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

Two sets of eucaryotic expression vectors encoding trans-acting hammerhead ribozymes and frans-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 (βAPP mRNA), 5′↓GUC↓3′. Ribozyme RNA transcribed from these vectors, which should cleave all six alternatively spliced forms of βAPP mRNA as well as βAPP pre-mRNA, was shown to cleave a βAPP RNA substrate analog in vitro. Stably transfected COS-7 cell lines bearing both vector types were prepared. Steady-state levels of βAPP 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 βAPP 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 α-actin mRNA and G3PDH mRNA levels in these cells remained constant. Western blotting of cell extracts revealed that all forms of βAPP 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 βAPP mRNA in COS-7 cells does not depend on ribozyme cleavage.