The Scripps Research Institute
  News Room Contacts  
  Information for Journalists  
  Calendar of Events  



News and Publications

The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999

Intracellular RNA Assembly and Catalysis

M.J. Fedor, A.J. Choi, C.P. Donahue, S. Nesbitt, T. Vivlemore, R. Yadava

Despite the biological importance of RNA-mediated reactions in RNA processing and translation, it remains unclear how reaction pathways defined for RNA enzymes through biochemical studies in vitro relate to RNA-mediated reactions in living cells. To address this gap, we are analyzing a simple RNA-catalyzed reaction in yeast. Because of its small size and simple mechanism, the hairpin ribozyme is an excellent model for more complex RNA-mediated processes that occur within cells.

A minimal hairpin ribozyme contains 2 helix-loop-helix segments with the reactive phosphodiester located in one of the loops (Fig. 1A). This ribozyme cleaves phosphodiester bonds in a reversible reaction that generates 5´ hydroxyl and 2´,3´-cyclic phosphate termini. In Nature, the hairpin ribozyme is part of a virus-associated RNA genome in which ribozyme-mediated self-cleavage and ligation reactions participate in processing intermediates of RNA replication. In the complete RNA genome, the catalytically essential helix-loop-helix segments combine with 2 additional RNA helices to create a 4-way helical junction (Fig. 1B). Our recent studies suggest that these additional helices, although not essential for catalysis, maintain the appropriate balance between cleavage and ligation activities.

To examine intracellular activity of the hairpin ribozyme, we express hairpin ribozymes in yeast as chimeric self-cleaving mRNAs. Chimeric mRNAs containing self-cleaving hairpin ribozymes are compared with the same mRNAs containing an inactivating mutation in the hairpin sequence. Chimeric mRNA containing mutant hairpin ribozymes decays through endogenous pathways of mRNA degradation. 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 RNAs corresponds to the intracellular cleavage rate (Fig. 2).

RNA catalysis depends on proper assembly of a functional RNA structure, so this system provides a unique op portunity to assess RNA structure and dynamics inside living cells. Under standard conditions in vitro, cleavage events catalyzed by minimal hairpin ribozymes are reversed by rapid religation when cleavage products remain bound to the ribozyme. Consequently, ribozymes have sharply reduced self-cleavage rates in vitro when products remain bound to the ribozyme long enough to undergo ligation. In contrast, ribozymes with products bound in very long, stable H1 sequences self-cleave at nearly normal rates in vivo. The failure of large products to inhibit self-cleavage could be explained if intracellular factors promote rapid helix dissociation. Alternatively, the balance between cleavage and ligation might not favor ligation in vivo as it does in vitro.

Our recent experiments allow us to distinguish whether the intracellular environment affects helix stability or the balance between cleavage and ligation. Ribozymes that contain a 4-way helical junction bind cleavage products more tightly and favor ligation over cleavage even more strongly than do minimal hairpin ribozymes. When we examined intracellular cleavage activity of ribozymes with 4-way helical junctions, we found normal intracellular cleavage rates for ribozymes with small cleavage products that are expected to dissociate rapidly. However, intracellular cleavage activity decreased when cleavage products were large and the H1 helix was expected to dissociate slowly. Therefore, intracellular cleavage has the same dependence on the size of the cleavage product and the stability of the RNA helix as in vitro cleavage reactions do. In vivo, the 4-way helical junction maintains the proper balance between cleavage and ligation.

Insights gleaned from these investigations will facilitate rational design of antisense ribozymes for specific and efficient cleavage of RNA targets in vivo. A hairpin ribozyme with a 4-way helical junction also might be used to repair defective RNAs in vivo through RNA-catalyzed ligation.


Fedor, M.J. Ribozymes. Curr. Biol. 8:R441, 1998.

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

Nesbitt, S.M., Erlacher, H.A., Fedor, M.J. The internal equilibrium of the hairpin ribozyme: Temperature, ion and pH effects. J. Mol. Biol. 286:1009, 1999.



Copyright © 2004 TSRI.