Transcripts containing a small anti-HIV hammerhead ribozyme that are active in the cell cytoplasm but inactive in vitro as free RNAs

Abstract

In order to study the activity of a hammerhead ribozyme in a cytoplasmic environment. HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase were contransfected with plasmids expressing the ribozyme and its target RNA (nucleotides (nt) +1 to +692 of HIV-1 RNA) under the control of a T7 promoter. Two ribozyme-containing plasmids were designed to express RNAs of respectively 181 nt (Rz181) and 132 nt (Rz132). The sequence of each of these RNAs contained a 35 nt hammerhead ribozyme which is known to cleave its minimal 14-mer RNA substrate efficiently in vitro at a site corresponding to position +115 of the HIV-1 RNA. Control transfections were carried out with the parental plasmid pET3, which expressed a 134 nt RNA lacking the ribozyme sequence, and also with a plasmid expressing a 181 nt RNA (Rz181M) containing a single mutation known to inactivate the in vitro cleavage activity of the ribozyme. As detected by RT-PCR, the amount of target RNA was reproducibly reduced at a ribozyme/target ratio higher than 50 with Rz181 and Rz132 whereas it remained unaffected with Rz181M, thus eliminating the possibility of antisense inhibition. Rz132 proved to be more efficient than Rz181. Competitive RT-PCR indicated that, at ribozyme/target ratio of 300, the amount of residual target RNA was reduced by approximately 85% in the presence of Rz181. In contrast to these in vivo effects, Rz181 and Rz132 obtained by in vitro transcription were inactive against the minimal 14 mer (or longer) substrate under a variety of conditions. In conclusion, although in vitro studies of ribozymes are essential to learn their catalytic mechanism, they cannot be used to predict the efficiency of RNAs containing a ribozyme sequence when it is expressed in cells.