From the Departments of Biological Sciences and
Biochemistry, Purdue University, West Lafayette, Indiana
47907 and the § Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School,
Boston, Massachusetts 02115
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ABSTRACT |
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A human cDNA was isolated encoding a protein
with significant sequence similarity (41% identity) to the BVP RNA
5'-phosphatase from the Autographa californica nuclear
polyhedrosis virus. This protein is a member of the protein-tyrosine
phosphatase (PTP) superfamily and is identical to PIR1, shown by Yuan
et al. (Yuan, Y., Da-Ming, L., and Sun, H. (1998)
J. Biol. Chem. 272, 20347-20353) to be a nuclear
protein that can associate with RNA or ribonucleoprotein complexes. We
demonstrate that PIR1 removes two phosphates from the 5'-triphosphate
end of RNA, but not from mononucleotide triphosphates. The specific
activity of PIR1 with RNA is several orders of magnitude greater than
that with the best protein substrates examined, suggesting that RNA is
its physiological substrate. A 120-amino acid segment C-terminal to the
PTP domain is not required for RNA phosphatase activity. We propose
that PIR1 and its closest homologs, which include the metazoan mRNA
capping enzymes, constitute a subgroup of the PTP family that use RNA
as a substrate.
The protein-tyrosine phosphatase
(PTP)1 superfamily includes a
large number of enzymes that dephosphorylate diverse substrates including proteins, nucleic acids, and lipids (1-6). Members of the
PTP superfamily are thought to use a common catalytic mechanism involving the formation and subsequent hydrolysis of a phosphocysteine intermediate (1-6). The essential Cys and Arg residues are located within an active site motif (HCX5R) that
characterizes all phosphatases of this superfamily.
The Autographa californica nuclear polyhedrosis virus
expresses a 19-kDa phosphatase of the PTP superfamily designated herein as BVP (also known as BVH1 and BVPTP) (7-9). BVP was originally characterized as a dual specificity protein phosphatase (7-9), but
subsequent studies have demonstrated that its RNA phosphatase activity
is several orders of magnitude greater than its activity with protein
substrates (3, 4). BVP shares significant sequence similarity with the
RNA triphosphatase domain of the metazoan mRNA capping enzymes
(2-4, 10). The bifunctional capping enzymes of metazoa contain an
N-terminal RNA 5'-triphosphatase domain and a C-terminal
GTP::RNA guanylyltransferase domain. The RNA triphosphatase
domain removes the Recently, Yuan et al. (19) identified and cloned human PIR1,
a nuclear phosphatase that interacts with RNA or ribonucleoproteins. To
identify a human homolog of BVP, we have independently cloned PIR1 and
have expressed the recombinant glutathione S-transferase (GST) fusion protein in Escherichia coli for further
enzymatic characterization. We demonstrate that at comparable substrate concentrations, the RNA triphosphatase and diphosphatase activities of
GST-PIR1 exceed its protein phosphatase activity by 2 or more orders of
magnitude. The strong preference for RNA substrates and its nuclear
localization suggest a potential role for PIR1 in RNA processing. We
also show that the C-terminal, noncatalytic domain of PIR1 is not
essential for its RNA phosphatase activity.
Plasmids and Site-directed Mutagenesis--
A cDNA
containing the PIR1 open reading frame was amplified by polymerase
chain reaction using oligonucleotide primers with 5'-restriction
linkers to facilitate cloning. This fragment was subcloned into the
BamHI/HindIII sites of pET21a-GST (20). A modification of a polymerase chain reaction-based site-directed mutagenesis method (21) was used to change PIR1 Cys152 Expression and Purification of Recombinant
Proteins--
E. coli BL21(DE3) cells transformed with the
GST-PIR1 constructs were grown in LB containing 100 µg/ml ampicilin
and 2% (w/v) glucose at 37 °C until the OD600 was 0.7. The cells were induced with 200 µM isopropyl
thio- Protein Phosphatase Assays--
Protein substrates were
phosphorylated on tyrosyl residues using recombinant GST-lyn
kinase and on seryl/threonyl residues using the catalytic subunit of
cAMP-dependent kinase as described (3). Phosphatase
reactions with p-nitrophenyl phosphate and protein
substrates were carried out in 50 mM Tris, pH 7.9, 10 mM KCl, 10 mM dithiothreitol for 30 min at
30 °C as described previously (20). Protein was determined by the
method of Bradford (22) using bovine serum albumin as a standard. The
Coomassie staining intensity of samples on a SDS-polyacrylamide gel
suggested that the amount of full-length GST-PIR1 but not GST-PIR1
(1-206) may have been overestimated by as much as 5-fold. However,
this apparent error in protein determination has not yet been confirmed by independent methods.
RNA 5'-Phosphatase Assays--
Triribonucleotide RNA substrates
were prepared with [
For the analysis of the 5' end, RNA was further incubated for 30 min at
37 °C with 2 ng of RNase T2 (purified from Aspergillus oryzae and supplied by Dr. S. Norioka, Institute For Protein
Research, Osaka University, Japan) in 20 mM ammonium
acetate, pH 5.8. After digestion, the products were analyzed by TLC on
PEI-cellulose plates with adenosine 3'-monophosphate (Ap) and adenosine
3',5'-diphosphate (pAp) (Sigma) as authentic markers.
An expressed sequence tag (GenBankTM accession number H60626)
encodes a polypeptide exhibiting 47% amino acid sequence identity to
an 87-residue segment of the BVP phosphatase (3). Using a probe derived
from the H60626 expressed sequence tag, a 1.6-kilobase cDNA was
isolated from a human placenta cDNA library
(CLONTECH). This cDNA encoded PIR1 (GenBankTM
AF023917) that was cloned by Yuan et al. (19) while this
work was in progress. In accord with Yuan et al. (19),
Northern analysis detected a single 1.7-kilobase PIR1 transcript in
eight different human tissues (data not shown). A sequence-tagged
site (GenBankTM accession number AA037694) identical to the PIR1
sequence has been mapped to the short arm of human chromosome 2.
PIR1 is a 38.9-kDa phosphatase that is localized to the nucleus, is
characterized by its ability to bind RNA in vitro and to
interact with the 9G8 and Srp30C splicing factors in the yeast two-hybrid system (19). As shown in the sequence alignment of Fig.
1A, an N-terminal segment of
PIR1 exhibits a high degree of sequence similarity to corresponding
segments from the BVP RNA phosphatase and the RNA triphosphatase domain
of the metazoan mRNA capping enzymes. This conserved region of PIR1
encompasses the hallmark PTP active site motif (HCX5R) (1).
PIR1 contains a C-terminal segment (residues 207-330) with a
polyproline-rich region (residues 276-285) (Fig. 1B) that
might serve as a binding site for SH3/WW domain-containing proteins
(23).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-phosphate from the 5' end of nascent mRNA to
leave a diphosphate terminus and the guanylyltransferase domain
catalyzes the subsequent transfer of a guanylyl group from GTP to
produce the unmethylated 5' cap structure, G(5')ppp(5')N (11, 12). BVP
and the triphosphatase domains of the capping enzymes contain the
signature HCX5R active site motif common to all PTPs and
are thought to employ a catalytic mechanism similar to that used by
PTPs to dephosphorylate proteins (2-4, 10, 13, 14). BVP differs from
the metazoan capping enzymes in that it lacks a guanylyltransferase
domain and releases both
- and
-phosphates from mRNA to yield
a monophosphate at the 5' end (3). BVP is unlikely to be involved in
the capping of viral messages because LEF-4, a subunit of the A. californica nuclear polyhedrosis virus RNA polymerase, is a
bifunctional capping enzyme with RNA triphosphatase and
guanylyltransferase activities (15-17). The function of BVP is
unknown, but it is not essential for viral replication (18).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Ser and introduce a silent mutation within the codon for
Arg158 to yield an ApaLI site that was used for
screening. The C-terminal truncation mutant was generated by amplifying
the open reading frame with an antisense primer containing a stop codon
after amino acid residue 206. Plasmids were sequenced to confirm their authenticity.
-D-galactopyranoside in fresh media, grown for 5-7
h at room temperature, and harvested. GST-PIR1 was affinity purified
from bacterial extracts using glutathione-Sepharose as described
previously (20). GST-BVP was expressed and purified as described
previously (3).
-32P]ATP (pppApCpC;
bold letter denotes labeled phosphate) and [
-32P]ATP
(pppApCpC) and used in RNA triphosphatase assays as
described previously (3). RNA diphosphatase assays were carried out
with diphosphate-terminated triribonucleotides
(ppApCpC), prepared with
[
-32P]CTP (3). Reaction products were analyzed by
thin-layer chromatography (TLC) on polyethyleneimine (PEI)-cellulose
plates (3). Phosphate (Pi) released from the termini of RNA
and monophosphate-terminated RNA (pApCpC) was
detected by using a Fuji BasX PhosphorImager and autoradiography.
Radioactive spots were cut from PEI-cellulose plates and quantitated by
liquid scintillation counting. Assays were linear with respect to time
and substrate concentration.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Multiple sequence alignment of PIR1
homologs. A, the following amino acid sequences were
aligned using the Clustal X computer program: PIR1 (GenBankTM accession
number AF023917, amino acid residues 30-199), T23G7 (Z68319, 52-225),
BVP (L22858, 1-166), BMVP (L33180, 1-166), MCE (AF025653, 5-173),
HCE (AB009022, 5-173), CEL-1 (AF003925, 2-171), and F54C8 (Z22178,
7-179). Solid black boxes identify residues that are
identical within all sequences. Shaded boxes enclose
residues that are either identical or conserved in six of the eight
sequences aligned. T23G7 and F54C8 designate protein sequences
predicted for the T23G7.5 and F54C8.4 genes of C. elegans,
respectively. BMVP designates a BVP-like protein encoded by
the B. mori nuclear polyhedrosis virus. B,
diagram illustrating the structural features of PIR1. The open
box designates the conserved catalytic domain, whereas the
cross-hatched and shaded boxes indicate the
position of the arginine-rich (19) and polyproline-rich regions,
respectively. The vertical line indicates the position of
residue 206, which is the C-terminal residue of the catalytic fragment
that was expressed as a GST fusion protein.
Protein Phosphatase Activity of PIR1-- GST-PIR1 dephosphorylates phosphotyrosyl-containing protein substrates and at least one substrate phosphorylated on serine/threonine residues (Table I). The activity of GST-PIR1 toward protein substrates is comparable to that of GST-BVP and the RNA triphosphatase domain of the Caenorhabditis elegans capping enzyme, CEL-1 (1-236). Of the four phosphotyrosyl substrates tested, the activity of GST-PIR1 was the highest toward the acidic substrate, poly(Glu4:Tyr1).
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RNA Phosphatase Activity of PIR1--
To test for RNA
5'-phosphatase activity, GST-PIR1 was incubated with an RNA
trinucleotide labeled at the -position of its 5'-triphosphate end
(pppApCpC). Analysis of the reaction products by TLC showed
that the labeled
-phosphate was released from this RNA trinucleotide
substrate (Fig. 2A,
lanes 9 and 10), indicating that PIR1 possesses
RNA triphosphatase activity. As shown in Fig. 2A, the
triphosphatase activity of PIR1 is comparable to that of BVP (3,
4).
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In the GST-PIR1 C152S mutant, the essential active site Cys has been replaced by Ser. At a concentration exceeding the wild type protein by 10-fold (Fig. 2A, lanes 11-13), GST-PIR1 C152S had no detectable RNA triphosphatase activity, confirming that this activity can be ascribed to PIR1 and not contaminating E. coli proteins. Both the RNA and protein phosphatase activities of PIR1 were inhibited by sodium vanadate, an inhibitor of tyrosine-specific and dual specificity protein phosphatases. The absolute requirement for Cys152 and its sensitivity to vanadate, suggest that PIR1 hydrolyzes RNA substrates via the mechanism utilized by the tyrosine and dual specificity protein phosphatases (24, 25).
RNA triphosphatases from yeast and viral capping enzymes do not contain the HCX5R hallmark motif of the PTPs, require Mg2+ for their activity and possess nucleotide phosphohydrolase activity (15, 16, 26-30). In contrast, the RNA phosphatase activity of PIR1 does not require Mg2+ and instead is inhibited about 50% by 100 µM MgCl2 (data not shown). PIR1 did not exhibit significant ATPase activity (Fig. 2C, lanes 5-7), suggesting that its phosphatase activity is specific to polynucleotides.
Using RNA radiolabeled at the -phosphate (pppApCpC), both
BVP and PIR1 produced a product that migrates almost as fast as
inorganic phosphate on PEI-cellulose plates (Fig.
3A, lanes 3-5 and
6-8). To determine if the observed product contained monophosphate-terminated trimeric RNA (not shown) or free phosphate (lane 1), the reaction products were further digested with
RNase T2 prior to analysis by TLC. As shown in Fig. 3B,
lanes 3-5 and 6-8, both BVP-treated and
PIR1-treated RNA released pAp. We conclude that PIR1, like BVP, leaves
a 5'-monophosphate end on RNA.
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PIR1 (Fig. 3C, lanes 6-8) and BVP (Fig.
3C, lanes 3-5) converted diphosphate-terminated
RNA (ppApCpC) to a species that migrated
similarly to 5'-hydroxyl-terminated RNA (ApCpC) produced by treatment with calf intestinal phosphatase (Fig.
3C, lane 1). To distinguish
5'-monophosphate-ended trimer from 5'-hydroxyl-ended trimer, the
reaction products were digested with RNase T2. This treatment revealed
that pAp was released from BVP-treated and PIR1-treated RNA
(Fig. 3D, lanes 3-5 and 6-8),
demonstrating that PIR1 also possesses potent RNA diphosphatase
activity. Its activity toward diphosphate-terminated RNA was more than
an order of magnitude higher than its activity toward
triphosphate-terminated RNA, whereas that of BVP is only 3-fold higher
(Table I). The presence of a mixture of diphosphate- and
monophosphate-terminated RNA in the products from
triphosphate-terminated substrate obtained at low concentrations of BVP
and PIR1 (Fig. 3, A and B, lanes 3-5
and 6-8), suggests that these two enzymes carry out the
triphosphatase and diphosphatase reactions sequentially. The RNA
diphosphatase and triphosphatase activities of both BVP and PIR1 are
also inhibited to the same extent by magnesium and vanadate (data not
shown). Both BVP and PIR1 can be distinguished from the mRNA
capping enzyme CEL-1 by their ability to remove the -phosphate from
the diphosphate termini of RNA substrates.
A comparison of the protein and RNA phosphatase activities of PIR1 (Table I) reveals that it dephosphorylates triphosphate- and diphosphate-terminated RNA with a specific activity that is 2 and 3 orders of magnitude greater than that obtained with a comparable concentration of poly(Glu4:Tyr1), the best phosphoprotein substrate tested. With artificial protein substrates, the specific activity of GST-PIR1 is about 5 orders of magnitude lower than that of PTP 1B (31), a typical tyrosine-specific phosphatase and at least 10-fold lower than that of the dual specificity protein phosphatase Cdc14 (20). In contrast, PIR1 dephosphorylates the 5'-end of RNA at rates comparable to CEL-1, which acts as a mRNA capping enzyme in vivo (2, 3). From these in vitro measurements, we conclude that PIR1 is a poor protein phosphatase and a more efficient catalyst with RNA substrates.
Role of the C-terminal Noncatalytic Domain-- A truncated form of GST-PIR1 containing only the catalytic domain (residues 1-206) was expressed in bacteria and purified in a manner similar to that of the full-length protein. When expressed on a molar basis, the specific activity of GST-PIR1 (1-206) toward p-nitrophenyl phosphate and the best protein substrate, poly(Glu4:Tyr1), was approximately 4- and 3-fold higher than that of the full-length enzyme, respectively (data not shown). The specific activity of GST-PIR1 (1-206) toward both triphosphate- and diphosphate-terminated RNA was 2-fold higher than that of the full-length enzyme (Fig. 2B, lanes 8-10, and data not shown). Like the full-length enzyme, the PIR1 catalytic domain did not dephosphorylate mononucleotides such as ATP (Fig. 2C, lanes 8-10).
Thus, the C terminus of PIR1 is not required for phosphatase activity
and its removal does not significantly alter the strong preference for
RNA or the level of RNA triphosphatase activity. The effects of
C-terminal truncation on phosphatase activity measured with the small
RNA and artificial protein substrates employed in these studies may not
reflect the properties of PIR1 with physiologic substrates. Moreover,
these findings do not eliminate a role for the noncatalytic domain in
modulating activity by serving as a binding site for regulatory
proteins or other effectors and/or by providing sites for
phosphorylation or other post-translational modifications.
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DISCUSSION |
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We demonstrate herein that PIR1 has both RNA tri- and diphosphatase activities like the viral BVP phosphatase (3, 4). The relatively poor protein phosphatase activity of PIR1 suggests that it is unlikely to be involved in controlling the level of protein phosphorylation. Instead, PIR1 has the potential to play a direct role in RNA metabolism by using RNA as its substrate.
The RNA Phosphatase Subgroup of PTPs-- At least 10 proteins exhibiting a high degree of sequence similarity to PIR1 (31-46% identity) have been identified. These include the metazoan capping enzymes from C. elegans (CEL-1) (2, 10), mice (MCE) (13, 32), and humans (HCE) (13, 33, 34); BVP and its homologs from four other nuclear polyhedrosis viruses2; and two predicted proteins (T23G7.5 and F54C8.4) of unknown function from C. elegans (Fig. 1A). We propose that these 11 proteins constitute a unique subgroup within the PTP superfamily because they exhibit much greater sequence similarity to one another than to other PTPs and all those that have been studied dephosphorylate the 5'-end of RNA much more efficiently than proteins.
Based on their common function in mRNA processing, similar domain
organization and high degree of sequence similarity within their RNA
triphosphatase domains (41% identity between enzymes from worms and
man), it is likely that the metazoan capping enzymes (CEL-1, HCE, and
MCE) are orthologs. On the other hand, PIR1, BVP, the four viral
BVP-like proteins and the T23G7 protein from C. elegans
appear to constitute a distinct group of orthologous proteins that
exhibit more similarity to one another (40-46% identity) than to the
capping enzymes (31-37% identity). Thus, like PIR1 and BVP, C. elegans T23G7 and the BVP-like viral orthologs are expected to
have the ability to remove - and
-phosphates from RNA and to
carry out similar cellular functions. This proposed evolutionary
relationship raises the possibility that the nuclear polyhedrosis
viruses may have acquired a PIR1-like gene from an insect host through
a mechanism involving horizontal gene transfer. The existence of a
cellular gene might explain why the BVP gene of A. californica nuclear polyhedrosis virus is not essential (18).
Potential Role for the PIR1 RNA Phosphatase-- It is unlikely that PIR1 functions in mRNA capping because its RNA diphosphatase activity leaves a 5'-monophosphate end on RNA, which is incompatible with the formation of the cap structure found on eukaryotic mRNAs. The possibility exists that a small subset of eukaryotic transcripts might form a mRNA cap structure using a 5'-monophosphate end to which GDP is transferred (35). To date, this unique RNA capping mechanism has only been found in some rhabdoviruses, a class of non-segmented, cytoplasmic RNA viruses (36-39).
Yuan et al. (19) found that PIR1 interacts with accessory splicing factors 9G8 and SRp30C in the yeast two-hybrid system and suggested that PIR1 might be localized to nuclear sites where RNAs are undergoing splicing. However, it should be noted that these interactions could not be confirmed by independent methods such as co-immunoprecipitation (19). To our knowledge, RNA intermediates containing 5'-tri- and diphosphate ends do not occur in splicing, making it unlikely that PIR1 plays a role in this process.
Two smaller splice variants (HCE1A and HCE1B) of the bifunctional human
capping enzyme HCE, were recently isolated and found to possess RNA
5'-triphosphatase activity but no guanylyltransferase activity (33).
Together with PIR1, these HCE variants constitute a group of RNA
phosphatases that might carry out RNA processing reactions other than
those involved in the capping and splicing of mRNAs. For instance,
they might be involved in processing of rRNA or tRNA or in the turnover
of RNAs. Elucidation of the precise role of PIR1 in RNA metabolism
must await further study.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Norioka for generously providing RNase T2. We also thank Drs. R. Kuhn and S. Rossie for suggestions and critical comments on this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA59935 and a Junior Faculty Research Award from the American Cancer Society (to H. C.). This is Paper 15935 from the Purdue University Agricultural Experimentation Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence encoding the PIR1 phosphatase characterized in this paper has been submitted to the GenBankTM/EMBL data bank with the accession number AF023917.
¶ Senior Postdoctoral Fellow of the American Cancer Society, Massachusetts Division, Inc.
** Recipient of an American Cancer Society Junior Faculty Research Award and a Pew Scholar in the Biomedical Sciences Award.
To whom correspondence should be addressed: Purdue University,
1153 Biochemistry, West Lafayette, IN 47907. Tel.: 765-494-4754; Fax:
765-496-6395; E-mail: charb{at}biochem.purdue.edu.
2 BVP homologs are encoded by the Bombyx mori, Rachiplusia ou, Anagrapha falciferia, and Orgyia pseudo tsugata nuclear polyhedrosis viruses. The corresponding nucleotide sequences have the GenBankTM accession numbers L33180, AF068270, U64896, and U75930, respectively.
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ABBREVIATIONS |
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The abbreviations used are: PTP, protein-tyrosine phosphatase; GST, glutathione S-transferase; PEI, polyethyleneimine; BVP, baculoviral phosphatase; PIR1, phosphatase that interacts with RNA and/or ribonucleoproteins; HCE, human capping enzyme; MCE, mouse capping enzyme.
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