Cloning and Characterization of the ArabidopsisCyclic Phosphodiesterase Which Hydrolyzes ADP-ribose 1",2"-Cyclic Phosphate and Nucleoside 2′,3′-Cyclic Phosphates*

In eukaryotic cells, pre-tRNAs spliced by a pathway that produces a 3′,5′-phosphodiester, 2′-phosphomonoester linkage contain a 2′-phosphate group adjacent to the tRNA anticodon. This 2′-phosphate is transferred to NAD to give adenosine diphosphate (ADP)-ribose 1",2"-cyclic phosphate (Appr>p), which is subsequently metabolized to ADP-ribose 1"-phosphate (Appr-1"p). The latter reaction is catalyzed by a cyclic phosphodiesterase (CPDase), previously identified in yeast and wheat. In the work presented here, we describe cloning of the Arabidopsis cDNA encoding the 20-kDa CPDase that hydrolyzes Appr>p to Appr-1"p. Properties of the bacterially overexpressed and purified Arabidopsis enzyme are similar to those of wheat CPDase. In addition to their transformation of Appr>p, both enzymes hydrolyze nucleoside 2′,3′-cyclic phosphates to nucleoside 2′-phosphates. For theArabidopsis CPDase, the apparent K m values for Appr>p, A>p, C>p, G>p, and U>p are 1.35, 1.34, 2.38, 16.86, and 17.67 mm, respectively. Southern analysis indicated that CPDase in Arabidopsis is encoded by a single copy gene that is expressed, at different levels, in allArabidopsis organs that were analyzed. Indirect immunofluorescence, performed with transfected protoplasts, showed that CPDase is localized in the cytoplasm. Based on substrate specificity and products generated, the plant enzyme differs from other known cyclic phosphodiesterases. The Arabidopsis CPDase does not have recognizable structural similarity or motifs in common with proteins deposited in public data bases.

Transcripts of many tRNA genes in eukaryotes contain a single short intron, located in a conserved position in the anticodon loop, which is excised by a different mechanism to that utilized during nuclear pre-mRNA processing ( Fig. 1; reviewed in Refs. 1 and 2). Splicing of pre-tRNA is initiated by endonucleolytic cleavages that result in removal of the intron and formation of two tRNA half-molecules, a 5Ј-half terminating in a 2Ј,3Ј-cyclic phosphate and a 3Ј-half bearing a 5Ј-hydroxyl group (3)(4)(5)(6)(7). In yeast and plants, these two tRNA exons are ligated to give an unusual 3Ј,5Ј-phosphodiester, 2Ј-phosphomonoester linkage. This reaction, catalyzed by the RNA ligase (8 -11), is a multistep process resulting in formation of the mature length tRNA containing a 2Ј-phosphate at the splice junction (1, 2) (Fig. 1). The ligation pathway leading to the formation of the 2Ј-phosphate-bearing tRNA molecules is also conserved in vertebrates (12), despite the fact that in these organisms most of the tRNA splicing appears to involve another RNA ligase, an enzyme which joins two tRNA halves by the regular 3Ј,5Ј-phosphodiester (3,4,13).
The 2Ј-phosphate present in the product of the spliced tRNA is removed by a specific phosphotransferase, previously identified in yeast and vertebrates (14,15). Culver et al. (16) found that this enzyme transfers the 2Ј-phosphate to an NAD acceptor molecule, to produce ADP-ribose 1Љ,2Љ-cyclic phosphate (ApprϾp). 1 However, ApprϾp is not the final product of this complex series of reactions. It has been found recently that ApprϾp is converted into ADP-ribose 1Љ-phosphate (Appr-1Љp) by the action of the cyclic phosphodiesterase (CPDase), identified in yeast and wheat (17). Although all partial reactions leading to the formation of ApprϾp and Appr-1Љp have, to date, only been demonstrated in yeast (16,17), the available evidence suggests that both compounds are also produced, as a result of the tRNA splicing reaction, in plants and vertebrates (12,(15)(16)(17). It has been suggested that ApprϾp, or its hydrolysis product, may perform some as yet unspecified regulatory function(s) in the cell (16). Conservation of the ApprϾp-forming pathway in vertebrates (12,15,16), despite the fact that most of the cellular tRNA in these organisms seems to be processed by another pathway (see above), offers some support for this hypothesis.
The plant CPDase was originally purified from wheat as an enzyme that hydrolyzes nucleoside 2Ј,3Ј-cyclic phosphates to nucleoside 2Ј-phosphates (18). The biological significance of this reaction is not known, but the ability of the enzyme to convert the 2Ј,3Ј-cyclic phosphate to the 2Ј-phosphate in mononucleotides but not in cyclic-phosphate-terminated oligoribonucleotides, together with its ability to hydrolyze ApprϾp, clearly distinguishes it from other known enzymes having the 2Ј,3Ј-cyclic phosphate 3Ј-phosphodiesterase activity (17,18; see "Discussion"). Although the yeast phosphodiesterase shares many characteristics with the wheat enzyme, it has a different substrate specificity, hydrolyzing ApprϾp to Appr-1Љp, but hav-ing no detectable activity on nucleoside 2Ј,3Ј-cyclic phosphates (17). Fractionation experiments performed with yeast and wheat germ extracts indicated that phosphodiesterases described above are, most probably, the only cellular activities converting ApprϾp to Appr-1Љp (17).
In this work we describe the molecular characterization of the plant cyclic phosphodiesterase. The cDNA encoding this protein in Arabidopsis has been cloned and the properties of the bacterially overexpressed and purified enzyme have been compared with those of its purified wheat counterpart. The Arabidopsis enzyme has no apparent structural resemblance to other known cyclic nucleotide phosphodiesterases.

EXPERIMENTAL PROCEDURES
Plant Material-Plantlets of Arabidopsis thaliana, ecotype Columbia C0, were grown in Petri dishes containing 0.8% agar, 1% sucrose, and MS salts (19) in a 22°C growth chamber under a 12-h light/12-h dark cycle. Three weeks after sowing, leaves, roots, and floral buds were harvested. For leaf strip incubation, the leaves were sliced and incubated in a culture medium described by Nagy and Maliga (20), containing 1 mg/liter of 2,4-dichlorophenoxyacetic acid. Aliquots were harvested at different times of culture for total RNA extraction.
Sequencing of the Wheat CPDase-About 5 g of wheat germ CPDase, purified as described previously (18), was applied to the SDS-PAGE 10% gel. After blotting to the polyvinylidene difluoride membrane (Bio-Rad) and staining with Ponceau S, the approximately 23-kDa CPDase band was excised and treated with trypsin. Proteolytic peptides were resolved by HPLC and sequenced by Dr. W. S. Lane (Harvard MicroChem, Cambridge, MA). Three peptides were sequenced (Fig. 2).
Screening of a cDNA Library-A ZAP cDNA library, prepared with a mixture of the poly(A) ϩ RNA isolated from 24, 48, and 72 h leaf strip cultures (a gift from J. Fleck, Institut de Biologie Moléculaire des Plantes du CNRS, Strasbourg, France), was screened with the partial cDNA clone (the Arabidopsis EST; GenBank™/EBI accession number T12916; kindly provided by the Arabidopsis Biological Resource Center at Ohio State University, Columbus, OH) as a probe. Hybridizations were performed overnight at 42°C in 5 ϫ SSPE (SSPE: 0.18 M NaCl, 10 mM NaH 2 PO 4 , 1 mM Na 2 -EDTA, pH 7.7) containing 50% formamide, 5 ϫ Denhardt's solution (100 ϫ Denhardt's solution is 2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin), 1% SDS, and 50 g/ml denatured salmon sperm DNA. Filters were subsequently washed in 2 ϫ SSC (SSC: 0.15 M NaCl, 15 mM Na 2 citrate) and 0.1% SDS for 30 min at 42°C and in 0.2 ϫ SSC and 0.1% SDS for 30 min at 42°C. Twentynine clones were isolated after screening 800,000 recombinant phages. After excision of the phagemids, the inserts were analyzed by restriction mapping and sequencing of the ends. The longest clones were subsequently sequenced on both strands.
Determination of the CPDase mRNA 5Ј Terminus by RACE-Primer extension was carried out (21) using a 25-mer-specific 32 P-labeled oligonucleotide 1 (ACGGTGACGTGAGGAACGAATCTTG), complementary to positions 203-227 of the cDNA (Fig. 2), 10 units of avian myoblastosis virus reverse transcriptase, and 100 g of RNA isolated from the Arabidopsis leaf strip cultures incubated for either 24 or 48 h. The resulting cDNA was purified on a 6% denaturing polyacrylamide gel and its 3Ј end was tagged with the 5Ј-phosphorylated oligonucleotide 2 (pCATCTCGAGCGGCCGCATCA) using T4 RNA ligase (22). The CPDase cDNA sequence was PCR-amplified using oligonucleotide 3 (GTGAGGAACGAATCTTGG; complementary to positions 202 to 219; Fig. 2) and oligonucleotide 4 (TGATGCGGCCGCTCGAGA; complementary to the 3Ј tag) as primers. The PCR products were cloned into pBluescript (Stratagene) and sequenced.
Cloning of the Promoter Region of the CPDase Gene-The fragment encompassing 226 base pairs of the promoter and upstream portion of the transcribed region was cloned by inverse PCR (23). The Arabidopsis DNA was digested with EcoRI and ligated using T4 DNA ligase. PCR was performed using oligonucleotide 5 (CGATTCCTCATCTGGTA-ATGC; complementary to positions 130 to 150 in Fig. 2) and oligonucleotide 6 (GCTAATGGAAGCTTTGAGATCC; positions 168 to 189 in Fig. 2) as primers. The amplified fragments were cloned into the SmaI site of pBluescript and sequenced.
Northern, Southern, and Western Blot Analysis-Total RNA from Arabidopsis organs and leaf strip cultures was isolated as described by Hall et al. (24). RNA (10 g/lane) was separated on a formaldehydeagarose gel, blotted onto Hybond-N nylon membrane (Amersham Corp.) by capillary transfer using 20 ϫ SSPE, and UV-cross-linked to the membrane. The integrity and the amount of RNA applied to each lane were verified by control hybridizations using a tomato 25 S rRNA probe (25). The cDNA fragment extending from positions 430 -741 ( Fig. 2) was used as a CPDase probe. The histone H4 probe corresponds to the 196-base pair restriction fragment AccI/DdeI of the coding region of the gene H4A748 (26). The actin probe corresponds to the 570-base pair PCR-amplified fragment of the Arabidopsis actin gene AAc1 (27). The genomic DNA was isolated from lyophilized Arabidopsis plants using a procedure similar to that of Murray and Thompson (28). The probes were labeled with [␣-32 P]dCTP (3000 Ci/mmol, Amersham) by the random priming method (29). RNA as well as DNA gel blots were hybridized overnight at 42°C in 5 ϫ SSPE, 50% formamide, 10% dextran sulfate, 1% SDS, and 50 g/ml denatured salmon sperm DNA. The blots were subsequently washed in 2 ϫ SSC and 0.1% SDS for 30 min at 42°C and in 0.2 ϫ SSC and 0.1% SDS for 30 min at 42°C and then at 60°C.
For immunoblot analysis, proteins were fractionated by SDS-PAGE and electroblotted onto the polyvinylidene difluoride membrane. The membrane was probed with a 1:1000 dilution of the hen polyclonal antibody. The immunoreactive proteins were detected using peroxidase-conjugated affinity-purified rabbit anti-chicken IgYs (Dianova) and the ECL Western blotting analysis system from Amersham.
Expression and Purification of the CPDase-A BamHI site was introduced 3Ј to the CPDase coding sequence by site-directed mutagenesis (30). The NcoI/BamHI fragment (the NcoI site is present at the AUG initiation codon of the CPDase cDNA) was cloned into the pQE-60 vector (Qiagen) yielding plasmid pQECPDase. In this construct, 10 additional amino acids (sequence GSRSHHHHHH) are placed in frame at the C terminus of the recombinant protein. The protein remained soluble during expression in the Escherichia coli strain BL21(DE3) and was purified in the native form, under nondenaturing conditions, using the nickel-nitrilotriacetic acid resin and following the Qiagen protocol. The purified CPDase was applied to a 10-ml Sephadex G-25 column equilibrated and eluted with 20 mM Tris acetate, pH 7.6, 0.5 mM dithiothreitol, 0.1 mM EDTA, 0.5 mM dithiothreitol, 5% (v/v) glycerol, 0.01% Triton X-100, and 10 M phenylmethylsulfonyl fluoride. The protein concentration was measured by the method of Bradford (31) using bovine serum albumin as a standard.
Preparation of Hen Antibodies-Two hens were immunized with the purified recombinant CPDase. For primary immunization, 20 g of the protein, in Freund's complete adjuvant, was used. After 4 weeks, 20 g of the protein with Freund's incomplete adjuvant was injected. Eggs were collected daily starting 2 weeks after the last immunization. Antibodies were purified from egg yolk according to the three-step method of Polson and von Wechmar (32). A 2 M (NH 4 ) 2 SO 4 precipitation was used for the complete removal of polyethylene glycol.
Sources of Nucleotides and Oligonucleotides-All nucleoside 2Ј,3Јcyclic and 3Ј,5Ј-cyclicphosphates, nucleoside 5Ј-, 3Ј-and 2Ј-phosphates, pGϾp and inositol 1,2-cyclic phosphate were obtained from Sigma and Pharmacia Biotech Inc. AϾp, GϾp, and CϾp were purified by reverse phase HPLC on Nucleosil C4 RP-300 column using 50 mM triethylammonium acetate-acetonitrile gradient. Products were collected as a single peak. ApprϾp was chemically synthesized as described elsewhere (33). The 32 P-labeled oligoribonucleotide AAAAUAAAAGϾp* (asterisk denotes the position of the label) was prepared as follows: the synthetic oligoribonucleotide AAAAUAAAAG (0.7 g), obtained from MWG-Biotech (Munich, Germany), was 3Ј-terminally labeled using 5Ј-[ 32 P]pCp (*pCp) and T4 RNA ligase. The ligation product was then digested with 5 units of RNase T1 yielding AAAAUAAAAGp*. The latter was quantitatively converted into AAAUAAAAGϾp* by incubation with the RNA 3Ј-terminal phosphate cyclase purified from HeLa cells (34). The oligonucleotide was recovered by phenol extraction and ethanol precipitation.
Assays of Cyclic Nucleotide Phosphodiesterase Activity and Thin Layer Chromatography (TLC)-For calculation of specific activities and kinetic analysis, a quantitative assay based on a measurement of the phosphatase-sensitive nucleotide product was used. All incubations (20 l) contained 50 mM Tris-HCl, pH 7.0, and 0.01% Triton X-100. Concentrations of CPDase and substrates and incubation times at 30°C were as indicated in the figure legends. Reactions were stopped by boiling for 2 min, and 80 l of 0.1 M Tris-HCl, pH 8.0, containing 0.2 unit of CIP was added. After incubation for 10 min at 37°C, liberated phosphate was assayed according to Hess and Derr (35). For determination of the K m and V max values, the assays contained substrates at concentrations of 1.38 -12.5 mM. All velocities were calculated from the initial linear rates. Values were fitted to the Lineweaver-Burk equation by the linear regression method assuming proportional errors.
Products of enzymatic digestions, performed as described previously (8), were analyzed by cellulose TLC in solvent A (saturated (NH 4 ) 2 SO 4 /3 M sodium acetate/isopropyl alcohol (80:6:2)) or by polyethyleneiminecellulose TLC in solvent B (0.75 M LiCl). The nucleotide standards and reaction products were visualized under UV light.
Transfection of Plant Protoplasts and Indirect Immunofluorescence-The CPDase coding sequence (the NcoI-BamHI fragment from pQECP-Dase; see above) was cloned into the NcoI and BamHI sites of the pGGS.5 expression vector (kindly provided by Gordon Simpson of this laboratory; the vector contains a duplicated cauliflower mosaic virus promoter and a cauliflower mosaic virus poly(A) signal), resulting in the plasmid pHATCPDase. The CPDase encoded by pHATCPDase contains the influenza hemagglutinin nonapeptide epitope tag (flu tag, amino acids YPYDVPDYA) at the C terminus. A similar plasmid (pNRBP43) expressing the nuclear RNA-binding protein N-RBP43 of Nicotiana plumbaginifolia with the flu tag fused at the C terminus, was used as a control for the nuclear-localized protein. Mesophyll protoplasts of N. plumbaginifolia were transfected by the polyethylene glycol method (36), using 20 g of plasmid per transfection. Transfected protoplasts were collected 24 h after transfection, washed twice with 10 ml of W5 solution (36), twice with 10 ml of 0.5% Mes, pH 5.7, containing 175 mM CaCl 2 , and suspended in 100 l of the same Mes/CaCl 2 buffer. The secondary antibody was diluted 1:100 with buffer B, containing 10 g/ml Hoechst 33258 dye. After washing four times with 1 ml of solution A and four times with 1 ml of solution C (0.1 M glycine-KOH, pH 8.5), samples were overlaid with a drop of the embedding material and covered with a cover glass. Samples were examined with a Zeiss Axiophot microscope and a Leica TCS 4D confocal scanning laser microscope, using a 63ϫ objective. Images were recorded using the Leica software (SCANware 4.2) provided with the system and analyzed with the Imaris software on a Silicon Graphics work station.

RESULTS
Cloning of the cDNA Encoding Arabidopsis CPDase-The previously purified wheat germ CPDase (18) was subjected to tryptic digestion, and three peptide sequences were obtained. One of these, the 20-amino acid-long pep3, showed 80% identity and 100% similarity to an open reading frame of the Arabidopsis EST, present in the GenBank™/EBI data base (accession number T12916). The EST cDNA was used as a probe to screen the ZAP cDNA library made with the poly(A) ϩ RNA obtained from the Arabidopsis leaf strip culture. Twenty-nine positive recombinant phages were isolated, and their inserts were analyzed by restriction mapping and sequencing of the ends. The longest cDNA obtained from this screening started approximately 85 nucleotides downstream from the transcription initiation site, as determined by primer extension (data not shown). To determine the 5Ј-terminal mRNA sequence, RACE experiments were performed using RNA isolated from leaf strip cultures incubated for either 24 or 48 h. Both RNA preparations yielded cDNA clones of identical sequence and extending to the same position (position 1 in Fig. 2). The sequence of the region identified by RACE was independently confirmed by cloning, using inverse PCR, and sequencing the promoter region of the gene. The apparent full-length cDNA is 741 nucleotides long, without counting the poly(A) tail (Fig. 2). The sequence including the presumed AUG initiation codon (AUC-CAUGGA) is similar to the consensus (AACCAUGGC) established for plant genes (39). The 5Ј-terminal leader contains one additional AUG in a much less favorable context, followed by termination codons (Fig. 2). Conceptual translation of the cDNA yields a 20.5-kDa protein of 181 amino acids with a predicted isoelectric point of 4.82. The deduced Arabidopsis protein contains sequences showing significant similarities with all sequenced peptides derived from the wheat protein.
The greatest sequence homology is for pep3 (see above). Peptides pep1 and pep2 show 39 and 38% similarity and 39 and 31% identity, respectively.
Enzymatic Properties of the Overexpressed Arabidopsis CP-Dase and Its Comparison with the Wheat Enzyme-The coding region of the Arabidopsis cDNA was subcloned in the pQE-60inducible expression vector to yield a fusion protein containing six histidine residues at the C terminus. The tagged protein was overproduced in E. coli and purified using the nickelnitrilotriacetic acid resin (Fig. 3A). The protein was over 95% pure as judged by SDS-PAGE. The polyclonal antibodies, raised in chickens immunized with the overexpressed Arabidopsis CPDase, detected purified Arabidopsis protein on Western blots and also cross-reacted with the purified wheat CP-Dase (Fig. 3B). The antibodies did not detect the CPDase in crude cellular extracts prepared from the leaves of Arabidopsis, but the protein band likely to correspond to the CPDase could be detected after partial purification of the enzyme (data not shown). Hence, consistent with previous observations (18), the CPDase appears to be a nonabundant protein.
The wheat CPDase was previously shown to cleave the 1Љ,2Љcyclic phosphate linkage in the enzymatically produced 32 Plabeled ApprϾp to generate Appr-1Љp (17). We have used chemically synthesized ApprϾp (33) to demonstrate that the  2), GϾp (lanes 3 and 4), CϾp (lanes 5 and 6), UϾp (lanes 7 and 8). A total of 4 l of each sample was subjected to cellulose TLC in solvent A.

Positions of nucleotide markers are indicated.
Arabidopsis enzyme has similar activity. The availability of larger amounts of ApprϾp also allowed us to determine the kinetic parameters for its hydrolysis by the wheat and Arabidopsis CPDases (see below). Hydrolysis of ApprϾp by the Arabidopsis or wheat enzyme yielded products of identical mobility in two different TLC systems (Figs. 5, A and B, lanes 2 and 3). Following additional treatment with CIP, in both cases the compound comigrated with ADP-ribose (Fig. 5, A and B, lanes  4 and 5). Treatment of ApprϾp with RNase T2 yielded the Appr-2Љp isomer, which chromatographs more slowly than Appr-1Љp on the polyethyleneimine plate (Fig. 5B, lane 7; Ref. 17). As expected, the RNase T2 hydrolysis product was also sensitive to CIP, yielding ADP-ribose (Fig. 5B, lane 8).
Reaction requirements of the Arabidopsis CPDase were determined using AϾp as a substrate (Fig. 6). Triton X-100 stimulated the enzyme activity, and 0.01% detergent was included in all reactions. The amount of substrate hydrolyzed was linearly dependent on enzyme concentration up to 20 ng/20 l. The rates of AϾp hydrolysis were similar at 20, 30, and 37°C. The optimal activity was found at pH 7.0. These reaction requirements are similar to those of the wheat enzyme (18).
The effects of mono-and divalent cations (chloride salts) and EDTA, previously tested with the wheat enzyme, were determined. Addition of NaCl to 0.2 or 0.4 M inhibited AϾp hydrol-ysis by 15 and 31%, respectively. Cu 2ϩ and Zn 2ϩ at 0.5 mM inhibited AϾp hydrolysis by 93 and 87%, respectively. At 0.5 mM, Mn 2ϩ slightly stimulated (by 5%) enzyme activity, whereas Ca 2ϩ , Mg 2ϩ , Co 2ϩ , Ni 2ϩ , and EDTA showed no effect at 0.5 mM and were only weakly inhibitory at 10 mM (data not shown). All these results are consistent with those obtained for the wheat CPDase (18).
The K m and V max for the Arabidopsis CPDase were estimated for four nucleoside 2Ј,3Ј-cyclic phosphates and ApprϾp and compared with those obtained with the purified wheat enzyme (Table I). K m values for individual nucleotide substrates were comparable for both enzymes (but see legend to Table I). However, the V max values obtained with the Arabidopsis CPDase were 10 -25 times lower than those measured with the wheat enzyme. It is possible that only a fraction of the Arabidopsis protein overexpressed in E. coli is enzymatically active. Alternatively, activity of the overexpressed enzyme may be lower due to the presence of the histidine tag or the absence of some essential modification of the protein. AϾp, CϾp, and ApprϾp over UϾp and GϾp.
Expression of the CPDase Gene-The CPDase gene copy number was estimated in a Southern blot analysis. Only one hybridizing band was detected in DNA digests carried out with three different restriction enzymes, consistent with the existence of a single-copy gene (Fig. 7).
Expression of the CPDase gene in various tissues of Arabidopsis plants and in germinating seeds and young plantlets was analyzed by Northern blotting (Fig. 8A). The CPDase mRNA is relatively low in abundance, consistent with the results of Western analysis (see above). Roots contained slightly higher levels of mRNA than other tissues analyzed. Still higher levels of mRNA were found in 3-week-old Arabidopsis plantlets.
Expression of the CPDase gene was also investigated during re-initiation of mitotic activity in leaf strip cultures. When Arabidopsis leaf strips are incubated in a culture medium containing 1 mg/liter of the auxin analogue 2,4-dichlorophenoxyacetic acid, the cells start to proliferate very rapidly. [ 3 H]Thymidine incorporation and Northern blot hybridization, performed with the histone H4 cDNA as a probe, have shown that 48 h after starting the culture most of the cells are in the S phase (40) (see also Fig. 8B). Using this experimental system, the highest CPDase mRNA level was found at 72 h (Fig. 8B), a time when a high number of cell divisions is observed. 2 RNase A/T1 mapping, performed with the antisense RNA probe covering the coding region of the CPDase cDNA, also indicated that after 72-h incubation, the level of the CPDase mRNA is approximately five times higher than at the start of the culture (data not shown). The significance of this mRNA accumulation is not understood at present. Cellular Localization of the CPDase Protein Studied by Indirect Immunofluorescence-The intracellular localization of the Arabidopsis CPDase was determined by an epitope tagging approach combined with indirect immunofluorescence. The coding sequence of the CPDase cDNA was cloned in a plant expression vector with an influenza hemagglutinin (flu) epitope fused in frame to the C terminus of the protein. The plasmid expressing the tagged protein was transfected into mesophyll protoplasts of N. plumbaginifolia. The protoplasts were processed for immunofluorescence microscopy, using a rabbit antiflu polyclonal antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Indirect immunofluorescence showed that the protein is cytoplasmic (Fig. 9B). No staining was seen in untransfected protoplasts visible in the same field (A and B) or in mock-transfected protoplasts (data not shown). Transfected protoplasts (B), in which the nucleus was localized by staining with Hoechst 33258 (A), were also examined by confocal microscopy. The expressed tagged protein was clearly excluded from the nucleus and the chloroplasts (C). In control experiments, in which the flu tag was fused to the protein N-RBP43 known to be targeted to the nucleus, 3 immunofluorescence was predominantly nuclear (D-F). These results indicate that the CPDase is a cytoplasmic protein.
The Arabidopsis CPDase Does Not Share Significant Sequence Similarity with Other Known Phosphodiesterases-A search of current sequence data bases did not reveal any proteins having significant sequence similarity with the Arabidopsis CPDase. The consensus signature motifs of 3Ј,5Ј-cyclic nucleotide phosphodiesterases (41,42) are not present in the Arabidopsis enzyme. Cyclic phosphodiesterases from brain (43,44), and tRNA ligase (45), two proteins having 3Ј-phosphodiesterase activity, also have no significant similarity with the plant protein.

DISCUSSION
Removal of the 2Ј-phosphate group present in the products of tRNA splicing (see Introduction) is catalyzed by a specific phosphotransferase, first identified in yeast and vertebrates by Phizicky and co-workers (14,15). In this reaction, the 2Ј-phosphate from tRNA is transferred to give a cyclic phosphate at the 1Љ-2Љ positions of NAD, yielding an unusual ADP-ribose derivative, ApprϾp, and nicotinamide (16). Culver et al. (46)   have recently cloned the gene encoding this enzyme in yeast and demonstrated that it is essential for viability. We describe here isolation of the Arabidopsis cDNA and properties of the encoded enzyme that metabolizes ApprϾp to Appr-1Љp. Phosphodiesterases that hydrolyze ApprϾp to Appr-1Љp have been previously characterized biochemically in both yeast and wheat (17). The purified wheat enzyme also hydrolyzes nucleoside 2Ј,3Ј-cyclic phosphates in addition to ApprϾp. The enzyme partially purified from yeast does not accept nucleoside 2Ј,3Јcyclic phosphates as substrates, but otherwise, its properties are similar to that of the wheat protein (17). Evidence presented here indicates that the protein encoded by the Arabidopsis cDNA and the protein purified previously from wheat (17,18) are equivalent. Both enzymes have similar substrate specificity, showing preference for AϾp, CϾp, and ApprϾp over UϾp and GϾp. Significantly, neither of the enzymes ring-opens the 2Ј,3Ј-cyclic phosphate group of pGϾp or at the terminus of an oligoribonucleotide (Ref. 18; this work), a property that distinguishes these enzymes from other known cyclic nucleotide 3Ј-phosphodiesterases (see below). The Arabidopsis and wheat proteins have similar molecular weights, show immunological cross-reactivity, and, based on the limited sequence information available for the wheat protein (Fig. 2), are structurally related. Finally, the reactions catalyzed by both enzymes have identical pH and temperature dependence and are similarily affected by various inhibitors.
Southern analysis has indicated that CPDase in Arabidopsis is encoded by a single copy gene (Fig. 7). In previous experiments, only single enzymatic activity for the hydrolysis of ApprϾp has been observed upon fractionation of yeast extracts, while chromatography of the wheat germ extract on DEAEcellulose yielded two pools of CPDase, having similar specificity toward NϾp and ApprϾp substrates (17,18). It is not known whether the two active pools represent products of two separate genes or two forms of the same protein.
With respect to substrate specificity, the plant CPDase studied in this work is clearly different from that of other known proteins possessing cyclic 3Ј-phosphodiesterase activity. The RNA ligase involved in tRNA splicing, extensively characterized for yeast and wheat (see Introduction), efficiently hydrolyzes terminal 2Ј,3Ј-cyclic phosphates in oligoribonucleotides  strip cultures (B). A, total RNA extracted from leaves, stems, roots, floral buds, 7-day-old germinating seeds, and 3-week-old plantlets was blotted and hybridized with the Arabidopsis CPDase-specific cDNA probe (upper panel) and the 25S rRNA probe (lower panel). B, total RNA extracted from Arabidopsis leaves (L; lane 1) and from leaf strips (LS) incubated in a medium suitable for cell division for 0, 24, 48, and 72 h (lanes 2-5, respectively). The blot was probed successively with the Arabidopsis CPDase, histone H4, and actin cDNA probes and with the tomato 25 S rRNA probe.
FIG. 9. Immunolocalization of the CPDase in protoplasts of Nicotiana plumbaginifolia. Protoplasts were transfected with constructs pHATCPDase (A-C) and pNRBP43 (D-F) expressing the fluepitope-tagged CPDase and the RNA binding nuclear protein, N-RBP43, respectively. The tagged proteins were immunoprobed with the polyclonal rabbit antibody HA-11, specific for the flu epitope. The fluorescein isothiocyanate-conjugated goat anti-rabbit antibody was used as a secondary antibody. Slides were examined with the Zeiss Axiophot microscope (A, B, D, and E) and the Leica confocal scanning laser microscope (C and F). A and D represent the same fields as B and E, respectively, but were visualized by staining with Hoechst 33258. The cell analyzed in C by confocal microscopy represents the transfected cell present in A and B. Bar represents 10 m.