IDENTIFICATION OF FUNCTIONAL RNAS THROUGH CYCLIC-PHOSPHATE CAPTURE

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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.