From the Departments of Medicine,
Oncology,
and ** Microbiology and Immunology, McGill University, Lady Davis
Institute for Medical Research, Sir Mortimer B. Davis-Jewish General
Hospital, Montréal, Québec H3T 1E2, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Parathyroid hormone-related protein (PTHrP) is a
secreted protein that acts as an autocrine and paracrine mediator of
cell proliferation and differentiation. In addition to its biological activity that is mediated through signal transduction cascades, there
is evidence for an intracellular role for PTHrP in cell cycle
progression and apoptosis. These effects are mediated through a
mid-region nuclear targeting sequence (NTS) that localizes PTHrP to the
region of the nucleolus where ribonucleoprotein complexes form in
vivo. In this work, we show that endogenous, transfected, and
in vitro translated PTHrP proteins bind homopolymeric and total cellular RNAs at salt concentrations up to 1 M. A
peptide representing the PTHrP NTS was effective in competing with the wild-type protein for RNA binding, whereas a similar peptide
representing the nucleolin NTS was not. Site-directed mutagenesis
revealed that the binding of PTHrP to RNA was direct and was dependent on preservation of a core GXKKXXK motif,
embedded in the PTHrP NTS, which is shared with other RNA-binding
proteins. The current observations are the first to document RNA
binding by a secreted cellular protein and predict a role for PTHrP in
regulating RNA metabolism that may be related to its localization in
the nucleolus of cells in vivo.
Parathyroid hormone-related protein
(PTHrP)1 is a secretory
protein that is both structurally and functionally related to PTH, the
major regulator of calcium homeostasis (1, 2). As a consequence of
sequence homology in amino acids 1-34, PTH and PTHrP bind to a common
G-protein-coupled receptor in bone and kidney through which they elicit
a spectrum of biological activity related to calcium and phosphate
homeostasis (3). In addition to these effects elicited through signal
transduction cascades, we (4, 5) and others (6, 7) have shown that some
of the biological activity of PTHrP is mediated through amino acids 87-107, which constitute a nuclear/nucleolar targeting sequence (NTS).
The PTHrP NTS bears some similarity to both the lysine-rich bipartite
sequences seen in proteins such as nucleolin (8) and the arginine-rich
sequence in the retroviral protein Tat (9) that mediates binding of the
protein to RNA as well as nucleolar targeting. The PTHrP NTS also
contains, at its amino terminus, a lysine-rich motif similar to the
consensus core that has been identified in numerous double-stranded
RNA-binding proteins (10).
Nucleoli are the sites within the nucleus that are involved primarily
in the transcription and processing of ribosomal RNA and its assembly
into ribonucleoprotein complexes prior to export into the cytoplasm
(11). As such, they are prominent in interphase cells undergoing rapid
protein synthesis and disappear as distinct entities in mitotic or
metabolically inactive cells. Morphological analysis at electron
microscopic resolution defines the nucleolus as a membrane-free
organelle containing regions of varying electron density (12). In
previous work, we localized PTHrP, by immunoelectron microscopy, to the
dense fibrillar component of nucleoli in tissue sections from fetal rat
bone (4). This region represents complexes of newly transcribed 45 S
rRNA and protein that are subsequently processed and assembled into
ribosomes. The complex series of events involved in ribosome biogenesis
depends on the presence of numerous nucleolar proteins that have known
RNA binding properties.
In a manner similar to protein/protein interactions and protein/DNA
interactions, the specific and stable association of RNA and protein is
most often mediated through recognition motifs (13). In many cases, a
larger module containing several repeats of shorter motifs is required
for efficient interaction, whereas in other instances, a self-contained
motif appears to be sufficient. Perhaps the best characterized and most
widely recognized RNA-binding proteins are those that contain multiple
copies of either ribonucleoprotein (RNP) (14) or K homology (KH)
domains (15). These modules stretch over 80-100 amino acids and are
often found in conjunction with RGG boxes (13). Other proteins that
have been loosely categorized on the basis of structural homology and
potential biological function are those such as Tat that contain an
arginine-rich motif. Yet another large (>300) group of loosely
categorized proteins, to which TRBP
(TAR-binding protein)
(16), GCN2 (17), and PKR (18) belong, are those that bind to
double-stranded RNA. These proteins contain one or more copies of a
double-stranded RNA (dsRNA)-binding motif with a lysine-rich core.
To extend our previous observations toward defining the role played by
PTHrP in the region of the nucleolus engaged in rRNA synthesis and
processing, we investigated the possibility that PTHrP binds RNA. We
now demonstrate that both endogenous and transfected PTHrP proteins
bind poly(G) homopolymeric RNA, GC-rich double-stranded RNA, and total
cellular RNA. The interaction is of high relative affinity and is
dependent on the presence of a core lysine motif shared with other
RNA-binding proteins.
Plasmids and Modifications--
The previously described
plasmids pPTHrP/3 (rat PTHrP) and p Cell Culture and Transient Transfections--
All cell culture
reagents were purchased from Life Technologies, Inc., and all
plasticware (Falcon) was from Becton Dickinson Labware (Lincoln Park,
NJ). The PTr cell line (a kind gift of G. N. Hendy, Calcium Research
Laboratory, Montreal, Canada) was maintained in Dulbecco's modified
Eagle's medium supplemented with 10% heat-inactivated calf serum and
antibiotic/antimycotic. COS-1 cells were maintained and transfected as
described previously using a mock transfection for control (4).
Briefly, 48 h after transfection, cells were lysed with buffer for
binding assays and immunoblot analysis. PTr cells plated at a density
of 2 × 106/150 mm2 were grown to 80%
confluence, washed twice with phosphate-buffered saline, and harvested
by scraping into lysis buffer (150 mM NaCl, 20 mM Tris (pH 7.4), 20 mM NaF, 0.1 mM
sodium vanadate, 1% Triton X-100, 500 µM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 15 µg/ml
aprotinin). Cell lysates were cleared of debris by centrifugation prior
to use in binding assays.
In Vitro Transcription and Translation--
Transcription and
translation were performed in vitro using pPTHrP/3 or
p Binding to Homopolymeric RNA and DNA--
Ribonucleotide and
deoxyribonucleotide binding assays were performed essentially as
described previously (20, 21) using the following synthetic
homopolymers: poly(A), poly(C), poly(G), poly(U), or double-stranded
poly(G)·poly(C) and poly(dG)·poly(dC) coupled to Sepharose beads
(Sigma). The beads were washed and reconstituted to 50% (v/v) in
phosphate-buffered saline. Binding reactions were carried out in 0.2 ml
of lysis buffer containing 2 µl of in vitro translated
product, 100 µg of total protein from transfected COS-1 cells, or 300 µg of total protein from PTr cells. Binding was performed at the
indicated NaCl concentrations for 1 h at 4 °C on a rocking
platform. The beads were pelleted by brief centrifugation and washed
three times in lysis buffer prior to resuspension in 20 µl of Laemmli
sample buffer. Bound proteins were eluted from the nucleic acid by
heating at 95 °C for 5 min, resolved on 15% SDS-polyacrylamide
gels, and analyzed either by autoradiography or by immunoblot analysis.
Immunoblot Analysis--
The polyclonal antiserum raised against
rat PTHrP-(67-86) has been previously characterized (22). The
monoclonal antibody 9E10, specific for the human c-Myc epitope (23),
was harvested as ascites fluid from mice 7 days after implantation of
the hybridoma (American Type Culture Collection). Horseradish
peroxidase-conjugated secondary antisera were purchased from Sigma
(goat anti-mouse IgG) and Pierce (goat anti-rabbit IgG). Proteins
denatured in Laemmli sample buffer were separated by size on 15%
SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
Membranes were blocked overnight with 5% nonfat milk protein in
Tris-buffered saline with 0.05% Tween 20 and incubated overnight with
either the anti-Myc or anti-PTHrP-(67-86) primary antibody. The
protein bands were detected using an appropriate horseradish
peroxidase-conjugated secondary antibody and the enhanced
chemiluminescence system (Amersham Pharmacia Biotech).
Binding to Total Cellular RNA--
Total cellular RNA was
labeled by incubating HeLa cells, grown to 50% confluence, with 500 µCi of [32P]orthophosphate (ICN) overnight (21) and
isolated using TRIzol (Life Technologies, Inc.) according to the
manufacturer's instructions. Proteins were immunoprecipitated, with
either control IgG or anti-Myc antibody, from COS-1 cells transiently
transfected with the various PTHrP constructs. 3 × 106 cpm of labeled RNA was incubated with the equivalent of
one-third of a cell lysate for 30 min at 4 °C. The beads were washed
three times in lysis buffer and counted in a Beckman scintillation
counter. Immunoblot analysis of the counted samples was performed as
described above using anti-Myc antibody to verify the expression level
of the immunoprecipitated proteins. To examine the profile of RNAs recovered in the binding assays, bound RNAs, eluted from the
precipitated proteins by denaturing in formamide buffer at 70 °C for
5 min, were subjected to formaldehyde-agarose electrophoresis and
transferred to a nitrocellulose membrane that was exposed to x-ray film
overnight. An aliquot of unlabeled cellular RNA was run in the presence
of ethidium bromide to identify 28 S and 18 S ribosomal bands on the membrane.
Peptide Synthesis and Competition Binding Assay--
Peptides
corresponding to amino acids 87-107 of PTHrP (PTHrP NTS) or amino
acids 281-301 of nucleolin (nucleolin NTS) were synthesized with a
carboxyl-terminal biotin tag at the Sheldon Institute for Biotechnology
(McGill University, Montreal). Competition assays were performed by
incubating 2 µl of in vitro translated PTHrP with
poly(G)-Sepharose in the presence of the peptides at concentrations of
10 In previous studies, we identified sequence homology between amino
acids 87-107 of the cellular protein PTHrP and amino acids 48-57 of
the retroviral regulatory protein Tat (4). In the case of Tat, the same
amino acids represent an arginine-rich RNA-binding motif. Taken
together with our previous observation that PTHrP localizes to the
dense fibrillar component of the nucleolus, we sought to determine if
PTHrP is an RNA-binding protein and if the interaction is mediated
through its NTS.
Binding of Transiently Expressed PTHrP to Cellular RNA and
Homopolymeric RNA--
We first examined the possibility that
transiently expressed PTHrP could associate with total cellular RNA.
COS-1 cells were transfected with an expression vector encoding
full-length rat PTHrP with the Myc epitope tag at the carboxyl terminus
(PTHrPmyc) to facilitate identification of the expressed protein.
Lysates of PTHrPmyc-transfected or mock-transfected COS-1 cells were
immunoprecipitated with control (IgG) or anti-Myc antibody. The
immunoprecipitated proteins were then incubated with
32P-labeled total cellular RNA and washed, and the bound
RNA was quantitated by scintillation counting. A significant proportion of the radiolabeled RNA was observed in anti-Myc immunoprecipitates of
PTHrPmyc-transfected cells, but not in mock-transfected cells or
IgG immunoprecipitates (Fig.
1A). Immunoblot analysis of
the counted samples was then performed, using anti-Myc antibody, to verify the equality of expression levels of the immunoprecipitated proteins (data not shown). To determine
whether the PTHrP NTS was required for this interaction, we transfected
COS-1 cells with an expression plasmid encoding PTHrPmyc devoid of the
NTS (PTHrPmyc-NTS) and analyzed the lysates for RNA binding. Despite an
equivalent level of PTHrP expression, Myc immunoprecipitates of
PTHrPmyc
To investigate whether PTHrP, like many other RNA-binding proteins
(13), associated with homopolymeric RNA, COS-1 cells expressing
full-length PTHrPmyc were lysed, and equal amounts of protein were
incubated with poly(A), poly(C), poly(G), or poly(U) homopolymeric RNA
covalently coupled to Sepharose beads. Immunoblot analysis of
PTHrPmyc-expressing COS-1 cell lysates reproducibly demonstrated two to
three distinct protein species that bound poly(G) homopolymeric RNA,
but not poly(A), poly(C), poly(U), or Sepharose beads alone (Fig.
1B). The proteins most probably represent prepro-PTHrP and
the mature protein. PTHrP protein devoid of the NTS (PTHrPmyc Profile of RNAs Recovered in Binding Studies--
To identify the
species of RNA that bound to PTHrP immobilized on the Sepharose beads,
the binding studies were repeated using total RNA harvested from
32P-labeled HeLa cells and protein expressed in COS-1 cells
from cDNAs encoding wild-type PTHrPmyc, PTHrPmyc Binding of Endogenous PTHrP to Homopolymeric RNA--
To further
explore the apparent specificity of the interaction between PTHrP and
poly(G) homopolymeric RNA, the PTr cell line was used as a source of
endogenous PTHrP. Lysates of PTr cells were incubated with different
homopolymeric RNAs conjugated to Sepharose beads, and the bound
proteins were separated by SDS-polyacrylamide gel electrophoresis and
analyzed by immunoblotting with PTHrP-(67-86) antiserum. As was true
of transfected PTHrP, endogenous PTHrP bound specifically to poly(G),
but not to poly(C)-, poly(A)-, or poly(U)-Sepharose or Sepharose beads
alone (Fig. 3A). The bound protein migrated with an apparent molecular mass of 27 kDa, and the
association was capable of withstanding salt concentrations up to 1 M, indicating that the interaction was specific and of apparent high affinity (Fig. 3B).
Effects of Amino Acid Substitutions within the PTHrP NTS on RNA
Binding--
To define the minimum sequence within the NTS required
for RNA binding activity, we tested several PTHrP constructs that
contain mutations in this region (Fig.
4A). We have shown previously
that mutation of 87GKKKK91 to
87GEEKI91 (M1PTHrPmyc) effectively blocked the
nuclear/nucleolar targeting function of the PTHrP NTS. Substitution of
102KKKRR106 for
102IIERG106 (M3PTHrPmyc) was only partially
effective in this respect, and mutation of
96KRREQ100 to
96KGTEL100 (M2PTHrPmyc) had little or no effect
on targeting the protein to the nuclear compartment (33). To determine
whether the proteins expressed from these cDNAs bound RNA, COS-1
cells were transfected with the expression vectors, and the lysates
were used for homopolymeric binding studies. All of the proteins bound
poly(G)-Sepharose at 250 mM salt, but considerable
differences were observed at higher salt concentrations (Fig.
4B). The binding of both M1PTHrPmyc and M3PTHrPmyc to
poly(G)-Sepharose was absent or severely impaired at 750 mM
salt, whereas the binding of M2PTHrPmyc was unaffected at that salt
concentration (Fig. 4B). These data suggest that the overall
composition of the basic residues in the NTS affects its interaction
with poly(G) homopolymeric RNA. We also examined the ability of the
mutant proteins to associate with total cellular RNA (Fig.
4C). M2PTHrPmyc and wild-type PTHrPmyc bound radiolabeled cellular RNA with similar apparent affinities. However, the ability of
M1PTHrPmyc to bind RNA was severely impaired, and that of M3PTHrPmyc was partially impaired (Fig. 4C). These observations were
consistent with the data demonstrating compromised binding of the
mutant PTHrPmyc proteins to poly(G)-Sepharose.
PTHrP NTS Peptide Competes with in Vitro Translated PTHrP for RNA
Binding--
We examined the possibility that the interaction between
PTHrP and RNA was direct by incubating in vitro translated
PTHrP with poly(G) RNA. Radiolabeled PTHrP was generated by incubating the full-length rat PTHrPmyc cDNA in the presence of
[3H]leucine in a rabbit reticulocyte lysate. In
vitro translated PTHrP bound poly(G)-Sepharose, and the binding
persisted at a 1 M salt concentration (Fig.
5A), suggesting that the
association between PTHrP and RNA was direct. The specificity of the
association was then tested by performing competition binding studies
in the presence of peptides corresponding to either the PTHrP NTS or the nucleolin NTS. Amino acids 87-107 of PTHrP and amino acids 281-301 of nucleolin are similar lysine-rich bipartite motifs that
target the respective proteins to the nuclear compartment (4, 8). When
in vitro translated PTHrPmyc was incubated with
poly(G)-Sepharose in the presence of the PTHrP NTS peptide at a
concentration of 10 Binding of PTHrP to Double-stranded Nucleotide Sequences--
TRBP
is one of a number of cellular proteins that associate with GC-rich
double-stranded RNA, including the stem-loop structure formed by the
Tat transactivating or TAR region of nascent viral RNA (16). In view of
the similarities between Tat and PTHrP, we investigated the possibility
that PTHrP might also bind to GC polynucleotide sequences. In the
presence of 250 mM salt, wild-type and mutant PTHrP
proteins bound poly(G)·poly(C) RNA, but did not demonstrate
significant binding to the corresponding deoxynucleotide sequences
(Fig. 6A). The association
between poly(G)·poly(C) and PTHrP was maintained in the presence of 1 M salt, indicating an apparent high affinity interaction
(Fig. 6B). The same pattern of compromised binding of
M1PTHrPmyc and M3PTHrPmyc was observed with poly(G)·poly(C)
compared with poly(G) alone, whereas M2PTHrPmyc had little effect.
These data suggest that PTHrP is capable of binding poly(G)-rich
sequences in the context of both single- and double-stranded RNAs, but
does not bind DNA.
We have shown that endogenous and transfected PTHrP proteins bind
to single- and double-stranded RNAs at salt concentrations up to 1 M. The binding is direct and is highly dependent on the presence of a core GXKKXXK motif within PTHrP
that is conserved among other RNA-binding proteins. A peptide
corresponding to the PTHrP NTS can bind alone to poly(G)-Sepharose and
can compete with wild-type PTHrP for binding to the homopolymer.
Site-directed mutagenesis revealed that the RNA binding activity of
PTHrP was direct and dependent on preservation of the core motif.
The endocrine, paracrine, and autocrine mechanisms of PTHrP action,
which are mediated through transduction cascades following the
interaction of secreted PTHrP with the common PTH/PTHrP cell-surface receptor, are well documented. On the other hand, little is known regarding the documented intracellular role played by PTHrP. The PTHrP
NTS is situated in a highly conserved region of the protein that has
also been identified as a site of endoproteolytic cleavage (25).
However, the presence of an intact NTS has been shown to have
biological relevance not only in protecting serum-deprived chondrocytes
from apoptosis (4), but also as a requirement for PTHrP-induced
mitogenesis in vascular smooth muscle cells (6). Others have shown that
PTHrP is targeted to the nucleolus of cells in G1 and
suggest that its subcellular distribution is cell-cycle dependent (7).
As a first step toward defining the molecular basis for these
intracellular actions of PTHrP, we now show that PTHrP is a
sequence-specific RNA-binding protein and that the association with RNA
is dependent on the presence of a core motif found in double-stranded
RNA-binding proteins.
In the absence of highly specific PTHrP antibodies, we modified all of
our expression constructs to include a Myc epitope tag. Using the
anti-Myc antibody, two major species of PTHrP were commonly detected in
immunoblot analysis of lysates prepared from PTHrPmyc-transfected COS-1
cells. They migrated with apparent molecular sizes slightly larger (due
to the 11-amino acid Myc tag) than those previously detected with an
antiserum raised against the amino terminus of PTHrP (4). An antiserum
that was raised against synthetic PTHrP-(67-86) recognized a major
species of endogenous PTHrP that bound to poly(G)-Sepharose and
migrated with an apparent size similar to that of prepro-PTHrP. Thus,
it appeared that more than one species of PTHrP could associate with homopolymeric RNA.
The strength of binding of a protein to a specific nucleotide sequence,
whether it be DNA or RNA, can be assessed by performing the binding
studies at high salt concentrations. If the binding is weak or requires
additional sequences outside of those being tested, then the
association will not withstand an elevation in salt concentration (13).
In this context, binding of the KH domain proteins hnRNP K and FMR1 to
RNA is mediated through a combination of KH domains and an RGG box (24,
26). Binding of in vitro translated, wild-type FMR1 to
poly(G)-Sepharose was severely compromised at a salt concentration of
500 mM. Binding of the prototypical hnRNP K protein to RNA,
on the other hand, was resistant to 1 M salt, as was
demonstrated for PTHrP in our own work. Homopolymeric RNA binding in
the presence of high salt concentrations was abrogated by deletion of
the RGG box or the carboxyl-terminal KH domain in FMR1 (24). Variable
alterations in binding to RNA at high salt concentrations resulted from
point mutations of critical Ile residues in the three KH domains in hnRNP K (26). In a similar manner, we have shown that deletion of the
PTHrP NTS results in abrogation of binding to poly(G)-Sepharose and
that conservative mutations within the NTS lead to variable alterations
in binding to RNA at high salt concentrations. It is interesting to
note that, despite their lack of structural similarities, PTHrP and
FMR1 both bind 28 S and 18 S species of ribosomal RNA with an apparent
equal affinity at low salt concentrations. As is true for many other
RNA-binding proteins, the physiological significance of the different
structural motifs and their RNA binding affinities and specificities
awaits further investigation.
In the absence of the NTS, there was no specific binding to total
cellular RNA or to homopolymeric RNA. However, deletion of a stretch of
20 amino acids could influence the overall structure of PTHrP, thus
preventing it from making contact with its RNA target. We therefore
sought to map the point of contact between PTHrP and RNA using a set of
NTS mutants that had previously been generated for immunofluorescent
localization of the protein. In those studies, it was shown that
substitution of the amino-terminal lysines was most effective in
preventing nuclear/nucleolar targeting of PTHrP (33). More recently,
others have demonstrated that proteins in which either the amino- or
carboxyl-terminal lysine tract was deleted failed to stimulate
proliferation when expressed in vascular smooth muscle cells, as did
intact PTHrP (6). We now show that substitution of these same basic
amino acids, particularly the amino-terminal stretch, compromises
binding of the protein to both synthetic and total cellular RNAs. This
is in keeping with the observation that PTHrP
87GXKKXXK93 constitutes a
core motif that is also found in the functionally heterogeneous group
of proteins containing dsRNA-binding domains. The general structure of
the dsRNA-binding domain is Another member of the family of dsRNA-binding proteins is TRBP, which
was isolated from a HeLa cell expression library on the basis of its
ability to bind with high affinity to the GC-rich double-stranded stem
of TAR RNA (16, 28). The prototypical TAR-binding protein, Tat, on the
other hand, appears to identify single-stranded loops in the context of
the double-stranded stem (29). We have demonstrated that PTHrP binds
avidly to G-rich sequences in both a single- and double-stranded
context. It is therefore possible that the cellular RNA target of PTHrP
might be a defined structure similar to that formed by TAR. In view of
evidence that suggests that some rDNA transcription takes place in the
dense fibrillar core of the nucleolus where PTHrP localizes in
vivo (30), it is possible that it might play some role in the
processivity of RNA polymerase I, as has been suggested for Tat in the
case of RNA polymerase II (31). PTHrP could inhibit rRNA synthesis and
allow the cell to arrest in G0/G1 in an
environment that could not support proliferation. In support of this
hypothesis is evidence that transfected PTHrP was found in the
nucleolus of only 10-15% of randomly cycling cells (4) and that
endogenous PTHrP localized to the nucleolus only in quiescent
keratinocytes (7).
An alternative role that PTHrP might play in the nucleus is in the
processing of pre-rRNA. However, despite its nucleolar localization,
the lack of repetitive RNP motifs or RGG boxes appears to exclude it
from the RNA recognition motif family, to which the large group of
small nuclear/nucleolar RNA-binding proteins involved in RNA processing
and transport belongs. One member of this family is nucleolin, which,
like PTHrP, has a classic bipartite nuclear/nucleolar targeting signal
composed of two polylysine tracts separated by a 10-amino acid spacer.
It has been suggested that the nucleolin NTS localizes the protein to
the nucleolus, where it is subsequently retained by binding to rRNA
through RNP and RGG motifs (8, 32). In our own studies, we have shown that the PTHrP NTS is both necessary and sufficient to localize the
protein to the nucleus/nucleolus (4). We now show that a peptide
corresponding to the PTHrP NTS binds directly, with specificity and
apparent high affinity, to poly(G)-Sepharose, whereas a similar,
equally lysine-rich peptide (nucleolin NTS) does not. In view of our
data that identify the PTHrP NTS as the motif that mediates binding of
the full-length protein to RNA, this observation strongly suggests that
PTHrP binds RNA directly, rather than through some other molecule.
Additionally, the inability of a peptide representing the nucleolin NTS
to compete with PTHrP for binding to poly(G)-rich RNA supports the
conjecture that PTHrP does not belong to the RRM family of RNA-binding
proteins. However, the possibility that PTHrP plays some as yet
undefined role in rRNA processing cannot be excluded.
It is evident from the above discussion that the classification
of RNA-binding proteins on the basis of structural similarities does
little to elucidate their biological function. Thus, the RNA binding
characteristics of PTHrP most closely resemble those of the
dsRNA-binding protein TRBP, whose biological function remains undefined. Our current work characterizing the RNA binding properties of PTHrP, combined with our previous observation that PTHrP localizes to the dense fibrillar core of the nucleolus, predicts a role for the
protein in the transcription or processing of rRNA. However, further
studies will be required to co-localize PTHrP with the subnucleolar
sites of rDNA transcription and early rRNA processing events to clarify
these issues.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
87-107PTHrP/3 (4) were modified
to include an in-frame Myc tag. Oligonucleotides myctag 3 (5'-cggaattcgcgtctatggaacaaaagctgattagcgaagagg-3') and myctag 4 (5'-ccgctcgagtcagttgttcaggtcctcttcgctaatcagc-3') were used to generate
a polymerase chain reaction product encoding the Myc epitope flanked by
a 5'-EcoRI site and a 3'-XhoI site. After
digestion with EcoRI and XhoI, the fragment was
ligated into the pcDNA3 expression vector (Invitrogen) to generate
pcDNA3myc. Oligonucleotides myctag 1 (5'-cgggatcccacgatgctgcggaggctg-3') and myctag 2 (5'-ggaattcatgcgtccttgagctggg-3') were used to generate PTHrP DNA
fragments with a 5'-BamHI site and a 3'-EcoRI
site using pPTHrP/3 and p
87-107PTHrP/3 as templates. The fragments
were digested with BamHI and EcoRI and ligated
into the BamHI/EcoRI site of pcDNA3myc. The
resulting plasmids, PTHrPmyc and PTHrPmyc-NTS, were verified by DNA
sequencing. Mutations were introduced into the target plasmid pPTHrP/3
using the Chameleon mutagenesis kit (Stratagene). The following series
of mutant cDNAs was generated with selectively altered residues
within one of three distinct basic domains in the PTHrP NTS: M1,
87GKKKK91 to 87GEEKI91,
M2, 96KRREQ100 to
96KGTEL100; and M3,
102KKKRR106 to
102IIERG106. The resulting cDNAs, M1PTHrP,
M2PTHrP, and M3PTHrP, were modified to include an in-frame Myc tag as
described above. Human FMR1 was used as a positive control to
demonstrate binding to ribosomal RNA proteins (19).
87-107PTHrP/3 as a DNA template with the TNT T7 coupled rabbit
reticulocyte lysate system (Promega) according to the manufacturer's
instructions. 1 µg of plasmid DNA was added to the rabbit
reticulocyte lysate containing T7 RNA polymerase and 500 µCi/ml
[3H]leucine (ICN). 2 µl of the translation product was
removed to represent the total cell lysate prior to dividing the
remaining product equally for RNA binding experiments.
7 to 10
3 M. The bound
proteins were separated on 15% SDS-polyacrylamide gels and analyzed by
immunoblotting with anti-Myc antibody. For the peptide binding studies,
10
7 M biotinylated peptides were incubated
either with poly(G)-Sepharose in lysis buffer containing up to 1000 mM NaCl or with poly(A)-, poly(C)-, or poly(U)-Sepharose in
the presence of 250 mM NaCl. The beads were washed, and the
bound peptides were eluted by heating in Laemmli sample buffer prior to
separating on 20% SDS-polyacrylamide gels. The peptides were detected
by immunoblot analysis with horseradish peroxidase-conjugated
streptavidin (Stratagene).
RESULTS
NTS-expressing cells showed no significant binding to
radiolabeled RNA (Fig. 1A), suggesting that the NTS is
required for the association of PTHrP with RNA.
View larger version (31K):
[in a new window]
Fig. 1.
Binding of PTHrP to total cellular RNA and
homopolymeric RNA. Total cellular RNA was harvested from
32P-labeled HeLa cells and incubated with protein
precipitated with anti-Myc antibody or with IgG as a control.
A, shown are the cpm of radiolabeled cellular RNA bound to
protein precipitated with anti-Myc antibody (hatched bars)
or IgG (white bars) from mock-transfected or PTHrPmyc
(PTHrP)-transfected COS-1 cells or from cells expressing
PTHrPmyc NTS (PTHrP-NTS). Bars represent the mean ± S.D. of triplicate wells from three different transfections.
B, COS-1 cells expressing full-length PTHrPmyc or
PTHrPmyc
NTS were lysed, and after removing an aliquot of total cell
lysate (TCL), equal amounts of protein were incubated in the
presence of 250 mM salt with Sepharose beads
(Seph) conjugated to synthetic poly(A), poly(C), poly(G), or
poly(U) RNA. Bound proteins were separated by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose membranes, which were
probed with anti-Myc antibody. Only poly(G)-Sepharose (lane
5) bound a significant quantity of wild-type PTHrPmyc, the
association being disrupted in the absence of the NTS (lane
7) The positions of molecular mass markers (in kilodaltons) are
shown on the left. C, incubation of equivalent amounts of
protein from COS-1 cells expressing wild-type PTHrPmyc with
poly(G)-Sepharose in the presence of increasing salt concentrations did
little to disrupt the association between PTHrPmyc and RNA.
View larger version (67K):
[in a new window]
Fig. 2.
Profile of cellular RNAs bound to transiently
expressed PTHrP. Total cellular RNA harvested from
32P-labeled HeLa cells was incubated with proteins
precipitated from COS-1 cells transfected with cDNAs encoding
PTHrPmyc, PTHrPmyc NTS (-NTSPTHrPmyc), or human FMR1myc
(hFMRmyc). A, bound RNA eluted from precipitated
proteins was resolved on formaldehyde-containing 1% agarose gels and
transferred to nitrocellulose membranes that were exposed to x-ray film
overnight. Positions of 28 S and 18 S ribosomal bands were verified by
ethidium bromide staining. Labeled RNA was eluted from anti-Myc
immunoprecipitates (IP) from lysates of cells expressing
PTHrPmyc and human FMR1myc, but not from
PTHrPmyc
NTS-expressing cells or when IgG was used in place of
antiMyc antibody. B, shown are the results from
immunoblot analysis of aliquots of Sepharose beads recovered
by immunoprecipitation.
NTS) was
unable to associate with homopolymeric RNA (Fig. 1B). To
assess the relative affinity of the PTHrPmyc/RNA interaction, we
performed binding studies in the presence of a high salt concentration,
which has been shown in the past to disrupt nonspecific protein/RNA
interactions (24). The association between PTHrPmyc and
poly(G)-Sepharose was stable and persisted at a salt concentration of 1 M (Fig. 1C). These data suggest that PTHrP is a
bona fide RNA-binding protein and that the association
requires all or part of the NTS.
NTS, and human
FMR1myc. RNAs eluted from the protein-bound beads were then subjected
to formaldehyde-agarose gel electrophoresis and autoradiography. As
shown in Fig. 2A, ribosomal RNA species bound to both
wild-type PTHrPmyc and human FMR1myc, but not to PTHrPmyc
NTS. Fig.
2B demonstrates immunoblot analysis of the proteins
harvested from transfected COS-1 cells that were used for RNA binding.
View larger version (44K):
[in a new window]
Fig. 3.
Binding of endogenous PTHrP to homopolymeric
RNA. A, after removing an aliquot representing the
total cell lysate (TCL), 300 µg of total protein from PTr
cell lysates was incubated with the different homopolymers, and the
bound proteins were visualized by immunoblot analysis as described
under "Results" using a polyclonal antiserum raised against
synthetic PTHrP-(67-86). The same pattern of exclusive binding to
poly(G)-conjugated beads (lane 4) was observed, with a major
band migrating around 27 kDa. B, when binding to
poly(G)-Sepharose was performed in the presence of increasing salt
concentrations, maximum binding of endogenous protein appeared to occur
at 500 mM salt (lane 3), although PTHrP bound to
poly(G) was still evident at a salt concentration of 1 M
(lane 5). Seph, Sepharose.
View larger version (33K):
[in a new window]
Fig. 4.
Effect of amino acid substitutions within the
PTHrP NTS on RNA binding. A, a series of mutant
PTHrPmyc cDNAs was generated with selectively altered residues in
one of three basic domains within the PTHrP NTS. The mutant proteins
were expressed in COS-1 cells, and their binding to poly(G) RNA was
assessed in the presence of increasing salt concentrations as described
under "Results." B, decreased binding of homopolymeric
RNA occurred at 500 mM NaCl for M1PTHrPmyc (lane
3), at 1 M for M2PTHrPmyc (lane 5),
and at 750 mM for M3PTHrPmyc (lane 4).
C, a similar pattern of affinity for radiolabeled cellular
RNA was evident in that binding of M1PTHrPmyc was reduced to near
background levels and that of M3PTHrPmyc was reduced to ~50% of that
seen for the intact protein or M2PTHrPmyc.
4 M, the interaction
between PTHrPmyc and RNA was effectively blocked (Fig. 5B).
The nucleolin NTS peptide, on the other hand, was ineffective in this
capacity even at 10
3 M (Fig. 5B).
The high concentration of peptide required to dislodge in
vitro translated PTHrPmyc from poly(G) homopolymeric RNA was a
further indication that the association was specific and of apparent
high affinity. These competition data suggested that the PTHrP NTS
peptide might bind RNA directly. This possibility was tested by
incubating biotinylated PTHrP NTS peptide with the different
homopolymeric RNAs. The bound peptides were separated by
SDS-polyacrylamide gel electrophoresis and detected by horseradish peroxidase-conjugated streptavidin. As was true of the full-length protein, the PTHrP NTS peptide bound specifically to poly(G)-Sepharose beads (Fig. 5C), and the binding withstood salt
concentrations up to 1000 mM. The nucleolin NTS, on the
other hand, was incapable of binding to any of the synthetic
homopolymers. These experiments confirmed that the interaction between
the PTHrP NTS and poly(G)-Sepharose was specific and direct and was not
merely a function of its ionic strength.
View larger version (45K):
[in a new window]
Fig. 5.
PTHrP binding specificity confirmed using
competition with NTS peptides. PTHrPmyc was transcribed and
translated in vitro in the presence of
[3H]leucine using a rabbit reticulocyte lysate.
A, equal volumes of lysate were incubated with the different
homopolymers at 250 mM salt and with poly(G)-Sepharose at
salt concentrations up to 1 M. Significant binding of
in vitro translated PTHrP was seen only for poly(G) and was
maintained up to 1 M salt (lanes 6-9). Peptides
corresponding to the PTHrP NTS or the nucleolin NTS were synthesized
for use in competition binding assays using the in vitro
translated PTHrPmyc. B, equal volumes of lysate were
incubated with poly(G)-Sepharose in the presence of increasing
concentrations of NTS peptides at a salt concentration of 250 mM. Despite their similar bipartite lysine motifs, the
PTHrP NTS effectively blocked binding of PTHrPmyc at a concentration of
10 4 M (lane 7), whereas the
nucleolin NTS was ineffective even at 10
3 M
(lane 10).C, 10
7 M
biotinylated PTHrP NTS peptide was incubated with the different
homopolymers in the presence of 250 mM NaCl and with
poly(G)-Sepharose in the presence of increasing NaCl concentrations.
The nucleolin NTS peptide was incubated with poly(G)-Sepharose in
buffer containing 250 mM NaCl. Bound peptides were
separated on 20% SDS-polyacrylamide gels and visualized with
horseradish peroxidase (HRP)-conjugated streptavidin.
Distinct bands of peptide alone were observed for the PTHrP NTS
(lane 1) and for the nucleolin NTS (lane 11). The
PTHrP NTS bound to poly(G)-Sepharose (lane 5 and lanes
7-10) in the presence of NaCl concentrations up to 1 M (lane 10). Seph, Sepharose;
TCL, total cell lysate.
View larger version (46K):
[in a new window]
Fig. 6.
Binding of PTHrP to double-stranded RNA.
Proteins expressed in COS-1 cells were used for binding to synthetic
double-stranded nucleotide sequences in the presence of 250 mM salt. A, in the presence of a low salt
concentration, PTHrPmyc, as well as the three mutant proteins, bound
poly(G) (lane 3) and poly(G)·poly(C) (lane 5)
with the same apparent affinity. None of the proteins demonstrated
significant binding to the corresponding deoxynucleotide sequences
(lane 6).B, binding of wild-type and mutant forms
of PTHrPmyc to double-stranded poly(G)·poly(C) RNA demonstrated the
same pattern of affinities as seen for single-stranded poly(G).
TCL, total cell lysate; Seph, Sepharose.
DISCUSSION
, and it is believed that
the junction between the third
-sheet and the
-helix plays a
critical role in RNA binding (10). PTHrP does not conform to these
structural requirements in that it has helix-breaking prolines at
positions 86 and 94 that flank the core motif and does not possess the
extended dsRNA-binding domains. Despite this lack of overall homology,
mutation of specific lysines within the core motif resulted in severely
compromised binding of PTHrP to RNA in our own studies as well as in
those of others examining binding of the dsRNA-binding domain proteins
PKR (27) and GCN2 (17) to RNA. In the case of PTHrP, substitution of other lysine or arginine residues in the NTS had only a modest or no
effect on binding.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Stephen Warren for the kind gift of the human FMR1 cDNA; Hugh Bennett, John Hiscott, Andrew Karaplis, and Antonis Koromilas for guidance and for critical reading of the manuscript; and Li Cheng for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Medical Research Council of Canada and by establishment funds from the Fonds de la Recherche en Santé du Québec (to J. E. H.).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.
§ Recipient of a studentship from the Medical Research Council of Canada.
¶ Supported by a challenge studentship from Manpower Canada.
Medical Research Council of Canada Scholar supported by grants
from the Medical Research Council of Canada and the Cancer Research
Society of Canada.
§§ Chercheur Boursier of the Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed: Lady Davis Inst., Rm. 602, 3755 Côte Ste. Catherine Rd., Montréal, Québec H3T 1E2, Canada. Tel.: 514-340-8260/4986; Fax: 514-340-7573; E-mail: jhenders{at}ldi.jgh.mcgill.ca.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PTHrP, parathyroid hormone-related protein; PTH, parathyroid hormone; NTS, nuclear targeting sequence; RNP, ribonucleoprotein; hnRNP, heterogeneous nuclear ribonucleoprotein; KH, K homology; dsRNA, double-stranded RNA.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|