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Inhibition of viral expression by the catalytic RNA subunlt o[ RNase P from Escherichia coli

Fenyong Liu and ~ Department of Biology, Yale University, New Haven, Connecticut 06520 USA

The catalytic RNA subunit (M1 RNA) of RNase P from Escherichia coil has been converted to an that specifically cleaves the mRNA that encodes thymidine kinase (TK) of 1 (HSV-1). Covalent attachment to the 3' end of M1 RNA of a sequence complementary to TK mRNA results in very efficient cleavage of the target RNA in vitro. This reaction can be stimulated by extracted from both E. coli and HeLa cells. When mouse cells in culture that express the novel RNA construct are infected with HSV-1, the levels of both TK mRNA and are reduced by -80% as compared with cells that either do not express the novel RNA construct or express constructs with certain deletions that are known to abolish the catalytic activity of M1 RNA. [Key Words: RNA ; gene inactivation; herpes simplex virus; thymidine kinase] Received November 3, 1994; revised version accepted January 20, 1995.

Ribonuclease P (RNase P) is a ribonucleoprotein that is Kikuchi et al. 1993; Frank et al. 1994) but have not, to essential for the biosynthesis of the 5' termini of tRNAs our knowledge, been exploited as sequence-specific en- (Altman et al. 1993). In Escherichia coli this enzyme donucleases in the manner described here. consists of a catalytic RNA subunit (M1 RNA) and a To further our understanding of catalysis by M1 RNA protein subunit (C5 protein) (Dart et al. 1992; Altman et and also to design strategies for efficient cleavage by al. 1993). M1 RNA itself cleaves precursors to tRNAs EGS-based RNase P, we constructed a derivative of M1 (ptRNAs) and other small in E. coli (Fig. 1A) in RNA, which we call M1GS RNA, by linking a guide vitro (Guerrier-Takada et al. 1983; Liu and Altman sequence (GS) to M1 RNA (Fig. 1C). We show that M1GS 1994). A minimal, model substrate for M1 RNA and the RNA can act as a sequence-specific and RNase P holoenzyme (M1 RNA plus C5 protein) con- can cleave target RNAs that base-pair with the GS just as tains only the equivalent of the acceptor stem and the T group I introns do (Zaug et al. 1986). We also demon- stem of a ptRNA molecule (McClain et al. 1987; Forster strate that a custom-designed M1GS RNA cleaves the and Altman 1990a). Moreover, the 5' leader sequence mRNA that encodes thymidine kinase (TK) of human and the 5' proximal sequence of the acceptor stem, when herpes simplex virus 1 (HSV-1) in vitro. Furthermore, hydrogen-bonded as a separate oligonucleotide to the 3' when M1GS RNA is expressed in mammalian cells in proximal sequence of the acceptor stem [a construct des- tissue culture, it reduces the level of expression of TK by ignated an external guide sequence (EGS)], can also be decreasing the amount of the target TK mRNA. cleaved by M1 RNA or RNase P (Fig. 1B). This observa- tion led us to a novel strategy for gene inactivation whereby an EGS that binds to and targets a specific Results mRNA (e.g., [3-galactosidase mRNA) such that the mRNA can be cleaved by M1 RNA or RNase P (Li et al. Cleavage of model substrates by M1GS RNA in vitro 1992). One limitation of this method is the relatively The HSV-1 TK gene has been well characterized (for re- weak binding of the target RNA to the enzyme, namely, view, see Roizman and Sears 1990). Although the TK M1 RNA. However, if the EGS is included as part of the gene product is not essential for viral repli