Key result
Expression of an appropriately designed external guide sequence in cultured human cells reduced chloramphenicol acetyltransferase activity by approximately 60% and decreased intact mRNA levels.
Why the study?
Does an appropriately designed external guide sequence induce targeted cleavage of mRNA by human RNase P in human cells?
Does an appropriately designed external guide sequence induce targeted cleavage of mRNA by human RNase P in human cells?
The use of external guide sequences to direct human RNase P to cleave specific mRNAs may provide a general technique for targeted gene inactivation.
May enable targeted mRNA cleavage in human cells; leaves open in vivo efficacy and safety for gene inactivation.
Ribonuclease P from Escherichia coli can cleave RNAs in simple, hydrogen-bonded complexes of two oligoribonucleotides that resemble the aminoacyl stem and 5' leader sequence of tRNA precursors. RNase P from human (HeLa) cells cannot catalyze the cleavage in vitro of the 5'-proximal oligoribonucleotide that contains the leader sequence in such simple complexes but can do so when the 3'-proximal oligoribonucleotide (external guide sequence) is altered to resemble three-quarters of a tRNA molecule. In such a complex, the efficiency of cleavage of the mRNA for chloramphenicol acetyltransferase, as the 5'-proximal oligoribonucleotide, depends on the structural details of the external guide sequence and on the choice of target site within the mRNA. The presence of the appropriately designed external guide sequence in cells in tissue culture reduces chloramphenicol acetyltransferase activity and the level of the corresponding intact mRNA in the cells. Thus, it appears that the use of such external guide sequences may provide a general technique for gene inactivation.
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Yuan et al. (1992) studied this question. External guide sequence (EGS) targeting CAT mRNA vs. Control cells transfected with plasmid lacking EGS was evaluated on Chloramphenicol acetyltransferase (CAT) activity and mRNA levels. Expression of an appropriately designed external guide sequence in cultured human cells reduced chloramphenicol acetyltransferase activity by approximately 60% and decreased intact mRNA levels.
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