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
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,