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Abstract

The B2 family represents a group of short repetitive sequences that are found throughout the rodent genome and are analogous to the human Alu sequences. Certain B2 subfamilies are transcribed by RNA polymerase III (pol III), and this transcription is in part controlled by the retinoblastoma protein. In addition to their putative role in retrotranspositional events, these actively transcribed B2 RNAs show a predicted highly stable secondary structure. Although B2 transcripts are normally confined to the nucleus, they demonstrate altered compartmentation after carcinogen treatment, in cancers, and in immortalized and/or transformed cell lines, the significance of which is unclear. Because modulation of B2 transcripts did not seem feasible with an antisense approach, we designed a triple ribozyme (TRz) construct to down-regulate B2 transcripts. The B2-targeted TRz undergoes efficient self-cleavage, resulting in liberation of the internal hammerhead Rz, which we targeted to a single-stranded region of the consensus B2 sequence. The liberated internal targeted Rz was 20 times more active than the corresponding double-G mutant construct that could not undergo self-cleavage, and 5 times more active than the same Rz flanked by nonspecific vector sequences. The B2-targeted TRz was used to develop stable transfectant clones from an SV40-immortalized hepatocyte cell line. These transfectant clones all showed variably reduced growth rates, accompanied by significant reductions in both cytoplasmic and nuclear B2 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 documented efficient self-liberation of the internal targeted Rz in vivo, and showed that the relative cytoplasmic expression levels generally paralleled the magnitude of the decrease in B2 transcripts. The RT-PCR analyses further demonstrated that up to 20% of the Rz was located in the nucleus, which presumably reflects competition between autocatalytic processing and nucleocytoplasmic transport of the initial TRz transcript