Growth inhibition by a triple ribozyme targeted to repetitive B2 transcripts

The B2 family represents a group of short repetitive sequences that are fou nd throughout the rodent genome and are analogous to the human Alu sequence s. Certain B2 subfamilies are transcribed by RNA polymerase III (poI III), and this transcription is in part controlled by the retinablastoma protein. In addition to their putative role in retrotranspositional events, these a ctively transcribed BZ RNAs show a predicted highly stable secondary struct ure, Although BZ transcripts are normally confined to the nucleus, they dem onstrate altered compartmentation after carcinogen treatment, in cancers, a nd in immortalized and/or transformed cell lines, the significance of which is unclear. Because modulation of BZ transcripts did not seem feasible wit h an antisense approach, we designed a triple ribozyme (TRz) construct to d own-regulate BZ transcripts. The B2-targeted TRz undergoes efficient self-c leavage, resulting in liberation of the internal hammerhead Rz, which we ta rgeted to a single-stranded region of the consensus BZ sequence. The libera ted internal targeted Rz was 20 times more active than the corresponding do uble-G mutant construct that could not undergo self-cleavage, and 5 times m ore active than the same Rz flanked by nonspecific vector sequences. The BZ -targeted TRz was used to develop stable transfectant clones from an SV40-i mmortalized hepatocyte cell line. These transfectant clones all showed vari ably reduced growth rates, accompanied by significant reductions in both cy toplasmic and nuclear BZ RNA levels: linear regression analyses showed that their growth rates were directly related to residual cytoplasmic B2 levels . Reverse-transcription polymerase chain reaction (RT-PCR) analyses documen ted efficient self-liberation of the internal targeted Rz in vivo, and show ed that the relative cytoplasmic expression levels generally paralleled the magnitude of the decrease in BZ transcripts. The RT-PCR analyses further d emonstrated that up to 20% of the Rz was located in the nucleus, which pres umably reflects competition between autocatalytic processing and nucleocyto plasmic transport of the initial TRz transcript.