们计划使用这种方法来识别自切割核酶从人类总RNA制剂。

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.