From the Departments of Human Genetics,
¶ Biological Chemistry, and ** Internal Medicine, University of
Michigan Medical School, Ann Arbor, Michigan 48109
Received for publication, July 21, 2000
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ABSTRACT |
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Incubation of HTC rat hepatoma cells with
8-bromo-cAMP results in a 3-fold increase in the rate of degradation of
type-1 plasminogen activator inhibitor (PAI-1) mRNA. We have reported
previously that the 3'-most 134 nt of the PAI-1 mRNA is able to confer
cyclic nucleotide regulation of message stability onto a heterologous transcript. R-EMSA and UV cross-linking experiments have shown that
this 134 nt cyclic nucleotide-responsive
sequence (CRS) binds HTC cell cytoplasmic proteins ranging
in size from 38 to 76 kDa. Mutations in the A-rich region of the CRS
both eliminate cyclic nucleotide regulation of mRNA decay and abolish
RN-protein complex formation, suggesting that these RNA-binding
proteins may be important regulators of mRNA stability. By sequential
R-EMSA and SDS-PAGE we have purified a protein from HTC cell polysomes
that binds to the PAI-1 CRS. N-terminal sequence analysis and a search
of protein data bases revealed identity with two human sequences of
unknown function. We have expressed one of these sequences in E. coli and confirmed that the recombinant protein interacts specifically with the PAI-1 CRS. Mutation of the A-rich portion of the
PAI-1 CRS reduces binding by the recombinant PAI-1 RNA-binding protein.
The amino acid sequence of this protein includes an RGG box and two
arginine-rich regions, but does not include other recognizable RNA
binding motifs. Detailed analyses of nucleic acid and protein data
bases demonstrate that blocks of this sequence are highly conserved in
a number of metazoans, including Arabidopsis, Drosophila, birds, and mammals. Thus, we have described a
novel RNA-binding protein that identifies a family of proteins with a
previously undefined sequence motif. Our results suggest that this
protein, PAI-RBP1, may play a role in regulation of mRNA stability.
Regulation of mRNA stability is an important component of the
regulation of gene expression and is known to have a significant role
in normal physiology and development (1-5). Our understanding of the
regulation of message degradation has been enhanced by the
identification of consensus cis-acting sequences that are involved in determining message stability and of some proteins that
interact with them (4, 6). Although it is known that many stimuli alter
mRNA stability and some cis-acting sequences responsible
have been identified, in few cases have trans-acting factors
been isolated (2, 7-10). In contrast, a broad spectrum of RNA-binding
proteins that are involved in RNA processing, cellular localization,
and translation have been identified, and structural domains involved
in RNA recognition have been described (11, 12). In many cases
RNA-binding proteins contain short signature domains that bind RNA and
anchor the protein such that functional domains align (13, 14). Much
less is known about the mechanisms by which RNA-binding proteins
regulate mRNA stability.
Plasminogen activators (PAs)1
are serine proteases that catalyze the conversion of plasminogen to the
broad spectrum protease, plasmin. Plasmin is the major fibrinolytic
enzyme in blood and also participates in a number of physiological and
pathological processes involving localized proteolysis such as tissue
remodeling and tumor cell invasion and metastasis (15, 16). PA activity is regulated in large part by type-1 plasminogen activator inhibitor (PAI-1), a 50-kDa glycoprotein found in plasma, platelets, and a
variety of cell types (17). PAI-1 expression is regulated by growth
factors, cytokines, and hormones, including agents that regulate
cellular cAMP levels (18-20).
HTC rat hepatoma cells synthesize tissue-type plasminogen activator
(tPA) and PAI-1. These cells respond to cyclic nucleotides with a
dramatic (50-fold) increase in tPA activity secondary to a 90%
decrease in PAI-1 activity and mRNA. The decrease in PAI-1 mRNA
is due primarily to a 3-fold increase in the rate of mRNA degradation (21). By transfecting HTC cells with chimeric constructs containing the Here we report the isolation and cloning of one of these proteins and
demonstrate its specific interaction with PAI-1 mRNA. Detailed
analysis of nucleic acid and protein data bases demonstrates that this
PAI-1 mRNA-binding protein includes blocks of sequence that are
highly conserved in a number of metazoans. Thus, we have identified and
cloned a novel RNA-binding protein that reveals a family of proteins
with a previously unidentified domain that may define a new RNA-binding
motif. Our results suggest that this protein may play a role in
regulation of mRNA stability.
Materials--
Eagle's minimal essential medium, calf serum,
fetal calf serum, restriction enzymes, RNase T2, and Benchmark®
protein markers were purchased from Life Technologies, Inc.
Benzamidine,
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), heparin (sodium salt), RNase TI, and tRNA were purchased from
Sigma. RNase A, leupeptin, 4-(2-aminoethyl)benzoylsulfonyl fluoride-hydrochloride (Pefabloc ®SC AEBSF), proteinase K, RNase inhibitor, T3 and T7 RNA polymerases, and the high fidelity PCR system
were obtained from Roche Molecular Biochemicals. Coomassie ®Plus
Protein Assay was purchased from Pierce. We purchased thrombin, anti-His antibody, and [32P]UTP from Amersham Pharmacia
Biotech. AmpliscribeTM T3 transcription kit was obtained
from Epicentre Technologies (Madison, WI), and Centricon concentrators
were from Amicon (Beverly, MA). The pET-15b vector, BL21(DE3)pLysS
competent Escherichia coli, IPTG, and His-binding resin were
purchased from Novagen. SDS-PAGE prestained protein markers were from
Bio-Rad.
Cell Culture and Polysome Preparation--
Monolayer cultures of
HTC cells were maintained in Eagle's medium with 5% fetal calf serum
and 5% bovine serum as described previously (21). Cells were grown to
confluence in T-150 flasks and harvested by trypsinization. HTC cell
polysomes were isolated as described previously (23).
Preparation of Radiolabeled and Unlabeled RNA--
All DNA
constructs used as templates to prepare radiolabeled or unlabeled RNA
have been described previously (23). 32P-Labeled sense
strand RNA was prepared by published methods (24) using as template
either plasmid DNA linearized with the appropriate restriction enzyme
or PCR products, including a T3 RNA polymerase site. The DNA template
was incubated for 60 min at 37 °C with T3 or T7 RNA polymerase in
transcription buffer containing RNase inhibitor, ATP, CTP, GTP, and
[32P]UTP (100 µCi of 800 Ci/mmol). The labeled RNA was
purified by electrophoresis through a 6% polyacrylamide, 8 M urea gel, eluted from the gel, and ethanol-precipitated.
Unlabeled competitor RNA was prepared by in vitro
transcription using the AmpliscribeTM T3 transcription kit
according the protocol provided by the manufacturer.
R-EMSA and UV Cross-linking Analysis--
R-EMSA and UV
cross-linking analyses were carried out essentially as described
previously (23). For R-EMSA, HTC cell polysomes or purified
recombinant protein were incubated with 32P-labeled RNA
(~200,000 dpm/reaction) for 20 min at room temperature in buffer
containing 10 mM Hepes (pH 7.6), 5 mM
MgCl2, 40 mM KCl, 5% gylcerol, 1 mM dithiotreitol, 8 units of RNase inhibitor, and 10-25
µg of tRNA. Following consecutive incubations with RNase T1 (1 unit/µl) and heparin (5 µg/µl), electrophoresis was carried out
at 4 °C in 5% nondenaturing polyacrylamide gels (40:1
acrylamide-bisacrylamide). For UV cross-linking analyses, proteins were
incubated with 32P-labeled RNA (~106
dpm/reaction) as described for R-EMSA. Following the heparin incubation, reactions were exposed to UV light (UV Statalinker 1800, Statagene) at a distance of 2.5 cm for 10 min (1.8 µJ/cm2) and incubated for 10 min with RNase A and RNase
T2. Reactions were subjected to SDS-PAGE and visualized by
autoradiography. For competition studies protein was incubated with
unlabeled RNA competitor for 10 min at room temperature prior to
addition of radiolabeled RNA.
Isolation and Cloning of PAI-1 RNA-binding Protein--
HTC cell
polysomes (60 µg/reaction) were incubated with
32P-labeled (~106 dpm/reaction) or unlabeled
(~7 ng/reaction) CRS RNA and R-EMSA carried out as described above.
In each gel, 4 of 16 lanes had R-EMSA reactions with radiolabeled RNA.
High molecular weight complexes were located by a 1-h exposure to x-ray
film at room temperature. Complexes from labeled and unlabeled
reactions were excised and eluted from the gel by overnight incubation
in 50 mM Tris (pH 7.9), 0.1 mM EDTA, 5 mM dithiotreitol, 150 mM NaCl, and 0.1%
SDS (25). Proteins from 32 such reactions were pooled, concentrated by
acetone precipitation, separated on 12% SDS-PAGE, and
electrophoretically transferred to a polyvinylidene difluoride membrane
(ProBlott, PE Applied Biosystems, Foster City, CA). The membrane was
stained with Coomassie Blue and submitted to the Protein and
Carbohydrate Structure Facility of The University of Michigan
Biomedical Research Core Facility, where N-terminal sequencing was
carried out, using a PE Applied Biosystems model 494 protein sequencer.
Expression and Purification of Recombinant Protein--
The
cDNA encoding "hypothetical protein" (DKFZp564M2423,
GenBankTM accession number AL080119) was kindly
provided by the Resource Center and Primary Data base of the German
Human Genome Project (Resource Center and Primary Database,
Berlin). The cDNA was amplified by PCR using a high fidelity PCR
system (Roche Molecular Biochemicals) to include the entire protein
coding sequence. The forward primer (DKFZ bp 71-95) was synthesized
with a mutation at bp 83-85 to create an NdeI site. The
reverse primer (DKFZ 1270-1244) has 1 bp altered to create a
BamHI site. The 1200-bp product was inserted into the
NdeI/BamHI sites of the vector pET-15b,
generating the construct pET-15b/DKFZ for expression of "hypothetical
protein" with an N-terminal histidine tag and thrombin cleavage site.
Ligation products were transformed into E. coli AG-1 cells,
and the subclone was verified by PCR and DNA sequencing. E. coli BL21(DE3)pLysS were transformed with purified pET-15b/DKFZ
plasmid, and protein expression was induced with 2 mM IPTG.
The recombinant protein was purified by Ni2+ chelation
using the His-binding resin according to the supplier's protocol and
was concentrated using Centricon 3 concentrators.
Data Base Search and Protein Sequence Alignment--
An
iterative search of the nonredundant protein data base with PSI-BLAST
(26) was carried out to identify proteins with statistically
significant similarity to the protein we have isolated. The data base
of expressed sequence tags was searched using the gapped version of
TBLASTN. Query sequence was filtered for low complexity regions using
SEG (27), since its RGG repeats nonselectively retrieve many unrelated
proteins with similar low-complexity regions. Gibbs sampling as
implemented in a computer program PROBE (28) was used to identify
conserved sequence regions shared by all members of the family. This
ungapped multiple alignment provided a seed for another round of BLAST
searches in an attempt to identify distantly related sequences.
Finally, we used HMMER programs (29) to produce a Hidden Markov Model,
which was also used to search the protein data base. Both BLAST seed
and Hidden Markov Model of this protein family are available to
interested researches upon request. Identified sequences were searched
against PFAM (30) and Blocks+ (31) to determine whether they have any
previously known sequence motif(s).
Affinity Purification and Identification of PAI-1 mRNA-binding
Protein--
To isolate proteins that interact with the rat PAI-1 CRS,
we have used an RNA affinity approach, using the 134-nt sequence of the
rat PAI-1 CRS shown in Fig.
1A. HTC polysomal proteins were incubated with 32P-labeled or unlabeled CRS and
separated by nondenaturing polyacrylamide gel electrophoresis. The high
molecular weight complex (Fig. 1B, brackets) was
excised from the gel, and the proteins were eluted from the gel slices.
Proteins from 32 such reactions, representing ~2 mg of starting
polysomal protein, were pooled, concentrated by acetone precipitation,
and separated by SDS-PAGE. Fig. 1C shows that three protein
bands are visible in the Coomassie Blue-stained gel. In a parallel
experiment, proteins from the SDS-PAGE gel were transferred to
polyvinylidene difluoride membrane (ProBlott, PE Applied Biosystems,
Foster City, CA), and stained with Coomassie Blue. N-terminal amino
acid sequencing of the major band (Fig. 1C,
arrow) yielded 19 amino acids of N-terminal sequence.
The sequence was submitted to a BLAST search of the nonredundant
protein data base and two entries with 18 identical N-terminal amino
acids were found: hypothetical protein (GenBankTM
accession number AL080119) and "CGI-55"
(GenBankTM accession number AF151813). Both
sequences represent human proteins with an as yet unknown function and
appear to be splice variants; CGI-55 has a 6-amino acid insertion after
position 202 in hypothetical protein (Fig. 1D). The CGI-55
sequence potentially codes for a 43.2-kDa protein, but is a composite
of a several ESTs (32) and is not yet cloned as a single sequence. The
cDNA for hypothetical protein was cloned by the German Cancer
Research Center (Deutsches Krebsforschungszentrum) and was
kindly provided to us by the Resource Center and Primary Data base of
the German Genome Project (Resource Center and Primary Database,
Berlin). While the amino acid sequence does not indicate an RNA
recognition motif (RRM) or a K-homology (KH) domain, the sequence does
include an RGG box at amino acid positions 343-359, an RG-rich region at amino acid 163-184, and an Arg-rich region at amino acid
126-137, motifs frequently found in RNA-binding proteins (11) (Fig.
1D). In addition, there is a potential protein kinase A
phosphorylation site a serine 74, indicating that the protein function
could be regulated by cyclic nucleotides.
Expression of PAI-1 RNA-binding Protein in E. coli--
The coding
region of the cDNA for hypothetical protein was amplified by PCR
and subcloned into the pET-15b vector downstream from the sequence
coding for a histidine tag and a thrombin cleavage site, and the
plasmid was expanded in E. coli AG-1. The subclone was
verified by PCR and DNA sequencing and transformed into E. coli BL21(DE3)pLysS. Expression was induced by 2 mM
IPTG and the protein partially purified by Ni2+ chelation
as described under "Experimental Procedures." Proteins in the
column eluate were separated by SDS-PAGE, and as shown in Fig.
2 (lane 3), the major product
appears as a triplet. To determine which protein band represents
hypothetical protein, we carried out a thrombin digestion, which is
expected to cleave at the thrombin site and remove the N-terminal
His-tag and associated amino acids. Thrombin digestion resulted in
approximately a 2000-dalton decrease in the size of each band in the
triplet (Fig. 2, lane 4), demonstrating that all three bands
represent the hypothetical protein. The different sizes most likely are
the result of premature translation termination due to differences in
codon usage frequency between bacteria and eukaryotes (24, 33). Western
analysis with anti-His antibody confirmed that all three bands carry
the His-tag. NorthWestern analysis demonstrated that a protein that migrates at the same location binds 32P-labeled PAI-1CRS
(data not shown).
Binding Activity of Recombinant Hypothetical Protein--
To
confirm that the recombinant human hypothetical protein binds to the
rat PAI-1 CRS, we have carried out both R-EMSA and UV cross-linking
experiments. Purified recombinant protein was incubated with
32P-labeled PAI-1 CRS and R-EMSA carried out as described
under "Experimental Procedures." Fig.
3A illustrates that the
recombinant protein binds in a concentration-dependent fashion
(lanes 1-5). Binding is competed by increasing amounts of
identical unlabeled sequence (lanes 6-8) but not by a
100-fold molar excess of the bacterial CAT RNA sequence (lane
11). The U-rich region of the PAI-1 CRS (nt 2926/3024), which by
itself does not form the major R-EMSA complex with HTC cell cytoplasmic
proteins (23), competes only weakly for CRS interaction with the
recombinant protein (lane 9). More significantly, a portion
of the rat PAI-1 mRNA (nt 2125/2296) that does not confer cyclic
nucleotide regulation of message stability (22) also fails to compete
for binding (lane 10).
Similar results were obtained by UV cross-linking experiments. As shown
in Fig. 3B, the RNA-protein complex migrates with an
apparent molecular mass of ~50 kDa, similar in size to the major band seen with HTC cytoplasmic proteins (23). Binding is
dependent on protein concentration (Fig. 3B, lanes
1-5) and is competed by unlabeled CRS (lanes 6-8),
but not by CAT (lane 11) or PAI-1 2125/2296 (lane
10). The presence of faster migrating bands observed in the
presence of unlabeled PAI 2125/2296 suggests that the apparent decrease
in the binding complex is probably caused by cross-linking between the
labeled probe and the unlabeled competitor RNA rather than by
competition for the same binding protein. The U-rich region of the
PAI-1 CRS (nt 2926/3024) at 100-fold molar excess competes somewhat,
but less well than the full sequence. These results confirm that the
protein isolated from an R-EMSA complex is a PAI-1 RNA-binding protein,
which we have named PAI-RBP1. Interestingly, the recombinant protein,
which migrates on SDS-PAGE as a triplet, forms only a single UV
cross-linked complex with PAI-1 CRS, suggesting that the smaller,
C-terminal truncated forms are not able to bind.
Delineation of Sequences Involved in Binding to Recombinant Human
PAI-1 RNA-binding Protein--
To further define the sequence within
the PAI-1 CRS required for binding to the PAI-RBP1, we carried out
binding experiments with portions of the CRS. Fig.
4A shows diagrammatically the
regions of the CRS used to generate radiolabeled probes. In R-EMSA the 3' portion of the CRS (nt 2926/2966, lane 4) and the entire
U-rich portion (nt 2926/3024, lane 6) fail to bind.
Mutations in the A-rich region of the CRS, which we have shown
eliminate binding to HTC cytoplasmic proteins (23), severely decrease
or abolish the ability of the RNA to bind to the recombinant RBP1
(lanes 8 and 10). As expected, neither labeled
CAT mRNA nor the PAI 2125/2296 appears to interact with the RBP1.
Thus, the PAI-RBP1 appears to interact primarily with the A-rich
portion of the CRS confirming that the newly isolated PAI-RBP1 has
sequence specificity and binding properties similar to that of the
major 50-53-kDa HTC cytoplasmic protein (23).
Protein Sequence Alignment and Identification of a Family of
Proteins--
The initial search of the protein data base using the
19-amino acid sequence produced only the two sequences with the
identical N-terminal amino acids. The data base has grown considerably
since, and we have now done an extensive search, as described under
"Experimental Procedures," for similarity to the entire
hypothetical protein and DKFZp564M2423 cDNA sequence. This search
has revealed several other proteins with a high degree of similarity,
particularly in the C-terminal portion. These similar proteins are
found in nine different species, including Arabidopsis,
Drosophila, chicken, mouse, and rat (Fig.
5). The availability of new members of
the family permitted us to generate a multiple alignment and to
identify their common motifs; the alignment revealed that these
proteins share several blocks of conserved sequence (Fig. 5). This
domain, which we propose has a function in RNA binding, is located at the C-terminal of all the proteins in this family and, apart from a
relatively short stretch of RGG repeats, appears to be a compact entity. The C-terminal RGG box is located between the last two sequence
blocks and the RG-rich and Arg-rich regions are N-terminal to
the conserved blocks. A number of the other proteins in the family have
the RGG box. Thus, PAI-RBP1/hypothetical protein is a member of a
family of proteins that share a putative novel RNA binding motif.
We report here the identification of a novel rat cytoplasmic
protein isolated based on its ability to form R-EMSA complexes with the
rat PAI-1 mRNA cyclic nucleotide-responsive sequence. N-terminal
sequence data reveal identity with a human sequence, hypothetical
protein (GenBankTM accession number AL080119) of
unknown function. We have expressed the human sequence in bacteria and
analyzed its binding properties. Our results demonstrate that the human
protein is a PAI-1 mRNA-binding protein; we now refer to this gene
product as PAI-1 RNA-binding protein or PAI-RBP1.
Recombinant human PAI-RBP1 binds the A-rich portion of the rat PAI-1
CRS and fails to interact with the isolated U-rich portion. Mutations
of the A-rich sequence severely decrease binding. In addition, the
specificity of PAI-RBP1 binding is demonstrated by its failure to form
a complex with either the bacterial CAT RNA or an upstream, noncyclic
nucleotide-responsive region of the PAI sequence. We have reported
previously that the PAI-CRS forms complexes with HTC cell cytoplasmic
proteins ranging in size from 38 to 76 kDa (23). The binding
specificity and the size of the complex seen in Fig. 3B
indicate that PAI-RBP1 is similar to the 50-53-kDa HTC cell
cytoplasmic protein and may, in fact, be the human homologue of that
rat protein. Because the PAI-1 mRNA binding site is a cyclic
nucleotide-responsive sequence, it would be reasonable to expect that
binding activity of PAI-RBP1 may be influenced by its phosphorylation
state. The recombinant protein was expressed in bacteria and,
therefore, not posttranslationally processed as it might be in
mammalian cells, possibly explaining the high concentration of protein
required for binding.
As seen in Fig. 2, the partially purified protein migrates on SDS-PAGE
as a triplet. The size of the smaller products is consistent with a
C-terminal truncation at the beginning of or within the RGG box. One of
the arginines and four of the glycines in this region are coded by rare
codons in bacteria and could cause premature termination (24, 33). Only
a single RNA-protein complex is detected in UV cross-linking
experiments (Fig. 3A), strongly suggesting that the most
C-terminal portion is required for RNA recognition.
A number of proteins that interact with RNA transcripts have been
identified and structural domains involved in RNA recognition described (11, 12). While the majority of known RNA-binding proteins function in RNA processing or translation, several proteins that interact with mRNAs and influence transcript stability have been identified. The iron response element-binding proteins were among
the first for which a clear relationship between binding and mRNA
stability has been demonstrated (10). Members of the Elav
family of proteins, including HuR and HuB (HelN1), bind RNA through
RRMs and have been reported to stabilize AU-rich element-containing RNAs (8, 34, 35). In contrast, the AUF-1 family of proteins (also
called hnRNP D), bind AU-rich element regions and enhance degradation
of the transcript (36, 37). AUF-1 proteins have two RRMs, as well as a
C-terminal RGG that appears to be important for binding (38-40).
PAI-RBP1 does not have a recognizable RRM or KH domain. It does,
however, have an RGG box at amino acid positions 343-359 (Fig.
1D), as well as an RG-rich (amino acids 126-137) and an Arg-rich (amino acids 163-184) motif, which places it in
the general category with RNA-binding proteins (11, 12). It is of
particular interest that this protein, which may be involved in a
cyclic nucleotide-mediated regulation event, has a potential protein kinase A site (RKES) at serine 74.
We have identified several additional proteins from the data base that
share significant similarity with PAI-RBP1 in the C-terminal region
(Fig. 5). Interestingly, UV cross-linking experiments, which show a
single protein-RNA complex, suggest that the most C-terminal region is
required for RNA binding. All members of the protein family shown in
Fig. 5 were used to search PFAM, a large data base of more than 2000 protein domain families (30), and failed to retrieve any previously
known protein domain. Furthermore, these proteins did not show any
significant matches when searched against a collection of short
sequence motifs contained in Blocks+ (31). Taken together, these
results argue that PAI-RBP1 and its homologues comprise a novel family
of proteins. Because PAI-RBP1 was isolated based on its RNA binding
property, and because most members of the family have a similar RGG box
in the C-terminal region and an additional Arg-rich motif, we
suggest they constitute a family of novel RNA-binding proteins. In
fact, two of the identified proteins are annotated in the protein data
base as nuclear RNA-binding proteins.
We also searched the data base of expressed sequence tags (ESTs) using
TBLASTN and found evidence that the C-terminal, putative RNA-binding
domain is conserved in more than 10 different metazoan species, which
include, in addition to those shown in Fig. 5, Xenopus,
zebrafish, and pig. A search of the human EST data base shows that the
mRNA is expressed in a wide range of tissues, including pancreas,
liver, lung, muscle, ovary, and brain. These findings strongly suggest
that PAI-RBP1 has a more general biological role involving regulation
of mRNA stability or processes requiring interaction with RNA. Thus
far homologues of RBP1 have been found only in metazoans, and their
role may be related to regulatory mechanisms that were developed after
the emergence of multicellular organisms. However, a protein of
statistically marginal similarity to PAI-RBP1 is found in yeast. This
protein, Stm1p, binds G-quartet DNA and purine motif DNA triplets, but
not double-stranded DNA (43). While it remains possible that Stm1p can
bind RNA, its limited similarity to PAI-RBP1 is probably a relic of
divergent evolution.
PAI-RBP1 appears to have four splice variants (two alternative splice
sites). CGI-55 differs from PAI-RBP1 (hypothetical protein) by the
insertion of 6 amino acids at position 202. An incomplete sequence for
a rat homologue of unknown function from intestinal epithelium (44)
(GenBankTM accession number U21718) has also been
reported. This cDNA codes for an additional 15 amino acid sequence
inserted after amino acid position 226 of PAI-RBP1 (Fig.
1D). Using primers complementary to the human PAI-RBP1, we
have cloned the rat homologue from an HTC cell cDNA library (45).
This cDNA sequence indicates a variant that includes both the 6 amino acid and 15 amino acid inserts. In addition, a scan of the human
genome found ESTs that include the 15-amino acid insertion with and
without the 6 amino acid insert. Thus, four variants of the rat and
human PAI-RBP1 are possible. Such variations could alter RNA binding
properties and/or the function of the RNA-protein complex. Experiments
are in progress to construct each of these variants and to analyze
their RNA binding properties.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin coding sequence and portions of the PAI-1 3'-UTR, we have shown that sequences in the PAI-1 3'-UTR are able to
confer cyclic nucleotide regulation onto the heterologous transcript. Analysis of deletion and insertion constructs demonstrated that the
3'-most 134 nt of the PAI-1 mRNA by itself is able to mediate this
response. This cyclic nucleotide-responsive
sequence (CRS) includes a 75-nt U-rich region at its 5' end
and a 24-nt A-rich region at its 3' end (22). RNA electrophoretic
mobility shift assay (R-EMSA) experiments have shown that the PAI-1 CRS
forms complexes with HTC cell cytoplasmic proteins, and UV
cross-linking studies have demonstrated binding proteins ranging in
size from 38 to 76 kDa. Most of these proteins interact with the A-rich portion of the PAI-1 CRS. Mutations in the A-rich regions abolish both
RNA-protein complex formation and cyclic nucleotide regulation of
message decay in transfected HTC cells, suggesting that these RNA-binding proteins may be important regulators of mRNA stability (23).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Isolation and identification of a PAI-1
mRNA-binding protein. A, PAI-1 CRS with the U-rich
and A-rich portions indicated by a light and heavy
underline, respectively. B, R-EMSA. Polysomes
were isolated from HTC cells, and R-EMSA was carried out using
32P-labeled PAI-1 CRS as described under "Experimental
Procedures." The brackets indicate the RNA-protein complex
excised for isolation of binding protein. C, SDS-PAGE
analysis of proteins eluted from RNA-protein complex. Gel slices were
taken from 32 reactions as shown in B, and proteins were
eluted in Tris/NaCl/SDS buffer. Eluted proteins were pooled,
concentrated by acetone precipitation, and separated on SDS-PAGE. The
figure shows the Coomassie Blue-stained gel, and the arrow
indicates the protein from which N-terminal sequence was obtained.
D, amino acid sequence of hypothetical protein. The amino
acid sequence was taken from GenBankTM locus CAB45718. The
RGG box is shown by an open box and the RG-rich and
Arg-rich regions are shown by a single underline.
The asterisk indicates the potential protein kinase A
phosphorylation site. The positions of the 6- and 15-amino acid inserts
found in variants are indicated.
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Fig. 2.
SDS-PAGE of partially purified PAI-1
mRNA-binding protein. The cDNA for hypothetical protein
(DKFZp564M2423), subcloned into the vector pET-15b (pET-15b/DKFZ), was
introduced into E. coli BL21(DE3)pLysS. Expression from the
vector was induced with IPTG, and the recombinant protein was purified
by Ni2+ chelation chromatography. Proteins were separated
by 0.1% SDS-10% PAGE and the gel stained with Coomassie Blue.
Lane M, prestained protein markers; lane 1,
untreated bacterial extract (10 µg); lane 2, IPTG-induced
bacterial extract (10 µg); lane 3, Ni2+ column
eluate (4 µg); lane 4, column eluate (4 µg) was
incubated for 3 h at 22 °C with thrombin (0.01 unit/µg of
protein, 0.04 unit total). The reaction was stopped by addition of
SDS-PAGE sample buffer.
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Fig. 3.
R-EMSA and UV cross-linking analyses of
PAI-RBP1. Recombinant human PAI-RBP1, expressed in E. coli and partially purified by Ni2+ chelation column
chromatography, was incubated at the concentrations indicated
(lanes 1-5) with 32P-labeled PAI-1 CRS and
R-EMSA (A) or UV cross-linking (B) carried out as
described under "Experimental Procedures." To examine specificity
of binding, PAI-RBP1 was incubated with unlabeled competitor RNA for 10 min prior to addition of the labeled probe. Unlabeled PAI-1 CRS was
used at 10-, 50- or 100-fold molar excess (lanes 6-8). The
U-rich portion of the PAI-1 CRS (nt 2926-3034, lane 9), a
portion of the PAI-1 3'-UTR that does not confer cyclic nucleotide
responsiveness (nt 2125-2296, lane 10), and bacterial CAT
RNA (lane 11) were each added at 100-fold molar
excess.
View larger version (53K):
[in a new window]
Fig. 4.
Analysis of RNA sequence requirement for
PAI-RBP1 binding. A, schematic representation of the
radiolabeled probes used. The full-length (134 nt) PAI-1 CRS and the
U-rich portions tested are shown schematically. The mutations in the
A-rich portion were created in the context of the full-length CRS.
B, and C, identification of the
protein binding sites. The 32P-labeled RNA sequence
indicated above each lane was incubated with buffer only ( ) or with
recombinant PAI-RBP1 (7 µg) (+), and RNA-protein interactions were
examined by R-EMSA (B) or UV cross-linking
(C).
View larger version (118K):
[in a new window]
Fig. 5.
Protein sequence alignment. An
iterative search of the nonredundant protein data base was carried out
with PSI-BLAST, and the multiple sequence alignment was produced using
Gibbs sampling function of PROBE (28). Some family members were added
from an alignment generated by HMMER (29). The figure was made using
ALSCRIPT (46). Numbers on the left are
GenBankTM identifier codes for proteins. The first and last
amino acid residues of the aligned region are numbered preceding the
first block and following the last block, respectively.
Numbers in parentheses indicate the size of gaps
between blocks. Common protein names from their data base annotations
are shown at the end of the alignment, followed by species names. The
sequence from Rattus Norvegicus (clone C426 intestinal
epithelium) has question marks in place of amino acids
numbers because the sequence is incomplete. Amino acids are colored
differently from the background when at least 90% of the residues
conform to a consensus. The following color scheme was used:
hydrophobic residues (ACFGHIKLMRTVWY) are dark blue;
aliphatic residues (ILV) are green; polar residues
(CDEHKNQRST) are red; small residues (ACDGNPSTV) are
purple; charged residues (DEHKR) are orange.
Individual residues with more than 90% identity across the whole
alignment are highlighted in yellow.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CP-1 and
CP-2 are KH domain (41) poly(rC)-binding proteins and
play an important role in regulation of
-globin and tyrosine
hydroxylase mRNA stability (2, 42). Vigilin is a KH-domain protein
that is induced in Xenopus oocytes by estrogen and binds to
and stabilizes vitellogenin mRNA (7).
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ACKNOWLEDGEMENTS |
---|
We thank Owen Wittekindt and Laura Thill for expert technical assistance. We thank The Resource Center and Primary Data base of the German Human Genome Project for providing the plasmid DKFZp564M2423.
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FOOTNOTES |
---|
* This work was supported by Public Health Service Grant CA22729 from the National Cancer Institute (to T. D. G.). We also acknowledge National Institutes of Health Grant 5 P60 DK-20572 to The University of Michigan Diabetes Research and Training Center for the support of core services.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.
§ To whom correspondence should be addressed: University of Michigan Medical School, Dept. of Human Genetics, 4909 Buhl Bldg., Box 0618, 1241 E. Catherine, Ann Arbor, MI 48109-0618. Tel.: 734-763-3460; Fax: 734-615-0465; E-mail: heatonj@umich.edu.
Supported by the AACR-Sidney Kimmel Foundation for Cancer
Research and is a Special Fellow of the Leukemia and Lymphoma Society.
Published, JBC Papers in Press, September 22, 2000, DOI 10.1074/jbc.M006538200
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ABBREVIATIONS |
---|
The abbreviations used are:
PA, plasminogen
activator;
tPA, tissue-type plasminogen activator;
PAI-1, type-1
plasminogen activator inhibitor;
UTR, untranslated region;
CRS, cyclic
nucleotide-responsive sequence;
RBP, RNA-binding protein;
PCR, polymerase chain reaction;
nt, nucleotide(s);
bp, base pair(s);
R-EMSA, RNA electrophoretic mobility shift assay;
PAGE, polyacrylamide
gel electrophoresis, IPTG,
isopropyl--D-galactopyranoside;
RRM, RNA recognition
motif;
KH, K-homology;
CAT, chloramphenicol acetyltransferase;
EST, expressed sequence tag.
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