Cleavage of bipartite substrates by rice and maize ribonuclease P. Application to degradation of target mRNAs in plants.

The objective of this investigation is to examine the potential for using plant ribonuclease P (RNase P) as a tool for cleaving target mRNAs and thereby disrupting gene expression in plants. RNase P is a ubiquitous ribonucleoprotein that is uniquely responsible for the 59 maturation of nearly sixty-odd precursor tRNAs (ptRNAs) in prokaryotes and eukaryotes (Fig. 1A; Altman and Kirsebom, 1999; Frank and Pace, 1998). Naturally occurring ribozymes, such as RNase P, have been adapted adroitly to cleave specific mRNAs by genetic engineering of either the ribozymes or their substrates (Forster and Altman, 1990; Hartmann et al., 1995; Tanner et al., 1999; Guerrier-Takada and Altman, 2000). The observation that a complex of two RNA molecules (Fig. 1B), which structurally resembles a typical ptRNA (Fig. 1A), is a good substrate for RNase P led to the idea that any cellular mRNA could be targeted for degradation by RNase P if the binding of the mRNA to an external guide sequence (EGS) forms a sequenceand structure-specific complex (Fig. 1C; Yuan et al., 1992; Yuan and Altman, 1994; Guerrier-Takada and Altman, 2000). Several recent studies have validated the use of this EGSbased approach to control gene expression (GuerrierTakada et al., 1997; Kawa et al., 1998; Plehn-Dujowich and Altman, 1998). For instance, RNase P-mediated degradation of viral mRNAs has been used successfully to inhibit influenza virus replication in cell culture (Plehn-Dujowich and Altman, 1998). Although the utility of the EGS approach has been demonstrated in mouse and human cells in tissue culture, similar possibilities in plant cells remain unexplored. A prerequisite for using plant RNase P in gene knockout procedures is the ability of plant RNase P to cleave bipartite substrates. This information is lacking, although partial purification and characterization of carrot (Daucus carrota) and wheat germ (Triticum aestivum) RNase P have been reported (Franklin et al., 1995; Arends and Schon, 1997). Therefore, we undertook this study with rice (Oryza sativa) and maize (Zea mays) nuclear RNase P. By successively employing ion-exchange chromatographic and density gradient fractionation procedures, nuclear RNase P was partially purified from rice (Pusa Basmati-1) and maize (Black Mexican Sweet) calli. Details of the purification procedures will be described elsewhere (M.L. Stephen Raj, D.K. Pulukkunat, J.F. Reckard III, G. Thomas, and V. Gopalan, unpublished data). Both of these partially purified preparations exhibited accurate ptRNA processing on three different substrates (namely Synechocystis sp. PCC 6803 ptRNA, Nicotina rustica nuclear ptRNA, and Nicotina tabacum chloroplast ptRNA. Using the ptRNA processing reaction of the wellstudied Escherichia coli RNase P as a standard, we first confirmed the accurate cleavage of ptRNA by rice and maize RNase P (Fig. 2). By using two criteria, we verified that these processing events were canonical RNase P-mediated cleavages. First, by using a reverse transcriptase-based primer extension method, we have established that the ptRNA substrate is cleaved between 21 (the last nucleotide of the leader) and 11 (the first nucleotide of the mature tRNA) of the substrate (data not shown; see Fig. 2A for location of cleavage site). Second, the functional group at the 59 end of the mature tRNA was established to be a monophosphate (data not shown). To use plant RNase P in gene knockout experiments, it is important to determine whether a substrate for plant RNase P can be constructed from two RNA molecules. To be specific, does plant nuclear RNase P behave like its mammalian counterpart? Can EGSs (Fig. 1C) be utilized for targeted degradation of mRNAs in plants? We have adopted the following approach to address these questions. The ptRNA substrate was dissected into two parts which when reconstituted will generate a ptRNA-like structure (albeit with a nick in the D loop; Fig. 3A). The “EGS” part of ptRNA was generated by transcription in vitro, whereas the 25-nt “model target 1 Research in V.G.’s lab is supported in part by the Ohio Agricultural Research and Development Center Research Enhancement Competitive Grants Program. D.K.P. was the recipient of a WoodWhelan Research Fellowship from the International Union of Biochemistry and Molecular Biology. J.R. was supported by the U.S. Army during his graduate studies at The Ohio State University. * Corresponding author; e-mail [email protected]; fax 614 –292– 6773.