News and Publications

Intracellular RNA Assembly and Catalysis

RNAs are dynamic molecules, and exchange among alternative RNA structures is an essential feature of virtually all biological processes mediated by RNAs. To understand these processes in detail, we must understand the relationship between RNA structures and the rates and equilibria of structural rearrangements. Because of the participation of many components in complex pathways, RNA structural transformations can be difficult to address experimentally in a biological context. RNA enzymes, also called ribozymes, are important model systems for studying RNA structure and function for the simple reason that ribozymes report their structure through the reactions that they catalyze.

The application of pre-steady-state kinetics methods to RNA cleavage and ligation reactions mediated by hairpin ribozymes in vitro has shown how cleavage kinetics are highly sensitive to ribozyme structure and to ionic conditions (Fig. 1). H1 is the helical secondary structure that forms between ribozyme and cleavage product RNAs. Increasing the stability of H1 slows product dissociation. Changes in the ribozyme sequence or in ionic conditions that stabilize tertiary interactions between loop A and loop B also stabilize product binding and accelerate ligation. Thus, hairpin ribozymes cleave at maximum rates when secondary and tertiary structures are stable enough to assemble into a functional ribozyme but not so stable that cleavage is reversed by rapid religation of bound products.

We are doing quantitative studies of hairpin ribozyme activity in yeast to learn how structure-function relationships established through in vitro experiments translate to the behavior of RNA molecules in living cells. Hairpin ribozymes are expressed in yeast as chimeric self-cleaving mRNAs. Chimeric mRNAs containing self-cleaving ribozymes are compared with the same mRNAs containing an inactivating mutation in the ribozyme sequence. Chimeric mRNAs containing mutant ribozymes decay through endogenous mRNA degradation pathways. Self-cleaving mRNAs degrade through the normal pathway but also disappear through self-cleavage. Consequently, the difference in decay rates between mutant and self-cleaving mRNAs corresponds to the intracellular cleavage rate (Fig. 2).

Our initial comparison of intracellular decay rates for one active ribozyme and its corresponding mutant revealed an intracellular self-cleavage rate of 0.06 min^-1. This rate is 5-fold less than the cleavage rate determined for the same ribozyme sequence in vitro under standard conditions. In vitro, cleavage kinetics can be limited by rapid religation of bound products, trapping of ribozyme sequences in inactive structures, or instability of ribozyme secondary and tertiary structures under unfavorable ionic conditions. It was not clear at first whether any or all of these factors limited intracellular cleavage.

We next examined a series of ribozyme variants in which cleavage products bind the ribozyme through H1 sequences that range in length from 1 to 26 bp and vary in stability by almost 50 kcal/mol. H1 sequences with fewer than 3 bp do not support full activity in vivo or in vitro, arguing against any significant stabilization or destabilization of short RNA helices in the intracellular environment. When H1 contains more than 6 bp, most cleavage events are reversed by religation of bound products under standard conditions in vitro. However, ribozymes with as many as 26 bp in H1 continue to cleave at maximum rates in vivo. The failure of extended H1 sequences to inhibit intracellular cleavage suggests that ligation is not favored in vivo as it is under standard conditions in vitro or that some feature of the intracellular environment accelerates product dissociation.

We are now focused on determining how the intracellular environment modulates the stability of ribozyme secondary and tertiary structures. These results are expected to make important contributions to understanding more complex RNA-mediated reactions in vivo and to developing RNA-based therapeutic agents.

Publications

Donahue, C.P., Yadava, R.S., Nesbitt, S.M., Fedor, M.J. The kinetic mechanism of the hairpin ribozyme in vivo: Influence of RNA helix stability on intracellular cleavage kinetics. J. Mol. Biol. 295:693, 2000.

Fedor, M.J. Structure and function of the hairpin ribozyme. J. Mol. Biol. 297:269, 2000.

Fedor, M.J. Tertiary structure stabilization promotes hairpin ribozyme ligation. Biochemistry 38:11040, 1999.

Fedor, M.J., Donahue, C.P., Nesbitt, S.M. Hairpin ribozyme activity in vitro and in vivo. In: Ribozymes: Technology and Applications. Gaur, R.K., Krupp, G. (Eds.). Eaton Publishing, Natick, MA, 2000, p. 205.