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Plant Physiol, March 2001, Vol. 125, pp. 1187-1190 SCIENTIFIC CORRESPONDENCE Cleavage of Bipartite Substrates by Rice and Maize Ribonuclease P. Application to Degradation of Target mRNAs in Plants1Departments of Biochemistry (M.L.S.R., D.K.P., J.F.R., V.G.) and Plant Biology (V.G.), The Ohio State University, Columbus, Ohio 43210; and Center for Biotechnology, SPIC Science Foundation, Madras, India (D.K.P., G.T.)
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 5' maturation of nearly sixty-odd precursor tRNAs (ptRNAs) in
prokaryotes and eukaryotes (Fig. 1A;
Altman and Kirsebom, 1999 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 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elsewhere (M.L. Stephen Raj, D.K. Pulukkunat, J.F. Reckard III, G. Thomas, and V. Gopalan, unpublished data).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 ptRNAGln, Nicotina rustica nuclear
ptRNATyr, and Nicotina tabacum chloroplast
ptRNAGly. Using the ptRNA processing reaction of the
well-studied Escherichia coli RNase P as a standard, we
first confirmed the accurate cleavage of ptRNAGln 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
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 ptRNAGln 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 ptRNAGln was generated by transcription in vitro, whereas the 25-nt "model target substrate" RNA was chemically synthesized. When these two molecules are mixed together, they should generate a ptRNA-like structure on account of Watson-Crick base pairing (Fig. 3A). Such a bipartite substrate is cleaved accurately and efficiently by rice and maize RNase P in vitro (Fig. 3B, lanes 7-10). Various control experiments indicate that the cleavage event is mediated by RNase P only when the target substrate and EGS RNAs are both present in the cleavage reaction. If the target substrate RNA is incubated with either rice or maize RNase P in the absence of EGS RNA, there is no cleavage (Fig. 3B, lanes 4 and 5). Moreover, in the absence of RNase P, the target substrate RNA remains unchanged when incubated in the presence of EGS RNAs (lanes 2 and 3).
As observed with human and mouse RNase P, a single nucleotide
substitution in the T-loop region of the EGS RNA (Fig. 1D) renders the
mRNA-EGS complex resistant to cleavage by rice and maize RNase P (Fig.
3B, lanes 11-14). By using a gel-retardation assay, we verified that
the T-loop mutant EGS RNA did bind the substrate under our assay
conditions (data not shown). Therefore, the failure of plant RNase P to
cleave the target substrate RNA when the mutant EGS RNA was added
cannot be due to lack of Watson-Crick base pairing, which is essential
for generating a ptRNA-like bipartite structure. These observations,
taken together with previous reports (Kawa et al., 1998 Because the EGS RNAs are complementary to the target mRNA, it is
conceivable that an EGS-mediated decrease in expression of the target
protein in vivo is attributable to antisense effects and not due to
cleavage of the target mRNA by RNase P. As mentioned above, the T-loop
mutant EGS can bind the target mRNA but the resulting complex is not a
substrate for cleavage by RNase P. Hence, the disruption of gene
expression (if any) that is observed with the mutant EGS will indicate
the degree to which the disruption is due to antisense effects. In
studies that employed the EGS approach in vivo, the mutant EGSs were
completely ineffective relative to the wild-type EGSs in decreasing
mRNA expression, thus confirming that the efficacy of the EGS-based
approach is due to RNase P-mediated targeted degradation (Kawa et al.,
1998 Our results suggest that a plant RNase P-based gene knockout procedure
could be included in the repertoire of approaches available for
delineating gene function in plants (Chuang and Meyerowitz, 2000
We are indebted to Dr. Erich Grotewold and Xiaoyun Dong
(The Ohio State University) for providing maize cv Black Mexican Sweet cultured suspension calli. We thank Dr. Agustin Vioque (University of
Seville, Spain) and Dr. Hildbürg Beier (Institut für
Biochemie, Würzburg, Germany) for providing the plasmids pT7Gln
and pNtY
Received September 27, 2000; accepted October 21, 2000. 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 Wood-Whelan 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 gopalan.5{at}osu.edu; fax 614-292-6773.
This article has been cited by other articles:
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TOP
ARTICLE
LITERATURE CITED
1 (the last nucleotide of the leader) and +1 (the
first nucleotide of the mature tRNA) of the substrate (data not shown;
see Fig. 
-[32P]GTP in the
in vitro transcription reaction. For 5'-end labeling,
ptRNAGln with a 5'-guanosine triphosphate was
first dephosphorylated using calf intestinal alkaline phosphatase and
subsequently phosphorylated using T4 polynucleotide kinase and
-[32P]ATP. The 20-µL RNase P assay mixture
contained 20 mM Tris (pH 8.0), 5 mM MgCl2, 7 mM dithiothreitol, 0.6 mM
phenylmethylsulfonyl fluoride (PMSF), 20 units of RNasin (Promega,
Madison, WI), and 50 nM ptRNA substrate (5,000 cpm). The assay was initiated by adding partially purified rice or
maize RNase P. The reaction was carried out for 50 min at 37°C and
terminated by adding 10 µL of stop dye solution (9 M urea, 10% [v/v] phenol, 0.1% [w/v]
bromphenol blue, and 0.15% [v/v] xylene cyanol). The reference
molecules for verifying the accurate processing of
ptRNAGln by plant RNase P was generated by
digesting ptRNAGln with E. coli RNase
P (purified as described by Gopalan et al. [1997]). The reaction
products were resolved in an 8% (w/v) polyacrylamide/8
M urea gel and visualized by autoradiography.
Lane 1, ptRNAGln in the absence of enzyme; lane
2, internally labeled ptRNAGln cleaved by
E. coli RNase P; lane 3, 5'-end-labeled
ptRNAGln cleaved by E. coli RNase P;
lanes 4 and 5, internally labeled ptRNAGln
cleaved by rice and maize RNase P, respectively.
T7, respectively. Discussions with Dr. Sidney Altman (Yale
University, New Haven, CT, and Drs. Erich Grotewold, Sophien Kamoun,
and Lien Lai (The Ohio State University) are gratefully acknowledged.
