Self-Cleaving-Ribozyme-Mediated Reduction of βAPP in Human Rhabdomyosarcoma Cells

Abstract

A self-cleaving hammerhead ribozyme targeted to codon 47 in β-amyloid precursor protein (βAPP) mRNA was cloned as a eucaryotic transcription cassette into the 3′ UTR of enhanced green fluorescence protein (EGFP) mRNA, producing a C-terminal fusion mRNA. CMV promotor-driven vectors bearing this construct or a mutationally inactive ribozyme construct were transiently transfected into human embryonic rhabdomyosarcoma (A-204) cells and their effects studied. Ribozyme self-cleavage in vivo was demonstrated by Northern blotting and the site of self-cleavage was delineated using site-specific deoxyoligonucleotide probes and primer extension arrest. Using this ribozyme reporter we demonstrated that ribozyme expression correlated with lower βAPP levels in the transfected cells. Control studies with the inactive ribozyme construct showed that both ribozyme cleavage and antisense mechanisms combined to produce the observed effect. Furthermore, production of truncated EGFP mRNA via ribozyme self-cleavage reduced EGFP-reporter expression compared to full-length EGFP control mRNAs, indicating that truncation affects the translatability of the reporter. This occurred because of a slight decrease in the stability of the fusion mRNA. The results of these studies suggest that self-cleaving ribozyme vectors may be an effective means of delivering and visualizing the expression of small active ribozymes in vivo.