From the Department of Microbiology, New York University School of Medicine, New York, New York
Received for publication, February 3, 2003
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ABSTRACT |
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INTRODUCTION |
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Apart from hnRNPs C1/C2 and U, all of the other hnRNPs appear to undergo nucleocytoplasmic shuttling (1, 2). hnRNP A1 represents one type of shuttling protein that is a nuclear protein at steady state but shuttles rapidly and accumulates in the cytoplasm with transcription inhibition (3, 6), which is associated with the presence of an M9 domain (3). The requirement for ongoing transcription in the transport of M9 domain-containing proteins is not well understood and is unlikely to simply involve co-transport by binding to mRNAs (7). hnRNP K is another type of shuttling RNA-binding protein. It contains a canonical NLS in addition to a unique shuttling domain known as KNS. Unlike hnRNP A1, the nuclear localization of hnRNP K is independent of transcriptional activity unless the NLS motif is deleted (7).
The hnRNP D/AUF1 family, similar to many hnRNPs, is comprised of four related isoforms produced by alternate splicing of a single mRNA (Fig. 1A) (8, 9, 10). AUF1 consists of a 37-kDa core protein (p37), a 40-kDa protein (p40) with an N-terminal 19 amino acid insertion of exon 2, a 42-kDa protein (p42) with a C-terminal 49 amino acid insertion of exon 7, and a 45-kDa protein (p45) containing both exon 2 and 7 insertions (Fig. 1A) (reviewed in Ref. 10). The different AUF1 isoforms bind various types of ARE sequences, which are associated with rapid decay of short-lived cytokine and proto-oncogene mRNAs (10). The different AUF1 isoforms bind to different ARE sequences, which have been attributed to the presence of exon insertions that might alter AUF1 protein conformation (9, 11). Different members of the AUF1 family also appear to have multiple functions including transcriptional activation (12, 13) and binding to nucleolin (14), telomeric single-stranded DNA (15), and components of the translation initiation apparatus (16). Nevertheless, all four AUF1 isoforms associate with hnRNP A1 (17), which is involved in mRNA transport (18) and splicing (19).
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Although AUF1 proteins are found in both the nucleus and cytoplasm, steady state analysis demonstrates a much higher nuclear accumulation of all four proteins (16, 20). Heat-shock or down-regulation of the ubiquitin-proteasome network mediates even greater nuclear accumulation of AUF1 and the increased abundance of p37 and p40 proteins (16, 21). Therefore, we sought to characterize the mechanisms for AUF1 nucleocytoplasmic shuttling. We demonstrate that all four AUF1 proteins are capable of undergoing rapid nucleocytoplasmic shuttling and that the shuttling pathway used by the AUF1 proteins is transcription-independent and carrier-mediated. We found that the uninterrupted C-terminal domain (CTD) found in the p37 and p40 AUF1 isoforms possesses a strong nuclear import activity, whereas the CTD interrupted by exon 7 promotes cytoplasmic localization, which is inserted in the CTD of the p42 and p45 isoforms. We show that a subset of AUF1 proteins can directly interact in vitro and in vivo in the absence of RNA, which suggests that nuclear import is probably facilitated by the p37 and p40 isoforms, whereas nuclear export is facilitated by the p42 and p45 isoforms as a part of a larger AUF1 complex of proteins.
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EXPERIMENTAL PROCEDURES |
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Immunofluorescence AnalysisCells grown on coverslips were rinsed with phosphate-buffered saline, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then blocked in 3% bovine serum albumin (BSA) (23). Alternatively, cells were fixed-permeabilized with methanol at -20 °C for 5 min. Primary antibodies were diluted in 3% BSA and incubated for either 1 h at 25 °C or 1216 h at 4 °C. Anti-Myc polyclonal antibody was from Santa Cruz Biotechnology, and anti-FLAG M2 monoclonal antibody was from Sigma. Polyclonal rabbit anti-AUF1 antibody was produced against recombinant p37 protein (as described below). Monoclonal anti-hnRNP D (5B9) was generously provided by Gideon Dreyfuss (University of Pennsylvania). Following three washes with phosphate-buffered saline, cells were incubated with anti-mouse and anti-rabbit IgG secondary antibodies coupled to fluorescein isothiocyanate or rhodamine (Jackson Immunoresearch), diluted in 3% BSA for 1 h. Samples were washed again, and Hoechst dye 33258 was included in the final phosphate-buffered saline wash to stain nuclei. After mounting, samples were examined on a Zeiss Axiophot microscope. Images were captured using Axiovision, version 2 software. Typical images are shown that present roughly equal total fluorescence-staining intensity.
Cell ExtractAt 36 h post-transfection, cells were washed with ice-cold phosphate-buffered saline and then harvested using Nonidet P-40 lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.5, 0.5% Nonidet P-40, supplemented with Complete protease inhibitor (Roche Applied Science)). Cells were disrupted by four passages through a 29-gauge needle followed by incubation on ice for 20 min. This procedure disrupts nuclei and produces whole cell lysates that contain soluble nuclear and cytoplasmic protein. Lysates were cleared by microcentrifugation at 4 °C for 15 min. Protein concentrations were determined using the Bradford assay (Bio-Rad).
PlasmidsFLAG-AUF1 constructs were made by PCR amplification
of each AUF1 isoform cDNA to generate a HindIII site upstream of the
initiating AUG codon and an EcoRI site downstream of the termination
codon. PCR products were purified by gel-electrophoresis (Qiagen), digested
with HindIII and EcoRI, and then ligated into pFLAG-CMV-2
(Sigma) digested with the same enzymes. The resulting construct contains the
9-amino acid FLAG epitope fused in-frame to the N terminus of each AUF1 coding
region. The truncation mutants, FLAG-p37CTD and FLAG-p40
CTD,
were created by digesting FLAG-p37 or FLAG-p40 with HindIII and
XbaI and ligating the resulting fragments into pFLAG-CMV-2 digested
with the same enzymes. Flag-p37
12 was created by standard PCR overlap
extension using primers spanning the deleted sequence and primers containing
the 5'-HindIII and 3'-EcoRI sites. The resulting
fragment was cloned into pFLAG-CMV-2 as noted above. GFP-CTD was constructed
by ligating a purified, digested PCR product generated by amplifying either
FLAG-p37 or FLAG-p42 plasmids using a 5'-HindIII primer and the
3'-EcoRI primer into pEGFP-C3 (Clontech) digested with the same
enzymes. Plasmids encoding glutathione S-transferase (GST) fusions of
each AUF1 isoform were also constructed by PCR cloning. An EcoRI site
upstream of the initiating AUG codon was introduced as well as a XhoI
site downstream of the termination codon. PCR products were purified by
gel-electrophoresis, digested with EcoRI and XhoI, and then
cloned into pGEX-4TI (Amersham Biosciences) digested with the same enzymes. To
generate His-tagged AUF1 proteins, cDNAs were PCR cloned into pET-23b
(Novagen) using the BamHI and XhoI sites, allowing in-frame
fusion with the N-terminal T7 tag and the C-terminal His tag. Specific details
of any oligomer are available upon request. Each reading frame was confirmed
by DNA sequencing. Plasmids encoding Myc-tagged hnRNP A1, Myc-tagged hnRNP C1,
GFP fused to the SV40 nuclear localization sequence, GFP-NLS, or GFP fused to
the
-subunit of I
B (GFP-I
B
) have all been
described elsewhere (24,
25,
26). The GFP-p37 plasmid was
kindly provided by Dr. David Port (University of Colorado Health Science
Center).
Recombinant Protein PurificationAll of the recombinant proteins were expressed in BL21(DE3) Escherichia coli cells. Bacterial lysate preparation and purification with glutathione-Sepharose 4B was performed as recommended by the manufacturer (Amersham Biosciences). His-tagged AUF1 proteins were purified using TALON resin (Clontech). Protein concentrations and purity were determined by Coomassie Blue staining and comparison to a titration of BSA.
Antibody Production and PurificationRabbits were immunized with GST-p37 following a standard protocol (Cocalico Biologicals, Inc.). Thrombin cleavage of the GST moiety produced nearly full-length p37 protein, which was then coupled to Affi-Gel-10 (Bio-Rad). Crude antisera were passed over immobilized GST (Amersham Biosciences) and then affinity-purified using the p37 resin.
In Vitro AUF1 Protein Interaction AnalysisRecombinant proteins were incubated in 100 µl of binding buffer (10 mM Hepes-KOH, pH 7.5, 100 mM potassium acetate, pH 7.5, 5 mM magnesium acetate, 0.5% Nonidet P-40, 0.1 mg/ml BSA) for 1 h at 4 °C. 15 µl of glutathione-Sepharose 4B slurry was then added followed by incubation for 1 h at 4 °C. Bound proteins were washed three times with binding buffer, eluted by boiling in SDS-loading dye, and then resolved by 10% SDS-PAGE. Immunoblotting was performed using polyclonal anti-GST antibody from Santa Cruz Biotechnology and monoclonal anti-T7 antibody from Novagen.
In Vivo AUF1 Protein Interaction AnalysisCHO cells were
transfected with vectors expressing GFP-p37 AUF1 and FLAG-tagged p37, p40,
p42, or p45 AUF1 proteins as described above. Extracts were prepared using
buffer (100 mM NaCl, 20 mM Hepes, pH 7.5, 2.5
mM magnesium chloride, 1 mM dithiothreitol, 10
mM NaF, 1 mM sodium orthovanadate, 2.5 mM
-glycerophosphate, 0.5% Nonidet P-40, supplemented with Complete
protease inhibitor) and precleared using Protein G Plus-agarose (Santa Cruz
Biotechnology). FLAG-AUF1 proteins were immunoprecipitated using EZview
anti-FLAG affinity gel (Sigma) for 4 h with or without the addition of 100
µg/ml RNaseA. Reactions were washed, and associated proteins were resolved
by SDS-PAGE and immunoblotted using antibodies to GFP (Molecular Probes) or
FLAG (Sigma).
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RESULTS |
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Rapid and Facilitated Shuttling of All Four AUF1 Protein IsoformsThe heterokaryon fusion assay was used to determine whether all four AUF1 isoforms possess nucleocytoplasmic shuttling activity. HeLa cells were transfected with plasmids expressing individual FLAG-AUF1 isoforms fused to non-transfected murine NIH 3T3 fibroblasts in the presence of cycloheximide to block new protein synthesis, and heterokaryons were fixed and immunostained for transfected FLAG-AUF1 protein. Hoechst DNA staining was used to distinguish murine NIH 3T3 cell nuclei, which produce a distinct punctate staining pattern, from human HeLa nuclei, which stain diffusely (25). All four AUF1 isoforms displayed rapid shuttling activity, moving from HeLa cell nuclei to murine 3T3 cell nuclei within 2 h following cell fusion (indicated by arrows) (Fig. 2). These results are similar to Myc-tagged hnRNP A1, an established rapid shuttling protein (Fig. 2) (27). Phase-contrast images indicate the position of nuclei in fused cells. Myc-tagged hnRNP C1, which does not shuttle, remained in the original transfected HeLa cell nuclei. Identical results were obtained using COS-1 cells instead of HeLa cells to donate FLAG-AUF1 proteins (data not shown), indicating that AUF1-shuttling activity is a rapid and general phenomenon. FLAG-AUF1 protein, Myc-C1, and Myc-A1 proteins were identified in equal amounts of whole cell lysates and shown to be undegraded and of proper molecular weight.
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The nucleocytoplasmic movement of AUF1 isoforms could occur by diffusion or by facilitated transport. Because at 4 °C both receptor-mediated nuclear import and export are inhibited whereas diffusion is unaffected (3), nuclear localization at 4 °C indicates use of a receptor-binding pathway, whereas cytoplasmic localization implies passive diffusion. As a control for receptor-mediated transport, a plasmid expressing the GFP fused to the SV40 NLS was used (24). Nuclear import of a GFP-NLS control was blocked at 4 °C, and diffusion was unopposed, allowing cytoplasmic accumulation beyond nuclear borders (data not shown), whereas endogenous AUF1 protein localization at 4 °C was unchanged from that at 37 °C, indicating that AUF1 export is energy-dependent (data not shown).
Transcription and CRM1-independent Transport of AUF1 ProteinTranscriptional inhibition alters the subcellular localization of many but not all RNA-binding proteins. Several hnRNP family members, HuR, and poly(A)-binding protein 1, which contain non-canonical NLSs, are dependent on transcription for nucleocytoplasmic shuttling (1). Studies also indicate an active role for transcription acting on the M9 domain itself in hnRNP A1 (25). Therefore, cells were treated with actinomycin D or vehicle control for 4 h to inhibit transcription prior to immunostaining to determine whether endogenous AUF1 protein localization is dependent on active transcription. As expected, Myc-tagged hnRNP A1 demonstrated a clearly visible and significant increase in cytoplasmic accumulation with transcriptional inhibition (Fig. 3, compare actinomycin D (Act. D) -/+ panels). There was no such change observed in endogenous AUF1 distribution with transcription inhibition, although nucleolar exclusion was lost. Identical results were obtained for FLAG-AUF1 proteins expressed from transfected plasmids, which was independent of the concentration of actinomycin D or increasing the time of treatment to 6 h (data not shown). Therefore, localization of AUF1 proteins is independent of ongoing transcription.
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LMB inhibits CRM1-mediated nuclear export
(1). LMB was used to determine
whether AUF1 proteins are exported from the nucleus to the cytoplasm in a
CRM1-dependent manner. Cells were transiently transfected with vectors
expressing either FLAG-AUF1 or GFP-IB
whose localization has
been shown to be sensitive to LMB treatment
(26). Cells were then treated
with LMB or vehicle control for 1 h prior to immunostaining endogenous or
transfected AUF1 proteins as described previously
(26). Images were somewhat
overexposed to make cytoplasmic AUF1 protein more readily detectable.
Endogenous AUF1 and FLAG-p37 proteins displayed low level cytoplasmic
staining, which was unchanged by LMB treatment
(Fig. 4, AUF1 panels).
GFP-I
B showed a marked increase in nuclear staining and decreased
cytoplasmic staining compared with mock-treated cells. Collectively, these
results indicate that AUF1 is exported from the nucleus via a CRM1-independent
and transcription-independent mechanism.
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AUF1 Nuclear Uptake Is Mediated by the Uninterrupted C-terminal
DomainThe M9 domain of hnRNP A1 is sufficient to mediate
transportin binding and nuclear import
(28). It is loosely
characterized as a region that is rich in glycine, arginine, and asparagine
(25), although many
transportin-binding proteins contain little if any homology to the M9
consensus sequence (1,
28). Transportin reportedly
interacts in vitro with the CTD of AUF1 proteins, although only the
p42 and p45 isoforms were tested
(25). The role of the AUF1 CTD
in transport is unknown. All four AUF1 isoforms share a common CTD but it is
interrupted in the p42 and p45 isoforms by insertion of exon 7 (see
Fig. 1A). Therefore,
we created truncation mutants of FLAG-p37 and FLAG-p40 to determine whether
the uninterrupted 35-amino acid CTD mediates AUF1 nuclear uptake
(Fig. 5A). In
vitro binding studies showed that deletion of the CTD does not affect the
ARE binding ability of AUF1 proteins
(29). Deletion of the CTD in
FLAG-p37CTD and FLAG-p40
CTD shifted AUF1 distribution to a
predominantly cytoplasmic localization compared with predominantly nuclear
distribution for wild type p37 and p40
(Fig. 5B). Thus, the
CTD of the two smaller AUF1 isoforms promotes nuclear import or, less likely,
retention. It is very unlikely that deletion of the CTD altered AUF1 protein
localization because of misfolding of the protein, because similar mutational
analysis showed that AUF1 was still capable of binding RNA and therefore
retained activity (29).
Inspection of the CTD of AUF1 indicates a weak similarity to the M9 domain: a
12-amino acid glycine-arginine rich sequence (AUF1 M9-like motif,
WGSRGGFAGRAR; consensus M9 domain, (Y/F/W/X)XJXSXZG(P/K)(M/L/V)(K/R)) and
several adjacent RGG repeats that might serve as RNA-binding sites. Therefore,
a small deletion was introduced in FLAG-p37 to specifically excise the
potential transportin-binding site (Fig.
5A, Flag-p37
12). Compared with
wild type FLAG-p37 protein, FLAG-p37
12 displayed increased cytoplasmic
accumulation but was clearly still retained in the nucleus as well
(Fig. 5B). Immunoblot
analysis of equal amounts of whole cell lysates confirmed that each AUF1
mutant was expressed at levels similar to wild type
(Fig. 5C). Thus, these
results indicate that a potential transportin-binding site in the CTD is
probably involved in nuclear uptake of AUF1 proteins but that additional CTD
elements also participate in AUF1 nuclear import or retention.
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The entire CTD that was deleted in the FLAG-p37CTD mutant contains
three RGG motifs, which can act as accessory RNA-binding motifs. RGG motifs
can also undergo asymmetric arginine methylation
(30), and hnRNP D (AUF1) is
reportedly methylated in vivo
(22). Arginine methylation
alters the localization of some hnRNP proteins, although hnRNP D/AUF1 has not
been examined (31,
32). We investigated whether
the acquired cytoplasmic localization of AUF1 p37
CTD and p40
CTD
truncation mutants resulted from inhibition of methylation caused by deletion
of the RGG motifs. Cells were untreated or treated with methyltransferase
inhibitor AdOX for 24 h prior to metabolic labeling for 3 h with
L-[methyl-3H]methionine in the presence of
protein synthesis inhibitors as described previously
(22). Bulk-labeled proteins
were resolved by SDS-PAGE and analyzed by fluorography. Because protein
synthesis was inhibited during labeling, any incorporation of radiolabel is
attributed to methylation. Treatment with AdOX dramatically reduced overall
cellular protein methylation (Fig.
6A). Fixation of cells and immunostaining following
identical AdOX treatment demonstrated that AUF1 localization was unchanged
compared with untreated cells (Fig.
6B), which was independent of cell lines or choice of
methyltransferase inhibitor (e.g. 5-methylthioadenosine) (data not
shown). We can conclude that arginine methylation does not regulate the
nucleocytoplasmic distribution of AUF1 proteins.
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Nuclear Export of AUF1 Involves the Exon 7 Interrupted CTD, a p42/45 Isoform-specific ElementResults shown above demonstrate that the uninterrupted CTD of the p37 and p40 AUF1 isoforms directs nuclear accumulation or retention. However, the p42 and p45 AUF1 isoforms contain a 49-amino acid exon 7 insert, which interrupts the CTD. Therefore, GFP fusion proteins were created to determine whether interruption of the CTD by exon 7 affects its nuclear localization function. Full-length p37 AUF1 fused to the C terminus of GFP served as a control and was compared in transfected cells to GFP fused to the uninterrupted p37 CTD or the CTD containing the exon 7 insert (Fig. 7A). Immunoblot analysis of whole cell extracts indicated that all of the GFP proteins were expressed and accumulated to levels within 3-fold of each other (Fig. 7B). GFP-p37 localized largely to the nucleus (Fig. 7C) similar to native AUF1, whereas GFP alone was distributed in both the nucleus and cytoplasm. GFP-CTD, which contains the uninterrupted p37 C terminus, displayed a predominantly nuclear-staining pattern with slight cytoplasmic decoration as expected from the results of Fig. 5. In contrast, GFP-CTD/exon 7, which contains the p42/p45 C terminus, demonstrated strong cytoplasmic localization but retained some nuclear distribution. This pattern indicates that within the context of the CTD, exon 7 promotes either nuclear export or cytoplasmic retention. Because additional mutagenesis of the p37 AUF1 protein, including deletion of most of the N terminus, failed to reveal additional import or export signals (data not shown), these data in combination with those of Fig. 5 indicate that the interrupted CTD with exon 7 sequences promotes cytoplasmic accumulation, either by impairing the nuclear import activity of the uninterrupted CTD or by actively facilitating nuclear export.
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AUF1 Isoforms Interact both in Vitro and in VivoAll four
AUF1 isoforms display similar steady state cellular distributions and
nucleocytoplasmic shuttling behavior. The uninterrupted CTD in p37 and p40
isoforms promotes strong nuclear import or retention, and the CTD/exon 7
configuration promotes p42 and p45 nuclear export or interferes with nuclear
import. Thus, the nuclear import and export functions in the two smaller and
two larger AUF1 proteins are segregated. However, if either of the two smaller
AUF1 isoforms interacts with either of the two larger AUF1 isoforms, the p42
and p45 proteins could promote nuclear export of the p37 and p40 forms, which
could in turn provide a nuclear import signal in trans. This
possibility was investigated in vitro and in vivo. An in
vitro binding assay was performed using purified recombinant AUF1
proteins expressed in E. coli as GST fusions or tagged with a
hexahistidine and T7 epitope. A previous study
(46) shows that appending a
foreign epitope to the N terminus of AUF1 does not inhibit AUF1 interactions.
Following independent incubation of each GST-AUF1 isoform with each of the
four His/T7-tagged AUF1 isoforms, glutathione-Sepharose was used to recover
the GST-AUF1 isoform and any interacting AUF1 proteins, which were resolved by
SDS-PAGE and detected by immunoblotting for T7 or GST
(Fig. 8A). Equal
amounts of His/T7-AUF1 and GST-AUF1 isoforms were used as shown, although some
degradation of the GST fusion isoforms was evident. Proteins were identified
by immunoblotting with anti-T7 or anti-GST antibodies. In control studies, GST
alone failed to bind any AUF1 isoforms. Each GST-AUF1 fusion protein
interacted (to different extents) with other members of the AUF1 family in
vitro (Fig. 8A,
·T7 panels). The strongest interaction was for GST-p37
with the His/T7-p37 protein (i.e. p37 with itself), although each
isoform interacted with other isoforms. The trans-isoform interaction
was strongest between p37 and p42 or p45 (GST-p42 or GST-p45 with His/T7-p37).
p40 AUF1 was the least interactive isoform, the significance of which is not
known.
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Studies were next carried out to determine whether the smaller (p37and p40)
and larger (p42 and p45) AUF1 isoforms directly interact in vivo.
GFP-p37 was co-expressed with FLAG-p37, FLAG-p40, FLAG-p42, or FLAG-p45
proteins. There were lower levels of FLAG-p37 and FLAG-p40 proteins than
FLAG-p42 or FLAG-p45 (Fig.
8B) as expected because of the greater turnover of the
two smaller isoforms (16).
FlAG-AUF1 proteins were immunoprecipitated from equal amounts of whole cell
extracts with or without RNaseA treatment to destroy RNA bridging
(16). Proteins were resolved
by SDS-PAGE and probed with antibodies to FLAG to detect recovered AUF1
isoforms or GFP to detect interacting GFP-p37
(Fig. 8C). In
mock-transfected cells lacking FLAG-AUF1 proteins, there was a weak
nonspecific binding of GFP-p37 to beads (lanes 2 and 3).
However, GFP-p37 interacted much more strongly with all of the FLAG-AUF1
isoforms but to different extents (Fig.
8C, bottom panel, -GFP). GFP-p37
interacted most strongly with FLAG-p45 (lanes 10 and 11) and
FLAG-p42 (lanes 8 and 9), which was not significantly
reduced by digestion of RNA. Thus, most of the trans-interaction is a
direct protein-protein association. GFP-p37 interaction with FLAG-p37 was
reduced
23-fold by RNaseA treatment (lanes 4 and
5). RNaseA treatment most significantly impaired the interaction
between FLAG-p40 and GFP-p37 proteins, indicating that they do not strongly
and directly interact. It has not yet been possible to delete a specific
AUF1-AUF1 interaction site to abolish oligomerization of the proteins. Control
experiments performed with a GFP expression plasmid showed no interaction of
GFP with FLAG-AUF1, indicating that binding is not through the GFP moiety
(data not shown). Although a comprehensive characterization of the AUF1
isoform-binding preferences and hierarchy of interactions are currently in
progress, these data demonstrate interaction both in vitro and in
vivo between the two larger exon 7-containing AUF1 isoforms (p42 and p45)
and the two smaller AUF1 isoforms that lack exon 7 (p37 and p40). Thus, a
subset of the different AUF1 proteins form higher molecular weight
heterocomplexes that contain signals for both nuclear import and nuclear
export.
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DISCUSSION |
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We show that all four AUF1 proteins rapidly shuttle and do so in a transcription-independent manner (Fig. 2, 3), that nuclear export is LMB-insensitive and therefore CRM1-independent (Fig. 4), and that nuclear export is not regulated by arginine methylation (Fig. 6). We found that the uninterrupted CTD of the p37 and p40 isoforms facilitated nuclear uptake, whereas the p42 and p45 CTD, which is interrupted by exon 7, promoted cytoplasmic accumulation, either by impairing CTD nuclear uptake function or by directly facilitating cytoplasmic transport (Fig. 7). Mutational analysis also showed that a putative transportin-binding site in the CTD is involved in cytoplasmic accumulation but is not sufficient (Fig. 7). The previous observation that the CTD of p42 and p45 can bind transportin in vitro strongly suggests that the AUF1 proteins are also substrates of transportin (25). While our studies were in progress, Kawamura et al. (35) examined the localization of the hnRNP D-like JKT-binding proteins. Similar to our results, they identified a localization motif in a C-terminal 25-amino acid segment of the JKT-binding protein. This domain is 72% identical to the CTD of AUF1 and mediates transportin binding. However, in contrast to AUF1, the JKT-binding proteins exhibited differential shuttling activity that is sensitive to transcriptional inhibition. Thus, structural organization of the hnRNP D-like proteins may be significantly conserved, but different mechanisms exist for transport of these various proteins. Because transportin shuttling is independent of transcription (36), it is possible that transportin also mediates nuclear export of AUF1 but probably in conjunction with other export receptors.
One study (37) reported that only the p37 and p40 isoforms shuttle, that this activity is transcription-dependent, and that the p42 and p45 AUF1 proteins are bound to the nuclear matrix, scaffold attachment factor-B, which is itself a stationary hnRNP (38). It should be noted, however, that hnRNP proteins are generally associated with the nuclear matrix, including hnRNPs that shuttle (39). Thus, the association of hnRNP D proteins with scaffold attachment factor-B is not in itself indicative of nuclear retention. Moreover, a recent study (40) has shown that the class of nuclear messenger RNPs, which do not contain stationary hnRNPs, includes p37 and p40 AUF1 proteins (40), consistent with our results. Additionally, biochemical fractionation of AUF1 protein from a number of laboratories is consistent with our results, demonstrating some cytoplasmic accumulation of all four AUF1 isoforms, although the relative ratio of cytoplasmic to nuclear AUF1 can vary (20, 41, 42). Thus, we cannot explain the difference in results between our study, other studies consistent with ours (20, 41, 42), and that of Arao et al. (37). A caveat to the biochemical analysis of cellular protein distribution is that nuclear leakage to the cytoplasm typically occurs during fractionation. This contamination is less likely when cells are fixed and analyzed by microscopy, so we therefore investigated AUF1 shuttling activity using indirect immunofluorescence.
Another potential means for regulating protein shuttling is arginine methylation (31, 32), although the role of arginine methylation in protein shuttling is not clear. Arginine methylation has been shown to regulate the cellular localization of hnRNP A2 but not hnRNP A1, which is also asymmetrically methylated (32). Importantly, the in vivo arginine methylation sites in hnRNP A1 have been identified and found to be distinct from the M9 localization domain (43). Therefore, it may not be surprising that the AUF1 proteins, which are reported to undergo arginine methylation (22), do not relocalize following methyltransferase inhibition.
A unique feature of our work was the identification of AUF1 isoform interaction or heterocomplex formation as a probable means to facilitate nuclear-cytoplasmic shuttling of the family of AUF1 proteins. Oligomerization of the AUF1 isoforms not only serves to explain the identical shuttling kinetics of the four protein isoforms, but it also fits a general paradigm of shuttling hnRNP proteins. It has been shown using in vitro binding studies that hnRNPs E2, I, K, and L all form homomeric and heteromeric complexes (44). Moreover, hnRNP A1 interacts with the core A/B and C1 proteins and weakly with D, E, I, M, and K proteins (45). Thus, the interaction of p42/p45 AUF1 proteins with the p37 and p40 isoforms would be expected to promote their nuclear export, and p37/p40 AUF1 isoforms would be expected to promote nuclear import of p42 and p45 isoforms. The interaction of isoforms can account for an otherwise paradoxical symmetry that should segregate the p37 and p40 proteins in the nucleus and the p42 and p45 proteins in the cytoplasm. The exact composition and stoichiometry of the AUF1 heterocomplexes are currently under investigation, although the AUF1 oligomer would be of sufficient size to limit diffusion (Fig. 3). It is also not yet clear whether all AUF1 proteins interact and shuttle or whether only a subset do so, which would indicate that a proportion of the protein is stationary and segregated in different nuclear compartments. Studies are currently in progress to also determine how AUF1 protein shuttling activity is regulated, because this might serve as a key mechanism for separating nuclear from cytoplasmic functions.
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FOOTNOTES |
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Supported by National Institutes of Health Training Grant 5T32CA09161.
To whom correspondence should be addressed: Dept. of Microbiology, New York
University School of Medicine, 550 First Ave., New York, NY 10016. Tel.:
212-263-6006; Fax: 212-263-8276; E-mail:
schner01{at}popmail.med.nyu.edu.
1 The abbreviations used are: CRM1, chromosomal region maintenance protein 1;
p37, 37-kDa core protein; p40, 40-kDa core protein; p42, 42-kDa core protein;
p45, 45-kDa core protein; ARE, AU-rich element; AdOX,
adenosine-2',3'-dialdehyde; CTD, C-terminal domain; BSA, bovine
serum albumin; hnRNP, heterogeneous nuclear ribonucleoprotein; LMB, leptomycin
B; NLS, nuclear localization signal; PEG, polyethylene glycol; GFP, green
fluorescent protein; EGFP, enhanced GFP; CMV, cytomegalovirus; CHO, Chinese
hamster ovary; GST, glutathione S-transferase; RBD, RNA-binding
domain.
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ACKNOWLEDGMENTS |
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REFERENCES |
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