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The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998


Intracellular RNA Assembly and Catalysis


M.J. Fedor, C.P. Donahue, H.A. Erlacher, S. Nesbitt

The mechanisms used by some RNA enzymes to catalyze biochemical reactions in vitro are now as well understood as the mechanisms used by their protein counterparts. Despite the importance of RNA-mediated reactions in RNA processing and translation, however, it remains unclear how reaction pathways defined for RNA enzymes in vitro relate to RNA-mediated reactions in living cells. To address this gap, we have undertaken a quantitative analysis of a simple RNA-catalyzed reaction in yeast.

The hairpin ribozyme is composed of 2 helix-loop-helix segments with the reactive phosphodiester located in one of the loops (Fig. 1).

This ribozyme cleaves phosphoester bonds in a reversible reaction that generates 5´ hydroxyl and 2´,3´-cyclic phosphate termini. In contrast to the requirement for high concentrations of metal cations typical of other ribozymes, hairpin catalysis requires only buffer and counterions. Because of its unique catalytic strategy, the hairpin ribozyme is particularly well suited to structure-function studies in vivo where concentrations of metal cations are low.

We expressed hairpin ribozymes in yeast as chimeric self-cleaving mRNAs. Chimeric mRNAs containing self-cleaving hairpin ribozymes were compared with the same mRNAs containing an inactivating mutation in the hairpin sequence. Chimeric mRNAs containing mutant hairpin 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 RNAs allows us to calculate the intracellular cleavage rate (Fig. 2).

In vivo, ribozymes self-cleave at rates similar to the rates of self-cleavage measured in vitro. A ribozyme mutation that slows cleavage in vitro also slows intracellular cleavage by a similar amount. Thus, the reaction pathway and the rate-determining steps for hairpin ribozyme--mediated cleavage are fundamentally the same in vitro and in vivo.

Cleavage kinetics monitor assembly and dissociation steps as well as catalytic steps in the reaction pathway, so this system provides a unique opportunity to assess RNA structure and dynamics inside living cells. To assess the requirements for RNA helix assembly in vivo, we compared intracellular self-cleavage rates for a series of hairpin ribozymes with helix I sequences as small as 1 bp. Helices shorter than 3 bp are too weak to support full activity in vitro. We found that the same situation is true in vivo, arguing against any significant stabilization of short RNA helices in vivo. Under standard conditions in vitro, cleavage is reversed rapidly by ligation when cleavage products remain bound to the ribozyme. Consequently, ribozymes with products bound in helices with more than 6 bp have sharply reduced self-cleavage rates in vitro because slow release of product becomes rate determining. In contrast, ribozymes with products bound through as many as 18 bp 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. The propensity of the hairpin ribozyme to catalyze ligation is sensitive to the stability of tertiary structure, so this result might reflect the influence of the intracellular environment on the strength of hairpin ribozyme tertiary interactions. Experiments are under way to distinguish between these explanations.

Using this simple system, we hope to reveal mechanisms of intracellular RNA assembly and catalysis that are intrinsic to the RNA sequences and that reflect general features of the intracellular environment. Insights gleaned from investigation of intracellular hairpin ribozyme catalysis also will facilitate rational design of antisense ribozymes for specific and efficient cleavage of RNA targets in vivo.

Publications

Donahue, C.P., Fedor, M.J. Kinetics of hairpin ribozyme cleavage in yeast. RNA 3:961, 1997.

Fedor, M.J. Capturing a speeding locomotive. Cell 88:589, 1997.

Fedor, M.J. Ribozymes. Curr. Biol., in press.

Nesbitt, S., Hegg, L.A., Fedor, M.J. An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis. Chem. Biol. 4:619, 1997.

 

 







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