Nucleic Acids Research, Vol 26, Issue 15 3494-3504, Copyright © 1998 by Oxford University Press
AA Seyhan, J Amaral and JM Burke
Studies involving ribozyme-directed inactivation of targeted RNA molecules
have met with mixed success, making clear the importance of methods to
measure and optimize ribozyme activity within cells. The interpretation of
biochemical assays for determining ribozyme activity in the cellular
environment have been complicated by recent results indicating that
hammerhead and hairpin ribozymes can cleave RNA following cellular lysis.
Here, we report the results of experiments in which the catalytic activity
of hairpin ribozymes is monitored following expression in mammalian cells,
and in which post-lysis cleavage is rigorously excluded through a series of
biochemical and genetic controls. Following transient transfection,
self-processing transcripts containing active and inactive hairpin
ribozymes together with cleavable and non-cleavable substrates were
generated within the cytoplasm of mouse OST7-1 cells using T7 RNA
polymerase. Unprocessed RNA and products ofintracellular cleavage were
detected and analyzed using a primer-extension assay. Ribozyme-containing
transcripts accumulated to a level of 4 x 10(4) copies per cell, and self-
processing proceeded to an extent of >75% within cells. Cellular RNA
processing was blocked by mutations within the ribozyme (G8A, G21U) or
substrate (DeltaA-1) that, in vitro , eliminate cleavage without affecting
substrate binding. In addition to self-processing activity, trans -cleavage
reactions were supported by the ribozyme-containing product of the
self-processing reaction, and by the ribozyme linked to the non-cleavable
substrate analog. Ribozyme activity was present in extracts of cells
expressing constructs with active ribozyme domains. These results provide
direct biochemical evidence for the catalytic activity of the hairpin
ribozyme in a cellular environment, and indicate that self-processing
ribozyme transcripts may be well suited for cellular RNA-inactivation
experiments.
ARTICLES
Intracellular RNA cleavage by the hairpin ribozyme
Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, 306 Stafford Hall, The University of Vermont, Burlington, VT 05405, USA.
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