(Received for publication, June 10, 1997)
From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium and § Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria
NIPP-1 is a nuclear inhibitory subunit of protein phosphatase-1 with structural similarities to some proteins involved in RNA processing. We report here that baculovirus-expressed recombinant NIPP-1 displays RNA-binding properties, as revealed by North-Western analysis, by UV-mediated cross-linking, by RNA mobility-shift assays, and by chromatography on poly(U)-Sepharose. NIPP-1 preferentially bound to U-rich sequences, including RNA-destabilizing AUUUA motifs. NIPP-1 also associated with single-stranded DNA, but had no affinity for double-stranded DNA. The binding of NIPP-1 to RNA was blocked by antibodies directed against the COOH terminus of NIPP-1, but was not affected by prior phosphorylation of NIPP-1 with protein kinase A or casein kinase-2, which decreases the affinity of NIPP-1 for protein phosphatase-1. The catalytic subunit of protein phosphatase-1 did not bind to poly(U)-Sepharose, but it bound very tightly after complexation with NIPP-1. These data are in agreement with a function of NIPP-1 in targeting protein phosphatase-1 to RNA.
The type 1 protein phosphatases (PP-1)1 represent a conserved family of Ser/Thr-specific protein phosphatases with functions in various cellular processes including metabolism, intracellular transport, and protein synthesis (1, 2). PP-1 is particularly abundant in the nucleus, where it is involved in the regulation of both transcription and mRNA processing. As for the latter, PP-1 has a demonstrated role in the subnuclear distribution of pre-mRNA splicing factors, spliceosome assembly, and (alternative) pre-mRNA processing, which has been explained by dephosphorylation of components of small nuclear ribonucleoproteins (snRNPs), such as the "SR" proteins, by PP-1 (3-5). The SR splicing factors share a common motif of Ser-Arg repeats that is reversibly phosphorylated in vivo and is essential for spliceosome assembly and function.
All known species of PP-1 are dimeric or trimeric enzymes (1, 2). They contain an isoform of the same catalytic subunit (PP-1C), but differ in the regulatory subunits. In addition to an activity-controlling and substrate-specifying function, the regulatory subunits also have a "targeting" role. This multifunctional role is best understood for the "G" subunit that anchors PP-1 to glycogen and promotes the dephosphorylation of glycogen-associated proteins like glycogen synthase (6, 7). Similarly, the "M" subunit targets PP-1 to myofibrils and enhances the dephosphorylation of myosin (6, 7). Other noncatalytic subunits of PP-1 (e.g. inhibitor-2, NIPP-1, and sds22) are known to affect the activity and/or substrate specificity of PP-1 in vitro, but it is unclear at present whether they also have a targeting role (1, 2, 8, 9).
NIPP-1 is a nuclear polypeptide that potently and specifically inhibits
PP-1C (8). It can be extracted from the insoluble nuclear
fraction as a heterodimeric complex with PP-1C, termed PP-1NNIPP-1 (10). Phosphorylation of intact NIPP-1 (39 kDa) or its inhibitory core (16-18 kDa) by protein kinase A and/or casein
kinase-2 reduces its affinity for PP-1C (10-13). The
inhibitory site and all the phosphorylation sites for protein kinase A
and casein kinase-2 reside in the central third of the polypeptide (13).2 The cDNA cloning
of bovine NIPP-1 has revealed a limited structural similarity with the
70-kDa U1-RNA-associated protein (13), which is a component of the U1
snRNP complex in the spliceosome (14). Also, the COOH-terminal third of
NIPP-1 (13)2 is identical to a human polypeptide of 13 kDa
that can reverse the pleiotropic effects of deletion mutants of a
5-segment of the rne gene in Escherichia coli.
The latter protein was named Ard-1, for "activator of RNA decay,"
since it restored deficient RNA processing in the bacterial
rne mutants (15). The complementation by Ard-1 is not easily
understood at the molecular level, since the rne gene
encodes a multifunctional protein that not only displays an
endoribonuclease E (RNase E) activity, but also contains domains that
have been implicated in RNA binding, in macromolecular transport and in
the maintenance of the cytoskeleton.
The established role of PP-1 in RNA-processing and the structural similarities between NIPP-1 and known RNA-processing enzymes have prompted us to investigate the potential RNA-binding properties of NIPP-1. We report here that NIPP-1 binds with high affinity to RNA, in particular to U-rich sequences. By analogy with the anchoring role of other regulatory subunits of PP-1, we propose that NIPP-1 targets PP-1 to RNA-associated substrates.
NIPP-1 from bovine thymus was expressed in insect Sf9 cells under control of the polyhedrin promotor, using the baculovirus expression system.3 48-72 h after infection of the Sf9 cells with the recombinant virus, NIPP-1 could be detected as a major polypeptide in the cell extracts. The recombinant NIPP-1 was purified until homogeneity by successive chromatographies on heparin-Sepharose (Pharmacia Biotech Inc.) and Poros HQ (Perseptive Biosystems) columns.
The spontaneous and trypsin-revealed phosphorylase phosphatase activities were measured as described by Jagiello et al. (10). NIPP-1 was detected by Western blotting with polyclonal antibodies raised against the 11 carboxyl-terminal residues of NIPP-1, by an overlay with digoxygenin-labeled PP1C, and by the assay as an inhibitor of PP-1C (10). Protein concentrations were measured according to Bradford (16). Endoribonuclease activities, using AU4 and 9 S RNA as substrates, were measured as detailed in Wennborg et al. (17).
Preparation and Labeling of RNA and Oligo(deoxy)ribonucleotidesThe homoribopolymers poly(A),
poly(C), poly(G), and poly(U) were purchased from Sigma. The following
oligoribonucleotides were obtained from Eurogentec (Liège,
Belgium): AG1, 5-CUCUAGAGGAUGCAGGUAAGCUUGGGUACCG-3
; AU1,
5
-CUCUAGAGGAUGCAUUUAAGCUUGGGUACCG-3
; AU2,
5
-CUAGAGGAUGCAUUUAUUUAAGCUUGGGUAC-3
; AU4,
5
-AGGAUGCAUUUAUUUAUUUAUUUAAGCUUGG-3
; and AUMYC,
5
-CUUUAACAGAUUUGUAUUUAAGAAUUGUUUUUAAAAAAUUUUAAGAUUUACACA-3
(17).
Oligodeoxyribonucleotides were synthesized using a Cyclone DNA
synthetizer (New Brunswick): dAT4,
5
-AGGATGCATTTATTTATTTATTTAAGCTTGG-3
and dTA4,
5
-CCAAGCTTAAATAAATAAATAAATGCATCCT-3
. dAT4 and dTA4 were annealed by
incubation of both oligos (each at 0.5 mg/ml) in 10 mM
Tris/HCl at pH 7.5, 1 mM EDTA, and 10 mM
MgCl2 during 10 min at 95 °C, followed by a slow
acclimatization to room temperature.
E. coli 9 S RNA and mRNA encoding outer membrane protein
A (ompA) were transcribed from 2 µg of the
HaeIII-linearized plasmids pTH90 and p106B-64, respectively,
using the in vitro transcription kit from Stratagene and
[32P]CTP (16). Subsequently, the DNA templates were
removed by incubation (30 min at 37 °C) with 400 units/ml RNase-free
DNase I (Pharmacia) in the presence of 600 units/ml RNase inhibitor RNAsin (Promega). The labeled probes were extracted with a mixture of
phenol/chloroform/isoamylalcohol (25:24:1), precipitated with 70%
(v/v) ethanol and 10 mM sodium acetate, and purified on 8% urea-PAGE. The labeled RNA was visualized by autoradiography and extracted with 10 mM Tris/HCl at pH 7.5 and 1 mM EDTA. The probes were once more extracted and
precipitated as detailed above, lyophilized, and resuspended in 10 mM Tris/HCl at pH 7.5 and 1 mM EDTA.
The oligoribonucleotides AG1, AU1, AU2, AU4, and AUMYC were labeled at
the 5-end by phosphorylation with T4 polynucleotide kinase (Boehringer
Mannheim) during 45 min at 37 °C, according to the manufacturer's
instructions, in the presence of [
-32P]ATP and 600 units/ml RNAsin. The labeled oligonucleotides were purified on 20%
urea-PAGE and extracted as detailed above for RNA.
NIPP-1 (5 µg/ml) or bovine
serum albumin (25 µg/ml) were incubated in 10 mM Tris/HCl
at pH 7.5, 0.05% (v/v) Triton X-100, 40 mM KCl, 3 mM dithiothreitol, 8.5% (v/v) glycerol, 2 mM
MgCl2, and 5 mM EDTA with 5-end-labeled AU4 (2 µCi/ml), or the 5
-untranslated region of the ompA
mRNA (1 µCi/ml), or else 9 S RNA (1 µCi/ml) as described
previously (17). Aliquots of 20 µl were exposed to UV light (254 nm
at 250 mJ for 1 min) in a Stratalinker UV apparatus. Subsequently, the
samples were subjected to 7.5% Tricine-SDS-PAGE, and the dried gels
were exposed for autoradiography.
NIPP-1 was subjected to 7.5%
Tricine-SDS-PAGE and blotted onto nitrocellulose membranes (Hybond-C
extra, Amersham Corp.) Prehybridization was performed for 90 min at
44 °C in a buffer containing 10 mM Tris/HCl at pH 8.0, 1 mM EDTA, 50 mM NaCl, 1 mM
dithiothreitol, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%
bovine serum albumin, and 150 µg/ml preheated (10 min at 100 °C
followed by an incubation at 0 °C) salmon sperm DNA (Sigma). The
hybridization was done for 2 h at 44 °C in the same buffer
supplemented with in vitro transcribed 9 S RNA (0.2 µCi/ml) or 5-end-labeled AG1 or AU4 (0.4 µCi/ml). Subsequently,
the membranes were washed four times for 10 min in 10 mM
Tris/HCl at pH 8.0, 1 mM EDTA, 50 mM NaCl, and
1 mM dithiothreitol, and the radioactive bands were
visualized by autoradiography.
Various proteins (1-25 µg/ml) were
incubated for 30 min at 0 °C in a buffer containing 10 mM Hepes at pH 7.5, 40 mM KCl, 3 mM
dithiothreitol, 8.5% (v/v) glycerol, 2 mM
MgCl2, 0.05 mM EDTA, 0.05% (v/v) Triton X-100,
and one of the 5-end-labeled oligoribonucleotides, or in
vitro transcribed 9 S RNA, or else the 5
-untranslated region of
the ompA mRNA (1-2 µCi/ml). Electrophoresis was
carried out for 2 h at 4 °C at 200 V using 5% native PAGE in
0.1 M Tris plus 0.1 M glycine. The dried gels
were analyzed by autoradiography.
The
present study was performed with bovine thymus NIPP-1 that was
expressed in insect Sf9 cells, using the baculovirus expression system,
and purified until homogeneity (Fig. 1,
left panel). The properties of the isolated recombinant
inhibitor were similar to those of native NIPP-1 (10) and of the NIPP-1
fragments that were previously isolated from bovine thymus (8, 11-12),
with respect to the specificity and potency of its inhibition of
PP-1C, and the decreased inhibitory potency after its
phosphorylation by protein kinase A and casein kinase-2 (not
illustrated).
Since it has been reported that the COOH-terminal third of NIPP-1 can substitute for some functions of the bacterial RNase E (see Introduction), we have initially focused on known substrates of this endoribonuclease to investigate putative RNA-binding properties of NIPP-1. These substrates include "9 S RNA" (the bacterial precursor of 5 S ribosomal RNA), the ompA mRNA, and the oligoribonucleotides AG1, AU1, AU2, and AU4, containing 0, 1, 2, or 4 copies, respectively, of the destabilizing AUUUA motif. Using these RNAs, NIPP-1 was identified as an RNA-binding protein by several independent criteria. Thus, North-Western analysis showed a binding of denatured NIPP-1 to bacterial 9 S RNA and to the oligoribonucleotides AU4 and AG1 (Fig. 1, left panel). We were also able to obtain a UV-mediated cross-linking of native NIPP-1 to AU4 (Fig. 1, right panel), and to ompA and 9 S RNA (not shown). Following cross-linking of NIPP-1 to AU4, a radioactive band of 50 ± 1 kDa (n = 4) was detected, which corresponds to the combined masses of NIPP-1 (38.5 kDa) and AU4 (10 kDa). In addition, a radioactive band of 130 ± 4 kDa (n = 4) was detected, which might represent a cross-linked AU4-NIPP-1 dimer. No cross-linking was obtained under identical conditions when bovine serum albumin was used instead of NIPP-1 (Fig. 1, right panel).
A binding of NIPP-1 to RNA could also be reproducibly demonstrated by
assays of RNA-mobility shifts in native gels, as is illustrated for
ompA, 9 S RNA, and AU4 in Fig.
2. Under the same conditions, no RNA band
shifts were seen with the control proteins casein, myelin basic
protein, bovine serum albumin, and PP-1C (not
illustrated). The specificity of the interaction between NIPP-1
and AU4 is also illustrated by the effects of NIPP-1 antibodies and of
the antigenic peptide on the mobility shift (Fig. 2). Phosphorylation of NIPP-1 by protein kinase A or casein kinase-2, which decreases its
potency as an inhibitor of PP-1C (10-13), had no effect on its ability to induce RNA-mobility shifts (not shown).
Since NIPP-1 is associated with PP-1C in the nucleus (10),
it appeared important to determine whether the complex
(PP-1NNIPP-1) also displayed RNA-binding properties. In
Fig. 3A, it is shown that
PP-1C did not bind to poly(U)-Sepharose. In contrast, free NIPP-1 (Fig. 3B) as well as an in vitro
reconstituted heterodimeric complex of NIPP-1 and PP-1C
(Fig. 3C) was completely retained by the affinity column.
The binding of NIPP-1 and PP-1NNIPP-1 to poly(U)-Sepharose
was very strong, since they were not eluted at NaCl concentrations up
to 1.5 M. NIPP-1 and PP-1NNIPP-1 could subsequently be eluted, however, with 3 M KSCN.
Specificity for Binding of NIPP-1 to Nucleic Acids
The
nonlabeled ribohomopolymers showed a different potency in competing for
the binding of NIPP-1 to 5-end-labeled AU4 at 0.1 µg/ml (Fig.
4). Indeed, while poly(G) was not a
competitor at concentrations up to 2.5 µg/ml, poly(U) at only 0.02 µg/ml nearly completely prevented the binding of NIPP-1 to AU4.
Poly(A) and poly(C) were less potent.
At the same concentrations and specific radioactivities, the
AUUUA-containing oligoribonucleotides AU1, AU2, and AU4 bound more
NIPP-1 than did AG1, where the AUUUA motif is altered into AGGUA (Fig.
5, upper panel). We did not
observe a difference in the affinities for AU1, AU2, and AU4,
indicating that their binding to NIPP-1 was not affected by the number
of the AUUUA motifs. These findings were corroborated by competition
assays with nonlabeled probes, showing that AU2 and AU4 were 5-10
times better than AG1 as competitors for binding of NIPP-1 to AU4 (Fig.
5, lower panel). Combined with the data of Fig. 4, this
suggests that NIPP-1 preferentially binds to U-rich RNA.
We have also compared RNA and DNA as competitors for binding of NIPP-1
to AU2 at 0.15 µg/ml. In Fig.
6
it is shown that the oligoribonucleotide AU4 and the corresponding
deoxyribonucleotide dAT4 were equally efficient competitors. However,
the complementary strand of dAT4, i.e. dTA4, was 5-10 times
less efficient and the double-stranded dAT4/dTA4 was completely
inefficient at concentrations up 2.5 µg/ml.
The above experiments were all performed with bacterial RNAs or with
designed synthetic oligonucleotides. However, we found that NIPP-1
bound with similar affinity to AU4 and to AUMYC, which comprises the
destabilizing AUUUA-repeat motif of the 3-untranslated region of
c-myc mRNA (17), as determined by UV-mediated
cross-linking, by AUMYC-mobility shift assays, and by competition for
the binding of NIPP-1 to AU4 (not shown).
Since the carboxyl-terminal third of NIPP-1 (Ard-1) has been shown to restore the RNase E functions in some rne mutants in E. coli (see Introduction) and since NIPP-1, like bacterial RNase E (15, 17), has RNA-binding properties (this work), we have investigated whether NIPP-1 also displays an endoribonuclease activity. All tested preparations of purified recombinant NIPP-1 displayed a weak Mg2+-dependent RNase activity toward AU4 and 9 S RNA (not shown), which are known substrates for RNase E (15, 17). However, this RNase-E-like activity could be detected only at NIPP-1 concentrations above 250 nM (10 µg/ml), i.e. three orders of magnitude higher than the concentrations of NIPP-1 that are routinely used for its assay as an inhibitor of PP-1. Moreover, this RNase-E-like activity could be separated from NIPP-1 by gel filtration on Superdex-75. These data indicate that NIPP-1 itself is not an RNase, but co-purifies with trace amounts of an RNase-E-like activity from Sf9 cells.
We report here several independent lines of evidence indicating that NIPP-1 is an RNA-binding protein. Thus, NIPP-1 was able to induce RNA-mobility shifts (Figs. 2, 4, 5, 6) and could be covalently bound to RNA by UV-mediated cross-linking (Fig. 1). An interaction of NIPP-1 with RNA could also be demonstrated by NorthWestern analysis (Fig. 1) and by affinity chromatography on poly(U)-Sepharose (Fig. 3). Moreover, the RNA binding was shown to be due to a protein with the same size as NIPP-1 (Fig. 1) and was blocked by NIPP-1-specific antibodies (Fig. 2).
AU4 and dAT4 were equally efficient competitors for the binding of NIPP-1 to AU2 (Fig. 6), suggesting that NIPP-1 has the same affinity for RNA and single-stranded DNA. On the other hand, NIPP-1 showed a clear preference for U-rich oligonucleotides and displayed no affinity for double-stranded nucleic acids (Figs. 4 and 6). Analysis of the primary structure of NIPP-1 did not reveal the presence of known RNA-binding motifs such as the "RNP" motif, the arginine-rich motif, the RGG box, or the KH motif (18). However, it is striking that the amino- and carboxyl-terminal thirds of NIPP-1 are very basic (pI 9-10) and are not needed for the interaction with PP-1C (13). It is therefore tempting to speculate on an electrostatic interaction between nucleic acids and the basic extremities of NIPP-1. However, it is clear that additional binding determinants must exist to account for the specificity of the interaction of NIPP-1 with nucleic acids. It is also important to note that the binding of NIPP-1 to both PP-1C (10) and to RNA (Fig. 1) could still be observed after SDS-PAGE and blotting of NIPP-1, indicating that the RNA- and PP-1C-binding domains resist denaturing procedures.
Is NIPP-1 an RNA-targeting Subunit of PP-1?Since both NIPP-1 and PP-1NNIPP-1 (but not PP-1C) bind to RNA (Fig. 3), we propose that NIPP-1 functions as an RNA-anchoring subunit of PP-1 and enables the phosphatase to dephosphorylate RNA-associated substrates. The activity of PP-1NNIPP-1 would be regulated through phosphorylation of NIPP-1 by protein kinase A and casein kinase-2, which decrease its affinity for PP-1C (10-12), but has no effect on its association with RNA (present work). The proposed association of PP-1NNIPP-1 with RNA agrees with our findings that the holoenzyme is associated with the nuclear insoluble fraction (8, 10) and can be solubilized by the mere incubation of this fraction with ribohomopolymers.2 The targeting model of PP-1NNIPP-1 is also strikingly similar to that of glycogen-associated PP-1 (PP-1G). Indeed, the latter holoenzyme consists of PP-1C and a glycogen-anchoring G-subunit and the interaction between both polypeptides is controlled by reversible phosphorylation of the G subunit (1, 2, 6, 7).
The RNA-anchoring hypothesis implies that PP-1NNIPP-1 is a physiologically active enzyme. At first glance, this contrasts with findings that NIPP-1 is inhibitory to PP-1C (8, 10). However, it is well known that the effects of the regulatory subunits of PP-1 are substrate-dependent. Thus, the G subunit endows PP-1C with synthase phosphatase activity but decreases its phosphorylase phosphatase activity (1, 6, 7). It has also been reported that inhibitor-2 blocks the dephosphorylation of most, but not all substrates of PP-1C (19). Similarly, we have found that intact NIPP-1 inhibits the dephosphorylation of all five tested substrates of PP-1C, albeit to a different extent (10). Also, a 16-kDa fragment of NIPP-1 was inhibitory to the dephosphorylation of phosphorylase, but actually enhanced the dephosphorylation of histone IIA (8). In conclusion, it is possible that NIPP-1 is not a true inhibitor of PP-1, but rather acts as a "substrate-specifying" subunit in vivo. The effects of phosphorylation of NIPP-1 by protein kinase A or casein kinase-2 may have to be reinterpreted along the same lines. Indeed, phosphorylation of NIPP-1 reduces its affinity for PP-1C but, depending on the physiological role of NIPP-1, such phosphorylation may cause an activation or inactivation of PP-1NNIPP-1.
Among the most likely substrates of PP-1NNIPP-1 are the SR-splicing factors, which have been shown to be dephosphorylated by PP-1C in vitro and to reverse the inhibition of spliceosome assembly by PP-1C (3, 4). Interestingly, the described effects of PP-1C on spliceosome assembly and splicing in cell extracts and in permeabilized cells were all obtained at rather high concentrations of the phosphatase (3, 4), which may indicate a requirement for a substrate-specifying and -anchoring subunit. It will be interesting to investigate whether NIPP-1 might promote the dephosphorylation of SR proteins by PP-1C.
NIPP-1 displayed a particularly high affinity for AUUUA sequences (Fig.
5). These are destabilizing motifs in the 3-end of short-lived
mRNAs encoding human (proto)-oncogenes and growth factors (20). The
same sequences have recently also been identified as destabilizing
motifs in snRNAs (21). The AUUUA motif is specifically cleaved by RNase
E from E. coli as well as from mammalian cells (17). In
E. coli, RNase E is part of a large RNA-degradation complex,
called the degradosome (22). We speculate therefore that
PP-1NNIPP-1 takes part in the control of RNA processing in mammalian cells by the dephosphorylation of polypeptides, like RNase E,
that are part of a putative mammalian degradosome. It is also important
to point out here that, while NIPP-1 is highly enriched in the nucleus,
both cell fractionation (13) and immunofluorescence studies2 show the existence of a cytoplasmic pool of
NIPP-1. Thus, NIPP-1 could control RNA-processing in the cytoplasmic as
well as the nuclear cell compartment.
V. Feytons, N. Sente and P. Vermaelen are acknowledged for expert technical assistance. We thank Drs. D. Angerer and Dr. J. Kaszuba (Vienna) for helpful advice in setting up the initial RNA-binding experiments.
It was recently reported by Claverie-Martin et al. (Claverie-Martin, F., Wang, M., and Cohen, S. N. (1997) J. Biol. Chem. 272, 13823-13828) that Ard-1 is an RNA-binding protein but, in contrast to NIPP-1 (this work), also displays a Mg2+-dependent endoribonuclease activity.