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ARD-1 cDNA from human cells encodes a site-specific single-strand endoribonuclease that functionally resembles Escherichia coli RNase E.

The human ARD-1 (activator of RNA decay) cDNA sequence can rescue mutations in the Escherichia coli rne gene, which specifies the essential endoribonuclease RNase E, resulting in RNase E-like cleavages in vivo in rne-defective bacteria and in vitro in extracts isolated from these cells (Wang, M., and Cohen, S. N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10591-10595). Recent studies indicate that the 13.3-kDa protein encoded by ARD-1 cDNA is almost identical to the carboxyl-terminal end of the bovine protein NIPP-1, a nuclear inhibitor of protein phosphatase 1; separate transcripts formed by alternative splicing are proposed to encode the discrete ARD-1 and combined ARD-1/NIPP-1 products (Van Eynde, A., Wera, S., Beullens, M. , Torrekens, S., Van Leuven, F., Stalmans, W., and Bollens, M. (1995) J. Biol. Chem. 270, 28068-28074). Here we show that affinity column-purified protein encoded by human ARD-1 cDNA in E. coli is a site-specific Mg2+-dependent endoribonuclease that binds in vitro to RNase E substrates, cleaves RNA at the same sites as RNase E, and, like RNase E, generates 5' phosphate termini at sites of cleavage. Our results indicate that the ARD-1 peptide can function as a ribonucleolytic analog of E. coli RNase E as well as a domain of the protein phosphatase inhibitor, NIPP-1.

RNA degradation in eukaryotes, as in bacteria, appears to be a regulated process that involves multiple enzymatic steps (for reviews, see Refs. [3][4][5]. Although a variety of ribonucleases have been identified in eukaryotes, with few exceptions little or no information is available about the specific biological role of these enzymes or the genes that encode them (for reviews, see Refs. 6 -8). This is especially true of nucleases that degrade the mRNA of mammalian cells.
The Escherichia coli rne gene encodes an essential endoribonuclease, RNase E, which has a key role in the decay and processing of 9 S ribosomal RNA, RNA I, an antisense regulator of replication of ColE1-type plasmids, and a variety of messenger RNAs (for reviews, see Refs. 9 -11). A human cDNA sequence designated as ARD-1 (activator of RNA decay) was identified recently by its ability to reverse the pleiotropic effects of temperature-sensitive (ts) and deletion mutations in rne (1). Expression of ARD-1 cDNA in E. coli results in RNase E-like cleavages in vivo in rne ts cells shifted to a nonpermissive temperature, and extracts isolated from cells mutated in rne but expressing ARD-1 cDNA cleave 9 S RNA in vitro at locations attacked by RNase E (1). Collectively, these findings suggested that ARD-1 may encode an endoribonuclease activity similar to that of RNase E. Moreover, the 13.3-kDa ARD-1 protein sequence deduced from analysis of the cDNA has multiple structural features in common with the 118-kDa Rne protein, including similarity to domains of eukaryotic proteins implicated in endocytosis, macromolecular transport, and RNA binding (12)(13)(14).
Recently, a peptide sequence almost identical to the protein encoded by human ARD-1 cDNA was found at the carboxyl terminus of bovine NIPP-1 (38.5 kDa), a nuclear inhibitor of protein phosphatase 1 (2). 1 Comparison of the cloned cDNA sequences of ARD-1 and NIPP-1 showed that the 5Ј-untranslated region of ARD-1 contains a 470-bp 2 segment that is not present in NIPP-1 cDNA and that ARD-1 cDNA lacks a 220 bp segment of the coding region of NIPP-1 (2); these observations have led to the proposal by Van Eynde et al. (2) that ARD-1 and NIPP-1 mRNAs are generated by alternative splicing.
Using ARD-1 protein overexpressed in E. coli and highly purified by affinity-column chromatography, we show here that ARD-1 is a single-strand-specific Mg 2ϩ -dependent endoribonuclease that binds to RNase E substrates, cleaves short oligoribonucleotides and complex substrates in AϩU-rich regions at the sites cut by RNase E, and generates 5Ј-phosphate termini. Thus, the amino acid residues comprising the ARD-1 sequence appear to function both as a domain of the protein phosphatase inhibitor NIPP-1 and a human analog of E. coli ribonuclease E.

EXPERIMENTAL PROCEDURES
Expression Plasmid and Host Strain-For expression of the histidine-tagged ARD-1 protein in E. coli, two oligonucleotides, 5ЈACGTG-ACTCATATGGTGCAAACTGCAGTGGTC-3Ј (primer 1) and 5Ј-TCAC-GTCTGGATCCTCAAATCAGCAAGGAAGGTG-3Ј (primer 2), corresponding to the 5Ј-end and 3Ј-end of ARD-1 cDNA sequence, respectively, were synthesized for PCR amplification of the ARD-1 cDNA sequence. An NdeI site and a BamHI site were added to the 5Ј ends of primers 1 and 2, respectively. The PCR product, after digestion with NdeI and BamHI, was ligated to the NdeI-and BamHI-cleaved pET16 expression vector (Novagen, WI), which contains a polyhistidine region and Factor Xa protease-cleavable region, and introduced into the protease-deficient DE3 lysogenic strain BL21, which expresses T7 RNA polymerase under control of an IPTG (isopropyl-1-thio-␤-D-galactopyranoside)-inducible lacUV5 promoter (15,16).
Growth, Media, and Plate Assays-E. coli cells containing the expression plasmid or its derivatives were grown at 35°C in LB medium containing 200 g/ml ampicillin to A 600 ϭ 0.6 -0.8, except where indicated. Expression of the ARD-1 protein was induced by addition of IPTG to a final concentration of 1 mM.
Cell Lysate-Extracts of cells allowed to grow for 2 h at 30°C following induction of ARD-1 expression by IPTG were analyzed by lysing the cells directly in the loading buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described by Sambrook et al. (17). For purification of ARD-1 by affinity column chromatography, cells were harvested and sonicated in 20 mM Tris-HCl, pH 7.9, containing 0.5 M NaCl and 5 mM imidazole. Sonications were repeated for 20-s intervals, with 30-s rest periods at 0°C until the solution was no longer viscous. For some preparations, 6 M guanidine HCl was added. Samples were then centrifuged at 10,000 ϫ g for 10 min to remove debris. The lysate was filtered through a 0.45-m filter before applying to a column.
Purification of the ARD-1 Protein-The histidine-tagged fusion protein was purified by binding to divalent cations (Ni 2ϩ ) immobilized on His-Bind (Novagen) metal chelation resin at 0°C. The column was washed at 0°C in 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9, containing 60 mM, and then 100 mM, imidazole. The protein was eluted in buffer containing 400 mM imidazole at a flow rate of 0.4 ml/min. Buffers used for purification under protein-denaturing conditions contained 6 M guanidine HCl and 20 mM imidazole in the washing buffer. The eluted fraction was concentrated by centrifugation using a Centricon-10 (Amicon) concentrator and dialyzed for 24 h at 4°C against three changes of storage buffer (18) at 4°C. Further protein purification on a denaturing preparative gel and electro-elution was as described previously (19). Alternatively, ARD-1 protein was also purified under nondenaturing conditions using a imidazole gradient to elute the protein from the affinity column.
Factor Xa Cleavage of the Fusion Protein-Proteolytic cleavage of the ARD-1 fusion proteins was carried out using a ratio of Factor Xa (New England Biolabs) to fusion protein of 1 to 100 (w/w) in a storage buffer (50 mM Tris-HCl, 20% glycerol, 1 mM EDTA, 0.5 M NaCl, and 0.5% Triton X-100). Incubations were carried out for 2-4 h at room temperature or overnight at 16°C.
In Vitro Transcription and RNA Purification-32 P-Labeled 9 S RNA and GGG⅐RNA I were synthesized in vitro from plasmids pTH90 (20) and pM21 (21), respectively, using the T7 MEGAshortscript™ in vitro transcription kit (Ambion, Inc.) in the presence of [␣-32 P]GTP (DuPont NEN) as recommended by the vendor. The oligoribonucleotides BR10 and BR13, containing sequences present at the 5Ј end of RNA I and GGG⅐RNA I, respectively (18), were labeled by treatment with T4 polynucleotide kinase (Life Technologies Inc.) (17) in the presence of [␥-32 P]ATP. All radioactively labeled RNA substrates were purified on urea-polyacrylamide gels (6% for 9 S RNA and GGG⅐RNA I, and 20% for oligonucleotides) and extracted with phenol-chloroform prior to use.
Cleavage Assays-32 P-Labeled 9 S RNA, GGG⅐RNA I, and oligoribonucleotides BR10 and BR13 were synthesized as described above. Endoribonuclease activity was assayed in 20-l reaction mixtures containing 20 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, 10 mM MgCl 2 , 3 units/ml RNasin ribonuclease inhibitor, and 10,000 -30,000 cpm of the 32 P-labeled RNA substrate. The mixtures were incubated at 30°C for 1 h, and then extracted with phenol/chloroform. After adding 20 l of formamide sequencing loading buffer and heating for 3 min at 85°C, samples were loaded in 6% or 20% sequencing gels. Gels were dried and then examined by autoradiography. The purified NH 2 -terminal half of RNase E, which contains the catalytic domain (22), was provided by K. J. McDowall (Stanford University). Placental RNase A, mung bean nuclease, and placental RNase inhibitor (RNAsin™) were purchased from Ambion, Sigma, and Promega, respectively.
Northwestern Assays-Proteins from SDS-polyacrylamide gels were blotted onto nitrocellulose filter membranes as described by Dunn (23). Filters were then prehybridized in buffer containing yeast total tRNA or E. coli total RNA at 250 g/ml before being probed with radioactively labeled 9 S RNA or GGG⅐RNA I as described by Cormack et al. (19).
Gel Mobility Shift Assays-Purified ARD-1 protein (concentrations indicated in figure legends) was incubated in 20-l reactions with ϳ30,000 cpm of 32 P-radiolabeled 9 S RNA in the presence of 20 mM Tris-HCl (pH 7.6), 200 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl 2 , 240 g/ml tRNA, 3 units/ml placental ribonuclease inhibitor, and 240 g/ml bovine serum albumin for 15 min at room temperature. Reaction mixtures for RNase E binding studies were carried out under the same conditions except that the buffer contained 40 mM EDTA. The mixtures were then adjusted to 3% Ficoll (400) and loaded on 6% nondenaturing polyacrylamide gels with a cross-linking ratio of 37.5:1 in 100 mM Tris-HCl (pH 8.0), 50 mM glycine buffer. Electrophoresis was carried out at 4°C at 240 V for 3 h. The gels were dried and then exposed to x-ray film (DuPont) with an intensifier screen at Ϫ70°C.
For RNA competition, radiolabeled RNA was first mixed with varying amounts of unlabeled RNA competitor and then subjected to the gel mobility shift assay as described above. Total RNA and mRNA from B cells were purified using the RNA STAT-60™ reagent (Tel-Test "B", Inc., Friendswood, TX), and the Poly(A)Tract mRNA isolation system (Promega), respectively. Synthesis of unlabeled 9 S RNA was carried out as described for the 32 P-labeled 9 S RNA, but in the absence of radionucleotide. Poly(A) oligoribonucleotides and yeast tRNA were purchased from Pharmacia Biotech (Uppsala, Sweden) and Boehringer Mannheim, respectively.
Analysis of 5Ј Termini Generated by ARD-1 Cleavage-Unlabeled GGG⅐RNA I was synthesized and purified as described for 32 P-labeled GGG⅐RNA I but in the absence of [␣-32 P]GTP. GGG⅐RNA I (1 g) was incubated at 30°C with ARD-1 (2.5 g), full-length RNase E (0.5 g), or RNase A (2 pg) in 50-l reactions for 3 h, 10 min, and 15 min, respectively. The ARD-1 and RNase A reactions contained 20 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, and 10 mM MgCl 2 . The RNase E reaction contained 20 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 , 100 mM NaCl, 5% glycerol, 0.1% Triton X-100, and 0.1 mM dithiothreitol. The ARD-1 and RNase E reactions contained 100 units of ribonuclease inhibitor. After incubation, samples were extracted with phenol/chloroform, precipitated with ethanol, and split into two aliquots. One aliquot from each sample was treated with calf intestine alkaline phosphatase (Boehringer Mannheim) to dephosphorylate the 5Ј termini, extracted with phenol/chloroform, and precipitated with ethanol. Then all samples were incubated with T4 polynucleotide kinase in the presence of [␥-32 P]ATP (6000 Ci/mmol). After phenol/chloroform extraction and ethanol precipitation, the RNA was resuspended in 50 l of diethyl pyrocarbonate-treated H 2 O and passed through a MicroSpin S-200 HR column (Pharmacia) to remove unincorporated radionucleotides. Samples (5 l) were mixed with formamide sequencing loading buffer (5 l), heated for 3 min at 85°C, and loaded in 6% sequencing gels.

RESULTS
Purification of the ARD-1 Protein-A PCR-amplified cDNA segment containing the full-length ARD-1 translational open reading frame was introduced into plasmid pET16b (Fig. 1) and expressed from the bacteriophage T7 promoter in E. coli as a His/ARD-1 fusion protein. Total protein extracted from cells containing the fusion construct contained a 19-kDa band that increased approximately 20-fold in relative concentration 2 h after IPTG induction of T7 RNA polymerase. This protein species, which migrated more slowly than was predicted for a protein formed by joining of the 13.3-kDa ARD-1 peptide to the polyhistidine tag and Factor Xa-cleavable region, was purified by Ni 2ϩ -column chromatograply as described under "Experimental Procedures." The purified His/ARD-1 fusion protein migrated in SDSpolyacrylamide gels stained by silver nitrate as a single 19-kDa band (Fig. 2); treatment of this protein by Factor Xa generated a product migrating at 17 kDa, which was slower than expected from the calculated molecular mass of the 13.3-kDa protein encoded by the ARD-1 open reading frame. The observed retarded migration in gels of ARD-1, which has a pI of 10.43 and contains highly charged regions at both the NH 2 -terminal and COOH-terminal ends (pI ϭ 10.43) (1), parallels the anomalous migration properties of Rne, which is a highly charged 118-kDa protein that migrates in SDS-polyacrylamide gels at the 180-kDa position (12,24).
Binding of ARD-1 to RNase E Substrates-Both Northwestern and gel mobility shift assays showed that ARD-1 is an RNA-binding protein. In initial Northwestern blots using crude extracts from E. coli overexpressing the His/ARD-1 fusion (data not shown), a 19-kDa protein band was observed to bind to the RNase E substrates 9 S RNA, GGG⅐RNA I, and the BR13 oligonucleotide. Fig. 3 (lanes 1 and 4) shows binding of the affinity-purified His/ARD-1 fusion protein to 9 S RNA and RNA I. E. coli extracts of cells expressing RNase E but lacking ARD-1 cDNA contained bands that in Northwestern blots were observed to bind to these same RNAs (Fig. 3, lanes 3 and 6), as has been reported previously (19,25,22,26); however, these extracts did not show a 19-kDa RNA-binding protein species. Gel mobility shift assays showed that ARD-1 fusion protein purified under nondenaturing conditions and incubated with radioactively labeled 9 S ribosomal RNA retarded migration of the target in native polyacrylamide gels (Fig. 4, top); unlabeled 9 S RNA (lanes 3 and 4), total B cell RNA (lanes 5 and 6), which consists largely of ribosomal RNA, and poly(A) mRNA isolated from B cells (lanes 10 and 11) efficiently competed with the 32 P-labeled 9 S RNA probe for binding to ARD-1. While the presence of unlabeled polyadenylic acid (poly(A)) in 10-fold excess (lanes 7 and 8) or tRNA in 50-fold excess (lane 9) did not prevent binding of ARD-1 to 9 S RNA, both of these RNAs reduced 9 S-specific binding of ARD-1 when added in still greater excess. As shown in Fig. 4A (bottom), the RNA-binding specificity of ARD-1 was identical to that seen for RNase E.
ARD-1 Cleaves Oligoribonucleotides and Complex RNAs at the Same Sites as RNase E-ARD-1 protein purified under nondenaturing conditions by affinity chromatography and shown to migrate as a single band in silver nitrate-stained gels (see above) was assayed for endoribonuclease activity on 9 S RNA, GGG⅐RNA I, and the BR13 and BR10 oligoribonucleotides, all of which are cleaved by purified RNase E at highly specific sites (18,19,22,(27)(28)(29). Control reactions that show the cleavages produced by RNase A, a common eukaryotic single-strand-specific endoribonuclease, were included for comparison; these controls demonstrated that the cleavage patterns of ARD-1 and RNase A are different, and also indicated the absence of RNase A activity in our purified ARD-1 preparations.
As shown in Fig. 5A, the affinity-purified His/ARD-1 fusion protein produced the same cleavages in 9 S RNA as RNase E,

ARD-1 Encodes an RNase E-like Single-strand Endoribonuclease
although cleavage at the "a" site (30,31) was relatively more prominent for ARD-1; an identical cleavage pattern was observed for the discrete ARD-1 peptide generated by Factor Xa treatment of the fusion protein (results not shown). GGG⅐RNA I was cleaved by ARD-1 at a site located 8 nt from the 5Ј end, leaving a product of 103 nt (Fig. 5B), as has been observed for RNase E (18,26,32). While the major product resulting from RNase A digestion of GGG⅐RNA I was also a 103-nt species, RNase A additionally cleaved this substrate at other sites (Fig. 5B).
Analysis of ARD-1 and RNase E cleavages of two synthetic oligoribonucleotides, BR10 and BR13, containing the same sequence as the 5Ј single-strand region of RNA I and GGG⅐RNA I, respectively (18) provided further evidence of the identical cleavage specificity of the two enzymes. McDowall et al. (18) have shown that BR10 and BR13 are each cleaved by RNase E at the intranucleotide bonds cleaved in RNA I; as seen in Fig.  5 (C and D, lane 6 of both), purified ARD-1 cleaved these oligoribonucleotides at the same sites as E. coli RNase E. However, differences in relative amounts of the products generated by the two enzymes, as indicated by the intensity of gel bands corresponding to individual RNA species, were observed. Cleavage of BR10 by ARD-1 occurred approximately equally at phosphodiester bonds located 5 and 6 nt from the 5Ј end, whereas this oligonucleotide was cleaved by RNase E predominantly at a site 5 nt from the 5Ј end ( Fig. 5C; see also Ref. 18).
RNase A cleaved oligonucleotides BR10 and BR13 at different locations than either ARD-1 or RNase E (Fig. 5, C and D). Additionally, RNase A generated 5Ј-OH termini that could be phosphorylated without further treatment using radioactively labeled ATP (Fig. 6, lane 7), as has been shown previously (33,49); in contrast, both RNase E (cf. Refs. 29 and 34) and ARD-1 generate 5Ј-phosphate termini that cannot be phosphorylated in vitro unless treated with alkaline phosphatase (Fig. 6, lanes  3 and 5). Cleavages by ARD-1 and RNase E did not occur in reaction mixtures that contain 0.01 M EDTA and lack Mg 2ϩ , whereas cleavages by RNase A do not require divalent cations (35). DISCUSSION The human gene ARD-1 was identified by the ability of ARD-1 cDNA to rescue temperature-sensitive and deletion mutants of the E. coli rne gene (1), which is essential for bacterial viability as well as for RNA decay and processing (36, 37). rne-defective mutants expressing ARD-1 can carry out RNase E-like cleavages in vivo, and extracts of cells that express ARD-1 but are deleted for the rne gene segment encoding the catalytic domain of RNase E (22), were observed to cleave 9 S RNA in vitro (1). Like Rne, ARD-1 was inferred by DNA sequence analysis to be a highly proline-rich peptide that has similarity to segments of the small ribonucleoprotein RNA- . The arrow indicates the RNA product generated by RNase E and ARD-1. After the cleavage, samples were treated as in the legend of Fig. 5, and run on 6% sequencing gels. C, BR10 substrate, 5Ј-ACAGUAUUUG-3Ј, 32 P-labeled at the 5Ј-end (see "Experimental Procedures") was incubated with the following: lane 1, buffer only at 4°C for 1 h; lane 2, buffer only at 30°C for 1 h; lanes 3-5, 0.25, 0.5, and 1 g of ARD-1, respectively, at 30°C for 1 h; lane 6, 100 ng of NH 2 -terminal domain of RNase E; lane 7, 5 pg of RNase A. Lane 7 contains a ladder generated by partial digestion of the substrate with mung bean nuclease. The reaction products were extracted with phenol/chloroform and separated in a 20% sequencing gel. Numbers at the right of the figure indicate nucleotide length of some of the mung bean nuclease cleavage products. D, BR13 oligoribonucleotide substrate, 5Ј-GGGACAGUAUUUG-3Ј, 32 P-labeled at the 5Ј-end, as described under "Experimental Procedures," was incubated and treated as the BR10 substrate. The sizes of the cleavage products were based on the 1-nucleotide ladder generated by partial digestion with mung bean nuclease, which produces 5Ј-monophosphate termini on products 3Ј to the cleavage site (48). Placental RNase inhibitor, which inhibits RNase A but has no effect on cleavages produced by either ARD-1 or RNase E, was included in all ARD-1 and RNase E reaction mixtures as a precaution against possible adventitious contamination of assays by RNase A.  1 and 2) and GGG⅐RNA I cleaved with ARD-1 (lanes 3  and 4), RNase E (lanes 5 and 6), or RNase A (lanes 7 and 8) as described under "Experimental Procedures" was incubated with T4 polynucleotide kinase and [␥-32 P]ATP before (Ϫ; lanes 1, 3, 5, and 7) or after (ϩ; lanes 2, 4, 6, and 8) treatment with calf intestine alkaline phosphatase. The arrow indicates the RNA product generated by cleavage.
binding proteins (38) and to dynamin, an eukaryotic protein implicated in endocytosis, membrane trafficking, and microtubule-based organelle transport (39,40). Analogous features of the 13.3-kDa ARD-1 and 118-kDa Rne proteins are further evident from our demonstration that ARD-1 has the same RNA binding properties as RNase E. However, antibodies raised against RNase E did not cross-react with ARD-1 (data not shown), consistent with the observation that these two proteins do not show regions of amino acid sequence homology (1,12,24).
The studies reported here show that highly purified ARD-1 protein is an endoribonuclease that, like RNase E (19,28,41), cleaves single-strand RNA segments in AϩU-rich regions. ARD-1 cleavage of native substrates and synthetic oligonucleotides containing RNase E cleavage sites occurs at the same phosphodiester bonds cleaved by E. coli RNase E. However, the relative rate of ARD-1 cleavage and RNase E cleavage differed at different sites within some substrates. These differences provide additional evidence that the activity we assayed for affinity-purified ARD-1 preparations, which had been synthesized in E. coli and were Ͼ99% pure by silver stain analysis (Fig. 2), does not result from contamination by RNase E.
No ARD-1 activity was detected in reaction mixtures that lack Mg 2ϩ and contain 0.01 M EDTA, indicating that ARD-1 activity, like RNase E activity, requires divalent cations. Neither ARD-1 nor RNase E was inhibited by placental RNase inhibitor, which inhibits RNase A and its analogs (35). Both RNase E (18,31,32,41) and ARD-1 (this paper) cleave singlestrand segments of RNA 3Ј to both purines and pyrimidines in AϩU-rich regions, whereas RNase A cuts single-stranded RNAs predominantly 3Ј to pyrimidine residues (C or U nucleotides) (33).
Cleavage of RNA I by ARD-1 generates 5Ј-phosphate termini on the product 3Ј to the cleavage site (Fig. 6), as does cleavage of RNA I and 9 S RNA by RNase E (Refs. 29 and 34; also Fig.  6). Other endonucleases generating 5Ј-phosphate ends include the bacterial enzymes RNase III and RNase P (42,43). In contrast, the mammalian ribonuclease, RNase A, and analogous endoribonucleases of E. coli that degrade substrates by cleavage at a large number of sites (i.e. RNase M, I, I*, and R) generate products that contain 5Ј-OH ends (33,44).
During the course of these experiments, we observed that a small amount of ARD-1 was produced in the absence of IPTG treatment; presumably, this was due to incomplete repression of the T7 RNA polymerase gene that controls production of ARD-1 mRNA from the T7 promoter on the expression construct. Populations of cells containing the uninduced ARD-1 gene expressed from a high copy number plasmid lose the plasmid or show DNA rearrangements that inactivate ARD-1 production (1), suggesting that excess ARD-1 protein is detrimental to E. coli cells. Similar findings have been reported for E. coli expressing RNase E from a high copy number plasmid (13). By growing cells containing the ARD-1 expression construct at 30°C, we minimized these effects, and upon addition of IPTG, routinely achieved a 20 -30-fold induction of ARD-1 expression.
Recent studies have shown that three regions of homology exist between the cDNA sequences of ARD-1 and NIPP-1, a nuclear inhibitor of protein phosphatase 1 (2). Two of these regions are located in a segment that corresponds to the 5Јuntranslated region of ARD-1 mRNA, and are separated by a 470-bp fragment that is not present in mRNA encoding NIPP-1 (Fig. 7). On the other hand, NIPP-1 cDNA contains a specific 220-bp segment not found in ARD-1 cDNA. The remaining sequences of both cDNAs suggest that the ARD-1 polypeptide is identical to the COOH terminus of NIPP-1 (2). Chromosome mapping studies (45) have shown that the common 3Ј-untranslated regions of the gene(s) encoding ARD-1 and NIPP-1 are located on human chromosome 1. The sequences of the ARD-1 (1), NIPP-1 (2), and RNase E (13,12) proteins all encode regions that resemble the highly conserved 70-kDa RNA binding component of the U1 small ribonucleoprotein complex, which is involved in mRNA splicing in eukaryotes. Moreover, PP-1, which interacts with NIPP-1 and is the target of NIPP-1 inhibition, has been shown to have a role in both in the regulation of spliceosome assembly and in the splicing process itself (46). Thus, it is tempting to speculate that ARD-1, which we have now shown to be a site-specific single-strand endonuclease having the cleavage specificity of RNase E, may be implicated in mRNA splicing.
Recently, a ϳ65-kDa enzyme having RNase E-like cleavage specificity in vitro has been identified and partially purified from human cells (47). This enzyme and its E. coli counterpart were both shown to cleave in vitro the pentanucleotide motif (i.e. AUUUA), which has been proposed as a determinant of c-myc mRNA stability in mammalian cells. The relationship of this enzyme to ARD-1 is unknown. The diagonally lined and dotted boxes represent regions that are specific for each mRNA, and the empty boxes indicate regions that have more than 90% sequence identity. The solid bars under each mRNA indicate the size of ARD-1 and NIPP-1 polypeptides.