Ribozyme mediated degradation of β-amyloid peptide precursor mRNA in COS-7 cells

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Abstract
Two sets of eucaryotic expression vectors encoding trans-acting hammerhead ribozymes and trans-acting hairpin ribozymes were constructed. In one set of vectors ribozyme RNA transcription was placed under the control of a mouse mammary tumor virus long terminal repeat (MMTV-LTR). In the other set ribozyme expression was controlled by a metallothionein IIA (Mt-IIA) promoter. Each ribozyme was directed to the first target sequence in the Alzheimer amyloid peptide precursor mRNA (beta APP mRNA), 5' decreases GUC decreases 3'. Ribozyme RNA transcribed from these vectors, which should cleave all six alternatively spliced forms of beta APP mRNA as well as beta APP pre-mRNA, was shown to cleave a beta APP RNA substrate analog in vitro. Stably transfected COS-7 cell lines bearing both vector types were prepared. Steady-state levels of beta APP mRNA were reduced 25-30% in cells containing either active or mutant hammerhead ribozyme vectors driven by the MMTV-LTR promoter grown in the presence of glucocorticoids. In cell lines bearing Mt-IIA driven ribozymes steady-state levels of beta APP mRNA were reduced 67-80% in both hammerhead and hairpin ribozyme containing cell lines following promoter induction by glucocorticoids. These levels correlate with the appearance of low levels of induced ribozyme RNA. In contrast, steady-state alpha-actin mRNA and G3PDH mRNA levels in these cells remained constant. Western blotting of cell extracts revealed that all forms of beta APP were correspondingly reduced. Neither the RNA nor protein decreases observed in ribozyme transfected cell lines were observed in stably transfected control cells bearing the vector alone. These results suggest that ribozyme-mediated degradation of beta APP mRNA in COS-7 cells does not depend on ribozyme cleavage.