Intracellular Hairpin Ribozyme Catalysis: a Dissertation

Abstract

Ribozymes are catalytic RNA molecules capable of performing functions normally attributed to proteins. The hairpin ribozyme is derived from the (-) strand of the satellite RNA of Tobacco Ringspot Virus, where it functions in processing rolling circle transcription intermediates. The hairpin ribozyme catalyzes the breaking of a phosphodiester bond to form a 2'-3' cyclic phosphate and a 5' OH on the product termini. RNA substrates are recognized through Watson Crick base pairs. In theory, ribozymes are able to cleave any RNA that forms the correct secondary structure. Therefore, ribozymes have been designed to recognize specific targets through base pair interactions with the substrate recognition sequence of the ribozyme. This feature of catalytic RNAs gives them endless potential as antisense reagents. While tremendous effort has gone into elucidating the kinetic mechanism of ribozymes in vitro, very few studies have addressed ribozyme function in the intracellular environment. Previous studies have had varying success. And while in some cases ribozymes have clearly reduced gene expression, the experiments were not quantitative and did not provide any information regarding the kinetic pathway of catalysis in vivo. Improved understanding of intracellular cleavage reactions requires the development of a system that can directly measure cleavage rates in vivo. Utilizing a self-cleaving ribozyme cassette inserted into the yeast PGK1 gene we have developed a system to detect ribozyme cleavage products and directly measure the cleavage rates of the hairpin ribozyme in yeast. Furthermore, we have performed controls confirming detected cleavage activity is reflective of intracellular catalysis. As ribozyme activity requires the formation of a catalytically active structure, cleavage can act as a monitor for the assembly of a functional molecule. We have used this system to address the effect of helix stability on intracellular hairpin ribozyme activity. The results of these experiments have important implications for the design of antisense ribozymes. Furthermore, catalysis by small RNAs in vivo serves as a model system for more complex RNA catalyzed reactions that are implicated in mRNA processing and translation.