(Received for publication, April 10, 1995; and in revised form, May 12, 1995)
From the
The central nervous system deposition by neurons and glia of
Alzheimer's disease (AD)
Increased APP mRNA levels have been observed in central nervous
system neurons of AD patients(5) . As mRNA and protein levels
are often correlated, the overexpression of APP mRNAs may be an
important predisposing factor to
In mammalian cells mRNAs decay
with very heterogeneous rates. Most cytokine (tumor necrosis factor
All amyloidogenic APP mRNAs that are
produced from a single copy gene by alternative splicing share an
identical 3`-UTR. We have previously shown that APP mRNAs decay in
resting human peripheral blood mononuclear cells (PBMC) with a
half-life of 4 h(12) . Upon cellular activation by phorbol
ester and phytohemagglutinin, APP mRNAs were stabilized with a
half-life >12 h. Regulated decay was dependent on a 29 base region
located approximately 200 bases downstream from the stop
codon(12) . This region was shown to specifically interact with
multiple, cytoplasmic proteins(13) . These binding activities
were not detectable in resting PBMC but were rapidly induced upon
cellular activation with mitogens(12) . Therefore, we proposed
that mitogen-activated mRNA-binding proteins interacted with the
29-base element and participated in stabilization of APP
mRNA(12) . In an effort to better understand the mechanisms of
mitogen-dependent APP mRNA stabilization, we have purified these APP
mRNA-binding proteins from cytoplasm derived from a human T-cell
leukemia. Amino acid and Western blot analysis unambiguously identified
nucleolin and heterogeneous nuclear ribonucleoprotein (hnRNP) C
proteins as the previously described APP mRNA-binding proteins. These
data demonstrated that these dominantly (but not exclusively) nuclear
proteins are likely involved in regulating the stability of cytoplasmic
mRNAs through 3`-UTR site-specific interactions.
The supernatant was dialyzed against buffer A (15
mM HEPES, pH 8, 10% glycerol, 1 mM dithiothreitol,
and 0.1 mM phenylmethylsulfonyl fluoride), centrifuged at
15,000
For purification of the protein components of the 42-
and 47-kDa APP RNA
Figure 1:
Purification of APP RNA- binding
proteins on phenyl-Sepharose. Panel A, 12% SDS-polyacrylamide
gel protein profile of phenyl-Sepharose fractions. Fractions (numbered
along the top of the figure) possessing APP mRNA binding activities
were subjected to electrophoresis on a 12% SDS-polyacrylamide gel and
visualized by Coomassie Blue staining. Migration of molecular size
markers is shown to the right. Putative APP RNA-binding proteins are
denoted by arrowheads on the left. Panel B, gel
mobility shift assays of phenyl-Sepharose fractions shown in panel
A (numbered along the top). Radiolabeled APP RNA(2246-2313)
was incubated with the samples as described under ``Materials and
Methods'' before the addition of RNase T1 and continued incubation
for 45 min. Reaction mixtures were then UV cross-linked and resolved on
a 12% SDS-polyacrylamide gel and subjected to autoradiography. The
migration of molecular size standards is shown to the right. APP
RNA-protein complexes are denoted by arrowheads along the
left.
Figure 2:
Northwestern blot analysis of
phenyl-Sepharose fractions. Panel A, phenyl-Sepharose
fractions (numbered along the top of the figure) possessing APP RNA
binding activities were subjected to electrophoresis in duplicate on
12% SDS-polyacrylamide gels. Half was stained with Coomassie Blue (left panel) and the other transferred to nitrocellulose
membrane and Northwestern analysis with radiolabeled APP RNA
(2246-2313) was carried out as described under ``Materials
and Methods'' (right panel). Migration of molecular size
markers is shown in the middle. The 70-kDa APP RNA-binding protein is
denoted by an arrowhead on the left. Panel B,
fraction 18 of phenyl-Sepharose column was electrophoresed in duplicate
on a longer 12% SDS-polyacrylamide gel. One lane was stained with
Coomassie Blue (left lane), and the other was used to perform
Northwestern analysis with APP RNA as described above (right
lane). The position of 48- and 47-kDa APP RNA-binding proteins is
denoted by arrowheads to the left.
Figure 3:
Four of the APP RNA
Figure 4:
Purification of 38- and 39-kDa APP
RNA-binding proteins on heparin-Sepharose. Panel A, fractions
(numbered along the top of the figure) from heparin-Sepharose were
analyzed for protein by Coomassie-stained 12% SDS-polyacrylamide gel (upper panel) and for APP RNA binding activity by gel mobility
shift assay (lower panel). Arrowheads in the upper panel denote the 38-kDa protein and in the lower
panel 42- and 47-kDa APP RNA
Figure 5:
Purified proteins interact with wild type
but not mutant APP RNA. Phenyl-Sepharose fractions 17 (containing 70-,
48-, and 47-kDa proteins), 18 (enriched for 48- and 47-kDa proteins),
and heparin-Sepharose fraction 19 (containing 38- and 39-kDa proteins)
were subjected to gel mobility shift assay using wild type APP RNA or
mutant APP RNA. Gel mobility shift assay was performed as described in
the legend to Fig. 1. The migration of molecular size standards
is shown to the right. APP RNA
Figure 6:
Schematic representation of the 110-kDa
nucleolin protein. Position of the sequenced peptides are shown by vertical arrows. N and C denotes the amino-
and carboxyl-terminal of nucleolin. R1, R2, R3, and R4 are RNA-binding domains, and the
glycine-arginine-rich region is denoted GR.
Figure 7:
APP RNA-binding proteins react with the
monoclonal antibodies against human nucleolin and hnRNP C proteins.
Fractions 15 and 18 from phenyl-Sepharose and 19 from heparin-Sepharose
(denoted along the top) were electrophoresed on 12% SDS-polyacrylamide
gels, transferred to Immobilon PVDF membrane, and allowed to react with
anti-human nucleolin CC98 hybridoma supernatant (panel A) or
anti-hnRNP C proteins 4F4 (panel B) monoclonal antibodies. Panel C, Western blot of phenyl-Sepharose fraction 18 that was
resolved on a longer gel and reacted with CC98 antibody. Positions of
70-, 48-, 47-, 39-, and 38-kDa APP RNA-binding proteins are denoted.
Migration of molecular size standard for panel A and B is shown in the middle.
Figure 8:
Nucleolin and hnRNP C proteins are present
in the cytoplasm and nucleus. Panel A, radiolabeled SP1
oligonucleotides were incubated with 5 µg of nuclear (N)
or cytoplasmic S20 (S) extracts at room temperature for 20 min
prior to separation on 4% native polyacrylamide gels. Cold competitors
(SP1) or (AP1), 50-fold molar excess were added 5 min before the
addition of radiolabeled SP1 oligonucleotides. Assayed fractions are
shown along the top. Free probe, nonspecific, or specific complexes are
denoted on the left. Panel B, gel mobility shift assay using
radiolabeled APP RNA(2246-2313) was performed with 4 µg of
nuclear (N), cytoplasmic (S), or Jurkat cytoplasm (C) protein. Reaction mixtures were UV cross-linked, resolved
on 12% SDS-polyacrylamide gels, and subjected to autoradiography. The
migration of 6 APP RNA
We have shown previously that APP mRNA specifically interacts
with multiple cytosolic proteins from actively growing tumor cells,
activated human PBMC, and human brain(12, 13) . We
have purified these APP RNA-binding proteins from the cytosolic lysate
of a human T-cell leukemia. On the basis of amino acid sequence and
reactivity to the monoclonal antibodies, we demonstrated that these
proteins were nucleolin and hnRNP C proteins. We also demonstrated that
the purified proteins specifically interact with the wild type but not
the mutant APP RNA.
Nucleolin is a multifunctional protein
implicated in the transcription and processing of rRNA(26) ,
chromatin decondensation(27) , and transcriptional repression (28) presumably through DNA binding(29) . The 110-kDa
protein is functionally divided into three regions: an N-terminal
acidic domain, C-terminal ribonucleoprotein-like (RNP)
domain(18) , and glycine-arginine-rich domains that mediate RNA
and protein interactions(16, 30) . The protein is
subject to phosphorylation (31, 32) and
methylation(30) . The N-terminal domain of nucleolin binds DNA
and histone H1 (27) and is phosphorylated by cdc-2 (32) or nucleolar casein kinase NII(31) . These data
suggest post-translational modifications likely regulate the function
of nucleolin. In resting, non-cycling cells, nucleolin was unable to
bind APP mRNA(12) . Mitogenic stimulation with phorbol ester
induced nucleolin binding activity and stabilization of APP
mRNA(12) . As cycloheximide failed to antagonize the effects of
mitogens on nucleolin or APP mRNA decay, we infer that preformed
nucleolin was post-translationally modified, possibly through a protein
kinase C-dependent pathway. At present we have no data implicating
methylation in regulating nucleolin's APP RNA binding activity.
Based upon solution phase, RNA mobility shift assay, and
Northwestern blotting, we have demonstrated that nucleolin fragments
but not full-length protein interacted with APP mRNA. This suggests a
precursor-product relationship between intact nucleolin and those
fragments capable of APP RNA binding and implies proteolytic cleavage
is an obligatory, regulatory step. All RNA-binding protein fragments
contained the C-terminal RNP domains suggesting these regions mediated
APP RNA binding. Previously an RNP-containing 50-kDa fragment of
nucleolin was reported to bind the pre-mRNA 3`-splice site matching
sequence, UUAGG(33) . A 48-kDa single-stranded DNA-binding,
C-terminal fragment of nucleolin has been reported that contained the
four RNP domains and the glycine-arginine-rich region(34) .
Thus, the C-terminal half of nucleolin likely mediates APP mRNA
binding.
Nucleolin has been shown to bind nonspecifically to 28 S
and 18 S ribosomal RNA(18) . All of these binding assays were
carried out on a nitrocellulose solid support. Under these conditions,
predicted variation in nucleolin's
Both
full-length and nucleolin fragments are labile in resting cells (17) . When cell-free extracts derived from resting PBMC were
incubated in vitro, anti-nucleolin immunoreactivity
disappeared within 1.5 h(17) . Conversely, substantial
full-length nucleolin and fragments of 70 and approximately 50 kDa were
intact for 72 h in extracts prepared from
12-O-tetradecanoylphorbol-13-acetate/phytohemagglutinin-activated
PBMC or cycling T-cell tumor lines(17) . These data demonstrate
that nucleolin becomes resistant to repetitive cleavage upon cell
activation and acquires the ability to bind APP mRNA. Even in lysates
from activated cells, full-length nucleolin appears unable to bind APP
mRNA. Thus, cleavage to 70-, 48-, and 47-kDa fragments, all of which
contain the C-terminal RNP domains, appears necessary. As 70-, 48-, and
47-kDa fragments are also present in cytoplasmic extracts from resting
cells which lack detectable APP mRNA binding activity, a second
modification of nucleolin likely occurs. Based upon the effects of
phorbol esters, protein kinase C-dependent phosphorylation is most
probable.
Using Western blots and amino acid sequence analysis, we
demonstrated that 42- and 47-kDa APP RNA
Interaction of APP RNA with cytosolic proteins resulted in the
formation of 104-, 84-, 73-, 65-, 47-, and 42-kDa UV-cross-linked
RNA
Nucleolin has
been reported to shuttle between the nucleus and cytoplasm whereas
hnRNP C proteins were reported to be localized to the nucleus. However
a cytoplasmic AUUUA binding activity has recently been attributed to
the hnRNP C proteins(37) . We have purified APP RNA-binding
proteins from cytoplasmic lysates. To provide conclusive evidence that
our cytoplasmic preparations were free of nuclear proteins, we
demonstrated the absence of SP1 and Oct-1 DNA binding activities,
whereas APP RNA binding activities were observed in both nuclear and
cytoplasmic fractions. Inactivation of SP1 or Oct-1 DNA binding
activity by the cytoplasm after leakage during subcellular
fractionation is unlikely. First, Oct-1 has DNA binding activity when
synthesized in vitro using programmed reticulocyte lysates (22) . Second, recombinant SP1 produced in transfected SF9
cells or Escherichia coli exhibited sequence-specific DNA
binding and activated transcription(40) . Third, Jurkat cytosol
failed to inhibit the DNA binding activity of other transcriptional
factors such as AP1, NF-AT, Jun, and Fos(24) . Previous reports (37) and Western blots here using anti-nucleolin and hnRNP C
protein antibodies further confirmed the presence of these proteins in
the cytoplasmic fractions.
The shuttling of nucleolin and hnRNP C
proteins suggests that they may modulate cytoplasmic mRNA stability.
Adenosine-uridine-binding factor, an AU-specific mRNA-binding protein,
blocks the rapid decay of granulocyte macrophage colony-stimulating
factor mRNA(11) . The iron response element-binding protein
subserves an equivalent role in the stabilization of transferrin
receptor mRNA(41) . These proteins may protect the AUUUA motifs
or iron response element, respectively, from the action of specific
endonucleases. Thus we propose that the regulated binding of nucleolin
and hnRNP C proteins similarly modulates the cytoplasmic stability of
APP mRNA.
We thank Ning-Hsing Yeh for monoclonal antibody CC98
to human nucleolin, Serafin Pinol-Roma and Gideon Dreyfuss for 4F4
monoclonal antibody against hnRNP C, and the laboratory for stimulating
and insightful comments.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
A4 amyloid protein is an important contributing factor to the
development of Alzheimer's disease. Amyloidogenic cells
overexpress amyloid precursor protein (APP) mRNAs suggesting a
transcriptional or post-transcriptional defect may contribute to this
process. We have previously shown that APP mRNAs display regulated
stability which is dependent on a 29-base element within the
3`-untranslated region (UTR). This domain specifically interacted with
several cytoplasmic RNA-binding proteins. We have purified these APP
RNA-binding proteins from a human T-cell leukemia and demonstrate that
five cytoplasmic proteins of 70, 48, 47, 39, and 38 kDa form the
previously observed APP RNA
protein complexes. Amino acid sequence
analyses showed that the 70-, 48-, and 47-kDa proteins were fragments
of nucleolin and that the 39- and 38-kDa proteins were heterogeneous
nuclear ribonucleoprotein (hnRNP) C protein. Northwestern and Western
blot analyses of purified material further confirmed these data.
Nucleolin protein is known to shuttle between the nucleus and cytoplasm
but hnRNP C has not been reported within the cytoplasm. This report of
sequence specific, mRNA binding by nucleolin and hnRNP C suggests that
these proteins participate in the post-transcriptional regulation of
APP mRNA through 3`-UTR, site-specific interactions.
(
)is
characterized by the presence of
A4 amyloid protein deposits in
neuritic (senile) plaques found in the brains of affected individuals.
A4 amyloid protein is derived from the proteolytic cleavage of
amyloid precursor proteins (APP) encoded by a single copy gene
located on chromosome 21(1) . APPs are multifunctional,
transmembrane glycoproteins that are synthesized by a variety of cells
and tissues (2) . During normal processing, APPs are cleaved
within the
A4 sequence, releasing a non-amyloidogenic peptide into
the extracellular space(2) . Overproduced
A4 amyloid
protein deposits heterogeneously throughout the brain and is associated
with neuronal dysfunction and death. Transformed cells grown in
vitro with exogenous
A4 amyloid protein have shown altered
morphology, growth rates, and gene expression profiles(3) .
Recent reports of amyloid deposition in the brain of APP transgenic
mice (4) further strengthens the connection between
overexpression of
A4 amyloid and neuropathology characteristic of
AD.
A4 amyloid protein deposition.
Increased APP gene transcription and/or decreased APP mRNA degradation
could account for increased steady state levels. The APP promoter was
responsive to phorbol esters and phytohemagglutinin (6) suggesting endogenous mitogens such as cytokines might
enhance APP gene transcription. Similarly, mitogen-driven entry into
the cell cycle has been associated with substantial changes in mRNA
decay rates(7, 8) .
, interleukin 2, and granulocyte macrophage colony-stimulating
factor) and proto-oncogene (fos, myc, myb) mRNAs are very unstable with
half-lives of 15-45 min(8, 9) . Mitogenic
activation resulting in cell cycle entry often increased the steady
state levels of these mRNAs by decreasing their
degradation(7, 8, 10) . Mitogen-dependent
post-transcriptional regulation of cytokine mRNAs appears to be
dependent on cis-trans interactions between cytoplasmic
RNA-binding proteins, such as the adenosine-uridine-binding factor and
3`-UTR AUUUA reiterations(11) . Such regulation allows rapid
and transient alteration of the gene expression in response to changes
in environmental conditions.
Purification of APP mRNA-binding Proteins
Subcellular Fractionation
Log phase human Jurkat
(J32) cells (2 10
) were washed twice in ice-cold
fresh media. The pellet was resuspended in 2 ml of ice-cold buffer A
(10 mM Tris, pH 7.4, 1.5 mM potassium acetate, 5
mM MgCl
, 2 mM dithiothreitol, and 0.1
mM phenylmethylsulfonyl fluoride). All subsequent steps were
performed on ice. Cells were Dounce homogenized using a loose fit
pestle until no intact cells were observed under the microscope. Nuclei
were collected by centrifugation at 1,000
g for 10 min
at 4 °C. The supernatant was centrifuged at 20,000
g for 10 min, and the supernatant was saved as S20 cytoplasmic
fraction. Pelleted nuclei were resuspended in two-thirds volume buffer
B (20 mM HEPES, pH 8, 1.5 mM MgCl
, 25%
glycerol, 420 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl
fluoride), and nuclear proteins were extracted on ice for 30 min.
Nuclear debris was removed by centrifugation at 12,000
g for 5 min at 4 °C. Nuclear supernatant was then dialyzed for 4
h against buffer A and stored at -80 °C.
Protein Purification
Peripheral blood mononuclear
cells were obtained by leukopheresis with consent from a T-cell
leukemia patient. Approximately 10 cells were incubated in
phosphate-buffered saline containing 0.5% Nonidet P-40 for 5 min at
room temperature and lysed by grinding in a blender for 2 min. Nuclei
and intact cells were removed by centrifugation at 15,000
g for 15 min at 4 °C. The supernatant was carefully removed,
snap frozen, and stored at -80 °C. Protein concentration was
determined using the Bio-Rad Micro assay as described by the
manufacturer.
g for 15 min at 4 °C, and the supernatant
loaded onto a DEAE-Sepharose column. The column was washed with buffer
A until all the unbound material was removed. The bound proteins were
eluted with a linear gradient of 0-500 mM NaCl, and
fractions were dialyzed against buffer A. APP RNA binding activities
were followed by RNA mobility shift assays throughout purification
using radiolabeled APP RNA (nucleotides 2246-2313 as in (14) ). The highest activity fractions were pooled,
concentrated using Centricon 30 concentrators (Amicon), and applied
onto a Sephacryl-S300HR (Sigma) gel filtration column equilibrated with
buffer A containing 0.2 M NaCl. All fractions from gel
filtration chromatography were then dialyzed against buffer A and
assayed for activity. Fractions possessing the APP RNA binding
activities were pooled, dialyzed against buffer A containing 1.7 M (NH
)
SO
, and loaded onto a
phenyl-Sepharose CL4B (Pharmacia) hydrophobic interaction column.
Proteins were eluted with a decreasing gradient of 1.7-0 M (NH
)
SO
in buffer A.
Fractions were dialyzed and again assayed for APP RNA binding
activities.
protein complexes, cytosolic supernatant was
dialyzed against buffer A containing 250 mM NaCl and loaded
onto a DEAE-Sepharose column. The column was washed until all the
unbound material was removed. The bound proteins were eluted with a
250-500 mM NaCl gradient in buffer A. Fractions were
dialyzed against buffer A and assayed for APP RNA binding activity. Gel
filtration and hydrophobic interaction chromatography were performed as
described above. Active fractions were pooled and loaded onto a
heparin-Sepharose CL6B column. Proteins were eluted with a gradient of
0.15-2 M NaCl in buffer A. All fractions were dialyzed
against buffer A and assayed for activity.
In Vitro Transcription
Radiolabeled wild type APP RNAs (nucleotides 2246-2313)
or mutant derivative as described previously (12) were produced
by in vitro transcription from APP cDNA templates as described (12, 13) . Briefly, after transcription at 37 °C
for 1 h, cDNA templates were digested with 1 unit of RNase free-DNase
(Promega), extracted with phenol-chloroform and passed through a
Sephadex G-50 mini-spin column. P-UTP-labeled RNA was
scintillation counted, diluted to 5
10
counts/min/µl, and used as such in band-shift reactions or
Northwestern blot experiments.
RNA Band Shift Assay and Polyacrylamide Gel
Electrophoresis
RNA band-shift assays were performed essentially as described (13, 15) . Briefly, protein samples were incubated
with radiolabeled APP RNA (0.5 10
counts/min) in 10
µl of a solution composed of 15 mM HEPES, pH 8, 2 µg
of yeast tRNA, 10 mM KCl, 10% glycerol, and 1 mM dithiothreitol at 30 °C for 10 min prior to the addition of 20
units of RNaseT1 (Sigma). Following a 45-min incubation at 37 °C,
UV cross-linking of RNA
protein complexes was performed by
exposing reaction mixtures for 6 min on ice to 254 nm UV light in a
Stratalinker (Stratagene) on the automatic setting. Subsequently, the
cross-linked samples were boiled for 3 min and RNA
protein
complexes resolved by 12% SDS-polyacrylamide gel electrophoresis. After
electrophoresis, gels were dried and exposed to x-ray film for
15-20 h with intensifying screens.
DNA Gel Mobility Shift Assay
Nuclear or cytoplasmic lysates (5 µg) were incubated with
35 fmol of [-
P]ATP 5`-end-labeled SP1 or
Oct-1 ligand oligonucleotides (Promega, Madison, WI) in a buffer
containing 10 mM Tris, pH 7.5, 1 mM MgCl
,
0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM
NaCl, and 4% glycerol in the presence of 500 ng of double-stranded
poly(dI-dC) for 20 min at room temperature. For competition studies, a
50-fold molar excess of the cold AP1, SP1, or Oct-1 oligonucleotides
were added to the reaction mixture, which was then incubated at room
temperature for 5 min prior to the addition of radiolabeled
oligonucleotides. Protein
DNA complexes were resolved on a 4%
polyacrylamide gel with 0.5
Tris borate-EDTA (TBE) buffer.
After electrophoresis, the gel was dried and exposed to x-ray film for
3 h with intensifying screens.
Western Blotting
Samples were resolved by SDS-polyacrylamide gel
electrophoresis and transferred from gel to Immobilon PVDF membrane
(Millipore) at 15 V for 30 min with a TransBlot semi-dry apparatus
(Bio-Rad). The filter was blocked with 20 mM Tris-Cl, pH 7.4,
0.9% NaCl (TBS) containing 5% BSA. Filters were incubated with CC98
hybridoma supernatant (neat) or 4F4 monoclonal antibody (at 1:5000
dilution) for 1.5 h in antibody incubation solution (TBS containing 1%
BSA and 0.05% Tween 20) and then washed three times with TBS containing
0.1% BSA. Goat anti-mouse IgG diluted 5,000-fold in the antibody
incubation solution was added to the filter for 1 h. After washing
three times with TBS, antigen-antibody complexes were visualized by
reaction with a freshly prepared solution composed of 0.1 M Tris-Cl, pH 9.5, 0.1 M NaCl, 5 mM MgCl, 0.48 mM NBT (nitroblue tetrazolium),
and 0.56 mM 5-bromo-4-chloro-3-indolyl phosphate.
Northwestern Blot Analysis
Protein samples were resolved on 12% SDS-polyacrylamide gels
and transferred to nitrocellulose membranes as described above.
Transferred proteins were renatured overnight in a solution containing
15 mM HEPES, pH 8, 10 mM KCl, 10% glycerol, and 1
mM dithiothreitol at 4 °C. Membranes were then incubated
with radiolabeled APP RNA (5 10
counts/min/ml,
final concentration) for 1 h at 30 °C in renaturation buffer
containing 2 mg/ml tRNA. Unbound RNA was removed by washing twice at
room temperature with renaturation buffer followed by autoradiography.
Protein Sequencing
Protein samples were resolved on 12% SDS-polyacrylamide gels
and transferred to the PVDF protein sequencing membrane (Bio-Rad).
Proteins were visualized and excised after staining the membrane with
Ponceau S for 1 min. These pieces of membranes were then washed
thoroughly with deionized distilled water, air dried, and sent for
amino acid sequencing to Beckman Research Institute, City of Hope
Microsequencing Facility, Duarte, CA.
Purification and Characterization of APP mRNA-binding
Proteins
Cytosolic lysate prepared from a human T-cell leukemia
was loaded onto a DEAE-Sepharose anion-exchange column. APP RNA binding
activities were eluted between 250-350 mM NaCl. Gel
filtration chromatography was then performed using a column packed with
Sephacryl-S300HR. Active fractions were combined and hydrophobic
interaction chromatography carried out using phenyl-Sepharose CL4B.
Fractions were followed for activity by gel mobility shifts, and for
protein profiles by Coomassie staining of 12% SDS-polyacrylamide gels (Fig. 1).
To identify the binding protein's precise
molecular weight, Northwestern blots were also performed (Fig. 2). Phenyl-Sepharose fractions were resolved on 12%
SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and
incubated with radiolabeled APP RNA. Fractions 15, 16, and 17 contained
an abundant 70-kDa protein which bound radiolabeled APP RNA (Fig. 2A). These same fractions were enriched for high
molecular weight, APP RNA binding activity (Fig. 1B).
Fractions 17, 18, and 19 also included a closely migrating doublet of
48 and 47 kDa (Fig. 1A) which was resolved on a longer
gel and transferred to nitrocellulose. NorthWestern blotting showed
that both the 48- and 47-kDa proteins bound APP RNA with equal
affinities (Fig. 2B). Low molecular mass APP RNA
binding activity segregated with the 48- and 47-kDa proteins (Fig. 1).
To determine if the Northwestern blot reactive
proteins account for the observed solution phase APP RNA mobility
shifts, the 70- and 48- and 47-kDa proteins were individually eluted
from SDS-polyacrylamide gel electrophoresis and analyzed by gel
mobility shift assay using radiolabeled APP RNA. The 70-kDa protein
formed three complexes of 140, 104, and 84 kDa while the 48- and 47-kDa
proteins formed four complexes of 104, 90, 73, and 65 kDa (Fig. 3). APP RNAprotein complexes of >104, 90, and 73
kDa were probably due to cross-linking of more than one protein
molecule to the radiolabeled RNA. These data, together with the results
of Northwestern blotting strongly suggest that the 70-, 48-, and 47-kDa
proteins are capable of independently interacting with APP RNA and
likely represent the activities which we initially sought to purify.
protein
complexes are formed by 70-, 48-, and 47-kDa gel purified proteins.
Fraction 17 of phenyl-Sepharose column was electrophoresed on 12%
SDS-polyacrylamide gels. Bands denoted by arrowheads (left
panel) were excised and eluted, and gel mobility shift assay was
performed as described in the legend to Fig. 1and
``Materials and Methods.'' Panel on the left represents protein profile of fraction 17 on Coomassie-stained 12%
SDS-polyacrylamide gels. Panel on the right shows the results
of gel mobility shift assays. Shifts with total fraction 17 are
included for comparison (denoted 17). Lane A is the
shift with the 70-kDa eluted protein while lane B is with the
48- and 47-kDa eluted protein. Autoradiogram for sample 17 and A were
exposed overnight whereas B was exposed for 4 days. Arrowheads to the left of the right panel indicate APP
RNA
protein complexes. Migration of molecular size markers is
shown to the right.
For purification of the lower molecular weight APP RNA-binding
proteins, the cytosolic supernatant was adjusted to 250 mM NaCl and anion-exchange chromatography performed on a
DEAE-Sepharose column. Proteins were eluted with a linear gradient of
250-500 mM NaCl. Fractions active in forming 42- and
47-kDa APP RNAprotein complexes were concentrated and size
fractionated on Sephacryl-S300HR columns. The desired activities eluted
before the 70-, 48-, and 47-kDa APP RNA-binding proteins described
above suggesting they were multimers or associated with other proteins
while transiting the column. All active fractions were then loaded onto
heparin-Sepharose and eluted with a linear gradient of 0.15-2 M NaCl. The activity of the fractions correlated with a 38-
and 39-kDa protein doublet. Band shift assay with doublet containing
fractions generated a weak 47-kDa RNA
protein complex above a
dominant 42-kDa complex (Fig. 4A). To identify the
relevant RNA-binding proteins, fraction 19 was resolved on 12%
SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated
with radiolabeled APP RNA (Fig. 4B). As shown, both
proteins and a 32 kDa band bound radiolabeled APP RNA. The quantity of
the 32-kDa protein did not correspond to APP RNA binding activity by
gel mobility shift assay suggesting APP RNA binding was nonspecific.
protein complexes. Molecular
weight markers in the upper panel are shown to the right. Panel B, Northwestern analysis of fraction 19 from
heparin-Sepharose column. Left lane represents a
Coomassie-stained 12% SDS-polyacrylamide gel. Right lane is an
autoradiogram of the Northwestern carried out as described in the
legend to Fig. 2and under ``Materials and Methods.''
Positions of 39-, 38-, and 32-kDa proteins are shown to the
left.
Previously we mutated an APP mRNA within the essential 29 base
region required for protein interactions. As assessed by solution phase
band-shift assays, mutant APP RNA was unable to bind
proteins(12) . Therefore, we tested if the phenyl-Sepharose
fractions 17 and 18 (containing the 70-, 48-, and 47-kDa proteins) and
heparin-Sepharose fraction 19 (containing the 39- and 38-kDa proteins)
could discriminate between radiolabeled wild type and mutant APP RNAs
in mobility shift assays (Fig. 5). As expected, the wild type
APP RNA produced RNAprotein complexes, whereas mutant APP RNA did
not. This suggested that the purified proteins specifically interacted
with APP RNA. However, when these fractions were Northwestern blotted,
the 70-, 48-, 47-, 39-, and 39-kDa proteins bound both the wild type
and mutant APP RNA equally well (not shown). These data suggest that
the specificity of some RNA-binding proteins may vary when they are
immobilized on solid supports.
protein complexes are denoted by arrowheads along the left.
Amino Acid Sequencing and Protein
Identifications
The 70-, 48-, and 47-kDa proteins were
transferred to PVDF membranes, and multiple peptides sequenced by
standard, automated techniques. In all cases, they were completely
homologous to human nucleolin (Table 1)(16) . The peptide
masses of tryptic digests were also identical to those expected for
nucleolin. Nucleolin is a nucleolar protein and possesses
autocatalytic, self-cleaving activity(17) . It is known to
contain four C-terminal RNA-binding domains and interact with
single-stranded nucleic acids(18) . For the 70-kDa protein, we
obtained amino acid sequences on four fragments including the N
terminus, a CNBr peptide before the first RNA-binding domain, and two
other tryptic peptides located within the third and the fourth
RNA-binding domains (Fig. 6). These data suggested that the
70-kDa protein contains all of nucleolin's four RNA-binding
domains. Several of the tryptic digests of the 48- and 47-kDa proteins
had identical peptide masses to those from the 70-kDa protein. Two of
these were pooled with the corresponding tryptic fragment derived from
the 70-kDa protein and sequenced. As this analysis produced a single,
unambiguous sequence, the original peptides must have been identical.
One tryptic fragment was located before the first RNA-binding domain
and the other within the fourth RNA-binding domain. Taken together, the
48- and 47-kDa proteins also contained nucleolin's four
RNA-binding domains. Presumably, the 70-, 48-, and 47-kDa proteins were
derived from the full-length 110-kDa nucleolin molecule after
self-cleavage and/or post-translational modifications.
Similarly,
the 38-kDa protein was digested with trypsin, and five fragments were
sequenced. All fragments showed complete identity to human
heterogeneous nuclear ribonucleoprotein C (hnRNP C) (19) (Table 1). Several of the tryptic peptide masses
also corresponded to the computer predicted tryptic masses of hnRNP C
proteins. hnRNP C1 and C2 originate from the same gene and differ only
in the addition of 13 amino acids to the C1 protein due to differential
RNA splicing(19) .
APP mRNA-binding Proteins React with the Monoclonal
Antibodies against Human Nucleolin and hnRNP C Proteins
To
confirm that the 70-, 48-, and 47-kDa proteins were nucleolin and the
39/38-kDa doublet protein was hnRNP C protein, phenyl-Sepharose
fractions 15 and 18 and heparin-Sepharose fraction 19 were Western
blotted and incubated with monoclonal antibodies specific for human
nucleolin (CC98, (17) ) or hnRNP C1 and C2 (4F4, (20) ) (Fig. 7). The CC98 antibody specifically reacted with the 70-kDa
protein in fraction 15 and to 48- and 47-kDa proteins in fraction 18
but not to proteins in heparin-Sepharose fraction 19. The CC98 reactive
proteins between 70 and 48 kDa are likely degradation products of
nucleolin which have been reported by other
groups(16, 17) . These results confirm that the 70-,
48-, and 47-kDa proteins are nucleolin and share the epitope recognized
by monoclonal antibody CC98. The immunoblot also demonstrated that the
38- and 39-kDa proteins in heparin-Sepharose fraction 19 were hnRNP C1
and C2.
Subcellular Localization of APP RNA-binding Proteins and
Their Activity in Human Tumor Cells
Nucleolin is known to
shuttle between the nucleus and the cytoplasm(21) , whereas
immunocytochemical and immunofluorescence analysis have suggested hnRNP
C proteins are exclusively localized within the nucleus(20) .
Depending on the affinity and availability of the target epitopes,
immunodetection may substantially underestimate the actual quantity of
protein localized within a subcellular space. Our data show nucleolin
and hnRNP C exist in the cytoplasm. Because of these conflicting
results, we sought to establish that nucleolin and hnRNP C proteins
segregated with known, compartmental markers and were authentic
constituents of the cytoplasm, rather than nuclear activities which
leaked out during subcellular fractionation. Therefore, we chose to
measure the DNA binding activity of transcription factors SP1 and Oct-1
which are restricted to the nucleus(22, 23) . Log
phase T-cell leukemic cells (Jurkat cells) were fractionated into
nuclear and cytoplasmic S20 extracts. Equal amounts of total protein
from each fraction were used for DNA mobility shifts, APP RNA mobility
shifts, and Western blots with anti-nucleolin and hnRNP C antibodies.
As shown in Fig. 8A, DNA mobility shifts with nuclear
proteins and radiolabeled SP1 oligonucleotides produced two, slowly
migrating complexes (denoted with bracket). Both complexes
could be specifically competed by unlabeled SP1 but not AP1
oligonucleotides. Cytoplasmic extract failed to produce a specific
shift with radiolabeled SP1 oligonucleotides. The prominent nonspecific
complex observed with the cytoplasmic extract has been reported (24) and could be competed with all oligonucleotides used. We
observed identical results when mobility shifts with radiolabeled Oct-1
oligonucleotides were performed (not shown). When the same extracts
were used for APP RNA mobility shifts (Fig. 8B), both
nuclear and cytoplasmic preparations showed appropriately sized,
RNAprotein complexes. Based upon PhosphorImager quantification of
the shifted complexes, approximately 75% of the nucleolin activity was
nuclear and 25% was cytoplasmic, whereas 65% of the hnRNP C activity
was nuclear and 35% was cytoplasmic. Tight association of hnRNP C
proteins with nuclear hnRNA (25) may result in
underrepresentation of its activity in the nuclear fraction and further
decrease the likelihood of inadvertent nuclear leakage of this protein.
These results were confirmed by Western blot with anti-nucleolin and
hnRNP C antibodies (Fig. 8C). Cumulatively, these data
prove that APP RNA-binding proteins are normally present within both
the nuclei and the cytoplasm of log phase cells.
protein complexes is shown to the left. Panel C, nuclear (N) and cytoplasmic S20 (S)
fractions (60 µg of total protein in each) or 200 ng of fraction 18
were electrophoresed on 12% SDS-polyacrylamide gel, transferred to
Immobilon PVDF membrane, and allowed to react with anti-human nucleolin
CC98 hybridoma supernatant (left panel) or anti-hnRNP C
proteins 4F4 (right panel) monoclonal antibodies. Positions of
70-, 48-, 47-kDa nucleolin fragments and hnRNP C1 and C2 are
denoted.
-helixes which flank the
RNA-binding domains (16) may reduce the specificity of the four
RNA-binding domains. Herein is similar data demonstrating the loss of
APP RNA binding specificity by filter-bound proteins. In solution phase
the purified proteins specifically interacted with the wild type but
not the mutant APP RNA. If these data are general, filter screening for
RNA-binding proteins should be cautiously interpreted.
protein complexes were
formed by hnRNP C proteins. hnRNP C proteins are dominantly nuclear
with a brief transit into the cytosol during mitosis(20) .
During purification, these APP RNA-binding proteins were eluted from
size exclusion resins before the 70-, 48-, and 47-kDa fragments of
nucleolin. This is consistent with the existence of hnRNP C tetramers
((C1)
C
) which have been previously
identified(35) . hnRNP C proteins are also known to bind
reiterative U or AUUUA motifs often located in the 3`-UTR of labile
mRNAs(36, 37) . Previously, we have shown that poly U
and AUUUA RNA specifically competed with APP RNA for the 42- and 47-kDa
complexes (13) composed of hnRNP C1 and C2 proteins. Recently,
it has been demonstrated that hnRNP C proteins are sequence specific
and bind to UUUUU sequences with high affinity(38) . Thus, the
most likely location for C protein to bind the 29-base element in APP
mRNA is at the sequence CUUUACAUUUUG, present at the 5`-end.
protein complexes(13) . In the present study we
demonstrated that the 70-, 48-, and 47-kDa fragments of nucleolin
formed the 104-, 84-, 73-, and 65-kDa APP RNA
protein complexes.
The smaller complexes of 47 and 42 kDa were composed of hnRNP C
proteins and APP RNA. How the five proteins assemble on the APP RNA
ligand is unclear. RNA mobility shifts with an APP RNA truncated in the
middle of the 29-base element (UCUCUUUACAUUUUGG) produced 104-, 84-,
47-, and 42-kDa RNA
protein complexes(13) . Therefore, the
70-kDa fragment of nucleolin and hnRNP C proteins must bind the 5`-half
of the element. The smaller 47- and 48-kDa nucleolin fragments likely
interact with the 3`-end of the element. The different binding
specificity of the nucleolin fragments occurs despite the presence of
identical RNP domains. Specificity may result from additional peptide
sequence present in the larger fragment or differential, internal
modification by phosphorylation. As gel-purified 70- and 48- and 47-kDa
proteins formed higher molecular mass complexes (>104 kDa) (Fig. 3), more than one protein molecule likely binds to the
same RNA molecule. hnRNP C which have previously been reported as the
components of splicing complexes (39) may bind to the APP RNA
together with one or more of the nucleolin fragments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.