©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nucleolin and Heterogeneous Nuclear Ribonucleoprotein C Proteins Specifically Interact with the 3`-Untranslated Region of Amyloid Protein Precursor mRNA (*)

(Received for publication, April 10, 1995; and in revised form, May 12, 1995)

Syed H. E. Zaidi , James S. Malter (§)

From the Department of Pathology and Laboratory Medicine, Neuroscience Program and Institute of Aging, University of Wisconsin Medical School, Madison, Wisconsin 53792

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The central nervous system deposition by neurons and glia of 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 RNAprotein 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.


INTRODUCTION

Alzheimer's disease (AD)()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.

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 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) .

In mammalian cells mRNAs decay with very heterogeneous rates. Most cytokine (tumor necrosis factor , 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.

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.


MATERIALS AND METHODS

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.

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 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.

For purification of the protein components of the 42- and 47-kDa APP RNAprotein 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 RNAprotein 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 RNAprotein 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. ProteinDNA 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.


RESULTS

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).


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.



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).


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.



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.


Figure 3: Four of the APP RNAprotein 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 RNAprotein 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 RNAprotein 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.


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 RNAprotein 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.


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 RNAprotein 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.




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.



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.


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.



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.


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 RNAprotein 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.




DISCUSSION

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 -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.

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 RNAprotein 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.

Interaction of APP RNA with cytosolic proteins resulted in the formation of 104-, 84-, 73-, 65-, 47-, and 42-kDa UV-cross-linked RNAprotein 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 RNAprotein 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 RNAprotein 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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1-AG 10675 (to J. S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: A4/204-CSC, Dept. of Pathology and Laboratory Medicine, University of Wisconsin Hospital and Clinics, 600 Highland Ave., Madison, WI 53792-2472. Tel.: 608-263-6043; Fax: 608-263-1568.

The abbreviations used are: AD, Alzheimer's disease; UTR, untranslated region; APP, amyloid A4 precursor protein; hnRNP, heterogeneous nuclear ribonucleoprotein; PBMC, peripheral blood mononuclear cells; BSA, bovine serum albumin; PVDF, polyvinylidene difluoride.


ACKNOWLEDGEMENTS

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.


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