1 Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark
2 Institute of Molecular Biology, University of Copenhagen, Denmark
* Author for correspondence (e-mail: janchr{at}mermaid.molbio.ku.dk )
Accepted 25 February 2002
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Summary |
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Key words: Cytoskeleton, Microtubules, RNA localization, RNP granules, Zipcode-binding protein
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Introduction |
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During a search for trans-acting factors associating with IGF-II mRNAs, we
recently identified a family of three IGF-II mRNA-binding proteins (IMP1, IMP2
and IMP3), which exhibit multiple attachments to IGF-II leader 3 mRNA and the
reciprocally imprinted H19 RNA (Nielsen et
al., 1999; Runge et al.,
2000
). IMP3 is orthologous to the Xenopus Vg1 RNA-binding
protein (Vg1RBP/Vera), which mediates the localization of Vg1 mRNA to the
vegetal pole of the oocyte during oocyte maturation, and IMP1 is orthologous
to the chicken zipcode-binding protein (ZBP-1), which participates in the
localization ß-actin mRNA to the leading edge of motile cells
(Deshler et al., 1998
;
Havin et al., 1998
;
Ross et al., 1997
). Moreover,
IMP1 is orthologous to the mouse c-myc coding region determinant-binding
protein, which may play a role in stabilisation of c-myc mRNA
(Doyle et al., 1998
). IMPs
contain the unique combination of two RNA recognition motifs (RRMs) and four
hnRNP K homology (KH) domains. The vertebrate IMP family originated by two
gene duplications, shortly before the divergence of vertebrates
(Spring, 1997
), and IMP
homologues consisting of only the four KH motifs have been identified in
Drosophila (Nielsen et al.,
2000
) and Caenorhabditis elegans (accession number
T23837). Mouse IMPs are produced in a burst at embryonic day 12.5 followed by
a decline towards birth, and they are co-expressed with IGF-II mRNA and H19
RNA in developing epithelia, muscle and placenta in both mouse and human
embryos (Nielsen et al.,
1999
). IMP is mainly cytoplasmic and the immunocytochemical
appearance ranges from a distinct concentration in perinuclear regions and
lamellipodia to a completely delocalized pattern
(Nielsen et al., 1999
). H19
RNA colocalizes with IMP, and removal of the high-affinity attachment site led
to delocalization of the truncated RNA
(Runge et al., 2000
),
indicating that IMPs are involved in cytoplasmic trafficking of RNA.
In the present study, we have characterised the dynamic subcytoplasmic localization of GFP-IMP1 fusion proteins and determined the structural features that are sufficient for proper transport and cytoplasmic granule formation.
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Materials and Methods |
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Cell culture, transfections and imaging
Mouse NIH 3T3 embryo fibroblast cells were obtained from the American Type
Culture Collection and routinely maintained in RPMI 1640 medium supplemented
with 10% fetal calf serum or in Dulbecco's modified Eagle's medium containing
10% calf serum. Cells were transiently transfected with LipofectAMINE 2000
(Life Technologies Inc., USA) according to the manufacturer's instructions.
Briefly, 30,000 cells/cm2 were seeded on glass plates or
laminincoated plates 24 hours prior to transfection. Cells were transfected
with 2 µg/ml of the indicated plasmid and left for 48 hours before they
were examined. NIH 3T3 cells stably expressing GFP-IMP1 were generated by
retrovirus-mediated insertion of the pBABE-GFP-IMP1, as described previously
(Morgenstern and Land, 1990).
The distribution of GFP-IMP and cellular markers in living and fixed cells
(see below) was examined with a Zeiss LSM 510 confocal laser scanning
microscope. During time-lapse photography and photobleaching experiments,
cells were kept in HEPES-buffered RPMI 1640 medium supplemented with 10% fetal
calf serum at 36°C. Fluorescence recovery after photobleaching (FRAP) was
determined by assessment of the total fluroescence recovery in the bleached
area at 20 second intervals. In ATP depletion experiments, the cells were
incubated with 0.05% sodium azide and 25 mM 2-deoxyglucose for 30 minutes
prior to FRAP.
Immunocytochemistry
Cells were fixed for 10 minutes at room temperature with 3.7% formaldehyde
and washed three times with phosphate-buffered saline (PBS) with 0.1% Triton
X-100. After treatment with 0.1% BSA in PBS for 30 minutes, cells were
incubated with anti-vimentin antibody at 1:100 (Santa Cruz Biotechnology, USA)
or anti-tubulin antibody at 1:200 (Sigma-Aldrich, USA). After washing with
PBS, cells were finally incubated with donkey rhodamine red-x-conjugated
secondary anti-goat or mouse antibody (Jackson Laboratories, USA) for 30
minutes and washed. F-actin staining was performed with Texas-Red-conjugated
phalloidin for 1 hour in 0.1% BSA in PBS according to the manufacturer's
instruction (Molecular Probes, USA).
UV crosslinking
A frozen cell pellet of approximately 5x107 NIH 3T3 cells
or NIH 3T3 cells stably expressing GFP-IMP1 was resuspended in 1 ml of lysis
buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 1.5 mM MgCl2, 0.5%
Nonidet P-40 (NP-40), 0.5 mM dithiothreitol (DTT) and centrifuged at 14,000
g for 10 minutes at 4°C. Glycerol was added to the supernatant at
a final concentration of 5%, and the cytoplasmic extract was stored at
-80°C in aliquots containing 5-7 µg of protein per µl. Radiolabelled
IGF-II leader 3 segment C mRNA (Nielsen et
al., 1999) was synthesised to a specific activity of 30 Ci of
uridine/mmol. NIH 3T3 extract containing 15 µg of protein was incubated
with 100 nCi of leader 3 segment C RNA for 25 minutes at room temperature in
10 µl of 20 mM Tris-HCl (pH 8.0), 140 mM KCl, 4 mM MgCl2, 0.75
mM DTT, 0.1% NP-40, 0.1 µg/µl Escherichia coli tRNA. Samples
were irradiated with 254 nm wavelength light for 30 minutes at 5.4
J/cm2 on ice in a Stratalinker 1800 (Stratagene, USA). Excess probe
was removed by digestion with 0.5 µg of RNase A at 37°C for 25 minutes.
Samples were analysed by 10% polyacrylamide-sodium dodecyl sulfate (SDS) gel
electrophoresis followed by autoradiography.
Mobility-shift analysis
In a final volume of 10 µl, 0.5 pmol of radiolabelled RNA (2 nCi) were
incubated with recombinant IMP-1, RRM1-2, KH1-4, KH1-2 or KH 3-4 for 20
minutes at 30°C in 20 mM Tris-HCl (pH 8.0), 150 mM KCl, 2 mM
MgCl2, 5% glycerol, 0.1% Triton X-100, 0.004% bromophenolblue, 2
units RNasin and 0.01 µg/µl E. coli tRNA. Samples were chilled
on ice and 1 µl of 10% Ficoll-400 was added. The non-denaturing gel
electrophoresis was carried out in a 5% polyacrylamide gel containing 90 mM
Tris-borate (pH 8.3), 50 mM KCl and 2 mM MgCl2 at 4°C and 80 V
for 6 hours. The recombinant proteins were expressed and purified as described
(Nielsen et al., 1999), except
that washing of chitin-beads was carried out in the presence of 1 M NaCl
before cleavage with dithiothreitol.
Gradient centrifugations and blots
NIH 3T3 cells that stably express GFP-IMP1 (3x107 cells)
were lysed in 500 µl 20 mM Tris-HCl (pH 8.5), 1.5 mM MgCl2, 140
mM KCl, 0.5 mM DTT, 0.5% NP-40, 1000 U of RNasin (Promega) per ml and 0.1 mM
cycloheximide. The lysate was centrifuged at 10,000 g for 10 minutes,
and the supernatant was applied to a linear 20 to 47% sucrose gradient in 20
mM Tris-HCl (pH 8.0), 140 mM KCl, 5 mM MgCl2. Centrifugation was
carried out at 200,000 g for 2 hours 15 minutes in a Beckman SW 41
rotor.
5x107 frozen RD rhabdomyosarcoma cells were homogenized, and the undiluted lysate was centrifuged at 10,000 g for 5 minutes. The supernatant was applied to a linear 10-60% Nycodenz gradient in 20 mM HEPES-KOH (pH 7.3), 115 mM KCl. Centrifugation was carried out at 150,000 g for 16 hours at 4°C in a Beckman SW 41 rotor.
Fractions of 1 ml were collected, followed either by precipitation of
sedimenting proteins in 10% trichloroacetic acid and western analysis or by
precipitation of RNA and slot-blot analysis. Proteins were separated in a 10%
polyacrylamide-SDS gel and transferred to polyvinyl difluoride Immobilon-P
membranes (Millipore-Amicon, USA). After blocking, filters were incubated
overnight at 4°C with anti-calnexin antibody (Santa Cruz Biotechnology,
USA) or anti-IMP1 (Nielsen et al.,
1999) in blocking solution and with
horseradish-peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) in
blocking solution for 1 hour at room temperature. Bound antibody was detected
with enhanced chemiluminescence reagents according to the manufacturer's
instructions (Pierce, USA). Total RNA in each fraction was hybridised with an
IGF-II leader 3-specific probe as described previously
(Nielsen et al., 1995
).
Recombinant 32P-labelled IMP1
For labelling of recombinant IMP1, an HMK-site was inserted close to the
C-terminus. The open reading frame of human IMP1 was amplified by PCR using
the upstream primer 5'-GGAATACCATATGAACAAGCTTTACATC-3' and the
downstream
5'-GGACCAGCTCTTCCGCACTTCCTCCGTGCCACGGACGCG-CGACGCTGTCCCTTCTGATGCT-3'
primer, which contains the HMK-site. The product was inserted into the
NdeI and SapI sites of pTYB1 and IMP1-HMK was expressed in
E. coli and purified as described previously
(Nielsen et al., 1999).
Mobility-shift analysis confirmed that IMP1-HMK binds to RNA with the same
affinity as wild-type IMP1. HMK-catalysed 32P-labelling of IMP1-HMK
was performed while IMP1-HMK was attached to the chitin-beads
(Jensen et al., 1995
). The
beads were washed five times with 20 mM HEPES-KOH, pH 7.9, 200 mM NaCl and 20%
glycerol and the labelled protein was isolated as described
(Nielsen et al., 1999
).
Pull-down of 32P-labelled IMP1 with polymerised tubulin
and actin
Binding of HMK-IMP1 to microtubules was examined with a Microtubule
Associated Protein Spin-Down, Assay Kit (Cytoskeleton Inc., USA), according to
the manufacturer's instructions, except that 0.2% Triton X-100 was included
throughout the assay to prevent aggregation. Briefly, 1 nCi of
32P-labelled IMP1-HMK was prespun at 164,000 g in a
Beckman Airfuge for 15 minutes at room temperature in the presence of 6 µg
BSA. The supernatant was added to 10 nM polymerised pure tubulin (20 µM
dimers) in the absence or presence of MAPs (Cytoskeleton Inc. catalogue
reference: MAPF or MAP2) for 20 minutes at room temperature in a total volume
of 50 µl. Samples were centrifuged through a 100 µl 20% (w/v) sucrose
cushion at 164,000 g for 25 minutes at room temperature.
Pellets were resuspended in SDS load buffer and analysed by 10% SDS-PAGE
followed by staining with Coomassie brilliant blue and autoradiography.
IMP1-HMK association with polymerised actin was analysed using an actin polymerisation kit (Cytoskeleton Inc., USA). Briefly, 6 nCi of 32P-labelled IMP1-HMK in 50 µl 5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.002% chlorhexidine was centrifuged for 150,000 g for 1 hour at room temperature in a Beckman Airfuge. 10 µl of supernatant was mixed with either 40 µl actin polymerisation buffer (5 mM Tris-HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2, 1 mM ATP, 0.2 mM CaCl2, 0.002% chlorhexidine) or 40 µl of 1 µg/µl polymerised actin in actin polymerisation buffer. A positive control with actin and actinin at 0.2 µg/µl was included. Samples were incubated for 30 minutes at room temperature, followed by centrifugation for 1.5 hours at 150,000 g. Pellets were resuspended in SDS load buffer, whereas 15 µl of 4x SDS load buffer was added to the supernatant. Samples were analysed in 10% SDS-PAGE gels followed by staining with Coomassie brilliant blue and autoradiography.
Surface plasmon resonance
Binding of microtubule-associated proteins to recombinant
Drosophila IMP and human IMP1 domains was examined with a Biacore
surface plasmon resonance apparatus (Biacore, Sweden). The Drosophila
IMP (dIMP) or segments corresponding to RRM1-2 and KH1-4 were inserted in the
BamHI and EcoRI sites of pET28 (Novagen) and expressed in
E. coli BL21/DE3 cells containing pR1952
(Nielsen et al., 1999). The
His-tagged RRM1-2, KH1-4 and Drosophila IMP (dIMP) were purified
under native conditions according to the manufacturer's instructions, and
about 6000 resonance units were immobilized on a CM5 sensorchip by
carbodiimide coupling. Phosphocellulose-purified tubulin, MAP-enriched tubulin
and MAPs isolated by phosphocellulose chromatography were obtained from
Cytoskeleton Inc., USA. 1 µl of each of the protein preparations were
diluted in 100 µl 10 mM HEPES-KOH, pH 7.4, 150 mM NaCl, 3 mM EDTA and
0.005% (v/v) polysorbate 20. Samples were injected at a flow of 20
µl/minute over 5 minutes. The surface was reconstituted by washing in 0.2 M
glycine for 30 seconds.
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Results |
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We previously found that IMP1 in human rhabdomyosarcoma cells was located
in particles that sedimented at a rate of 40-150S
(Nielsen et al., 1999).
Western analysis with anti-IMP1 antibody of fractions from a 20-47% sucrose
gradient showed that GFP-IMP1 co-sedimented with endogenous IMP1
(Fig. 1B). From the western
analysis of the Mg2+-containing gradient, it is estimated that the
amount of GFP-IMP1 is about twice that of endogenous IMP1, which is expressed
at a relatively low level in NIH 3T3 cells. We infer that GFP-IMP1 exhibits a
similar mRNP distribution to endogenous IMP1.
Cytoplasmic trafficking of GFP-IMP1
The cytoplasmic localization and appearance of GFP-IMP1 in NIH 3T3 cells
stably expressing this protein were examined by confocal microscopy of living
cells. In resting cells, GFP-IMP1 was present in large granules, which were
diffusely distributed in about 90% of the cells
(Fig. 2A, frame 1). In the
remaining cells, GFP-IMP1 accumulated in lamellipodia, near the leading edge
and around the nucleus (Fig.
2A, frame 2). After seeding on laminin-coated plates, which
stimulate spreading and migration of the cells, GFP-IMP1 was localized in
about 40% of the cells. Both cells with localized and cells with evenly
distributed GFP-IMP1 exhibit granules that are often arranged in rows along
the cytoskeleton (Fig. 2A,
frame 3). The apparent optical diameter of the granules ranges from 200-700 nm
(Fig. 2A, frame 4). During
anaphase, GFP-IMP1 becomes concentrated on the polar microtubules in the
midzone of the mitotic spindle (Fig.
2B).
|
To examine the dynamics of GFP-IMP1 localization, cells were followed by
time-lapse microscopy (Fig.
3A). Localization in interphase cells changed rapidly within 15-20
minutes during extension of lamellipodia. Monitoring of GFP-IMP1 movements
showed that granules exhibited a characteristic saltatory and irregular
behaviour. The speed of the individual particles ranged from 0.04 to 0.22
µm/s, with an average speed of 0.12 µm/s
(Fig. 3B). Some granules
coalesced, creating larger granules that subsequently split up into smaller
particles. We also determined the speed and direction of movement by bleaching
16 µm of a cytoplasmic extension and subsequently followed granules moving
into the bleached area. Transport was bidirectional and, in agreement with the
single-particle measurements, the first particles arrived at the center of the
bleached area after 80 seconds, corresponding to a maximal speed of 0.2
µm/s (data not shown). Half-maximal FRAP was reached after 45 seconds, and
the total recovery was
50% (Fig.
3C). By comparison, the half-maximal recovery of a
GFP-ß-galactosidase fusion protein took place after 15 seconds, and total
recovery was 80%. The presence of sodium azide and 2-deoxyglucose reduced the
rate of recovery by a factor of three, suggesting that transport of GFP-IMP1
particles is ATP dependent.
|
IMP1 is associated with microtubules
As described above, GFP-IMP1 granules are often found in rows, suggesting
an association with the cytoskeleton. Moreover, the accumulation of GFP-IMP1
in the midzone during anaphase suggests an association with microtubules.
Co-staining of microtubules and GFP-IMP1 in interphase cells showed that
GFP-IMP1 granules were distributed along the microtubular network
(Fig. 4A,B). Moreover, in the
lamellipodia there was a partial overlap between the staining pattern of
phalloidin (F-actin) and GFP-IMP-1. We did not observe any particular
association with the intermediate filaments. Incubation with nocodazole (10
µg/ml) was followed by collapse and aggregation of GFP-IMP1 in the
cytoplasm, but it left a subpopulation of GFP-IMP1 in the lamellipodia
unaffected (Fig. 4C). The
GFP-IMP1 in the lamellipodia remains attached for more than an hour after
nocodazole treatment, suggesting stable microtubule-independent anchoring.
Incubation with cytochalasin D (2 µg/ml) caused a rapid (2 minutes)
disappearance of GFP-IMP1 from the lamellipodia
(Fig. 4C), indicating that
anchoring is connected to microfilaments. After longer incubations (15-20
minutes) a marked disruption of the cellular architecture was noted.
|
To confirm that IMP1 could associate with microtubules, 32P-labelled recombinant IMP1 was incubated with MAP-enriched and paclitaxel-stabilised polymerised tubulin from calf brain and precipitated by centrifugation (Fig. 5A). Whereas IMP1 was efficiently precipitated by MAP-enriched polymerised tubulin, neither polymerised tubulin in the absence of MAPs nor unpolymerised tubulin in the presence of MAPs was able to precipitate IMP1. To test whether the binding to microtubules was mediated by protein-protein interaction or whether IMP1 was indirectly bound through RNA, precipitation was also carried out in the presence of RNAse T1, which had no effect on IMP1 precipitation. Inclusion of an excess of a high-affinity RNA target from IGF-II mRNA actually prevented precipitation. Moreover, substitution of the MAP fraction with 90% pure MAP2 gave virtually identical results. Finally, we examined the binding of recombinant IMP1 to microfilaments (Fig. 5B). In contrast to actinin, which was efficiently precipitated by actin-filaments, IMP1 was almost exclusively detected in the supernatant. We infer that IMP1 does not associate directly with microfilaments.
|
In order to examine a possible colocalization of IMP1 with membranous
compartments in the mouse fibroblasts, CFP-IMP1 and YFP-linked markers of
membranous compartments were cotransfected
(Fig. 6A). Colocalization with
the markers was neither observed in the cell body nor in the lamellipodia. The
lack of membrane association was corroborated by centrifugation analysis of an
undiluted RD cellular lysate in a 10-60% Nycodenz gradient
(Fig. 6B), where endogenous
IMP1-containing RNPs exhibited an apparent density of 1.23 g/ml. The calnexin
marker for ER sedimented at an apparent density range of 1.09-1.16 g/ml.
Moreover, slotblot analysis of the Nycodenz gradient fractions with an IGF-II
leader 3-specific probe revealed that the translationally repressed 6.0 kb
transcript (Nielsen et al.,
1995) co-sedimented with endogenous IMP1
(Fig. 6B).
|
Granule formation and cytoplasmic localization of GFP-IMP1 deletion
constructs
IMP1 consists of six putative RNA-binding domains, so to identify the
structural determinants necessary for proper granule formation and
localization, a series of GFP-IMP1 deletion constructs were transiently
expressed in NIH 3T3 cells, and the cytoplasmic appearance was examined by
confocal microscopy (Fig.
7A,B). Whereas full-length GFP-IMP1 formed large granules and
localized to the lamellipodia, as described above, the two RRM domains were
diffuse and were distributed all over the cytoplasm. In contrast, KH domains
1-4 assembled in granules that were distributed in a manner similar to
full-length IMP1. Deletion of KH domain 1 or 4 was followed by loss of both
granular appearance and anchoring in lamellipodia. In order to examine whether
the KH1-4 deletion mutant assembles in the same granules as full-length IMP1,
we co-expressed CFP-IMP1 with YFP-KH1-4. Overlay of the two pictures revealed
that CFP-IMP1 and YFP-KH1-4 were embedded in the same granules
(Fig. 7C). We conclude that the
entity comprising the four KH domains is sufficient for the cytoplasmic
trafficking of full-length IMP1.
|
KH-dependent RNA-binding and microtubule association
We examined the RNA-binding of IMP1 deletions by electrophoretic
mobility-shift analysis. Fig.
8A shows an autoradiograph from electrophoresis of the
32P-labelled 173-nucleotide segment H of H19 RNA, which is a
high-affinity IMP1 RNA target (Runge et
al., 2000), in the presence of increasing concentrations of IMP1,
RRM1-2 and KH1-4. Whereas IMP1 and KH1-4 bound to segment H RNA with apparent
Kds of 0.4 nM and 2 nM, respectively, neither the two RRM modules
nor the KH1-2 or KH3-4 modules were able to bind to the RNA-target at
concentrations of 0.1 µM. We infer that the entity consisting of the four
KH domains is the major H19 RNA-binding determinant.
|
To examine whether the RRMs or the KH domains interacted with the MAP fraction, we tested the binding of RRM1-2 and KH1-4 to phosphocellulose-purified MAPs by surface plasmon resonance. Equimolar amounts of the domains were immobilized on the sensorchip, and a clear interaction was observed between the four KH domains and the MAP fraction (Fig. 8B, left panel). No binding was detectable with the two RRM modules. Finally, we examined the binding of MAPs to Drosophila IMP (dIMP), which consists of only the four KH motifs. Whereas immobilized dIMP did not bind to purified tubulin, a distinct binding was obtained with both the MAP-enriched tubulin and the purified MAP preparation (Fig. 8B, right panel), implying that the interaction between the four KH domains and the MAP fraction is phylogenetically conserved.
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Discussion |
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GFP-IMP1 was cytoplasmic at steady state and present in large granules that
were distributed along the microtubular network and in the cortical region of
the lamellipodia, similar to ß-actin mRNA and H19 RNA
(Hill et al., 1994;
Latham et al., 1994
;
Runge et al., 2000
). The
optical diameter range of 200-700 nm of the granules was similar to that of
Staufen- and myelin basic protein mRNA-containing granules. Trafficking in the
interphase cells was highly dynamic and changed within 15-20 minutes during
extension of lamellipodia and motility. The fraction of cells that exhibit
localization of GFP-IMP1 thus seems to reflect the fraction of the cells that
are actively migrating or spreading at a given time point, rather than merely
being a random subpopulation of the cells. During anaphase, GFP-IMP1
accumulated at the polar microtubules in the midzone of the mitotic spindle,
whereas little, if any, GFP-IMP1 colocalized with the kinetochore or astral
microtubules.
IMP1 was able to associate with microtubules, which was in agreement with
previous data from Elisha et al. (Elisha et
al., 1995), who showed that Xenopus Vg1-RBP associated
with microtubules, and this may rationalize the predominant localization of
GFP-IMP1 to the tightly packed polar microtubules during anaphase. The size of
the mobile fraction in the FRAP experiment implies that about half of the
GFP-IMP1 is immobilized on microtubules, whereas the other half is in transit
by a mechanism that is likely to involve motor proteins. Granules moved in a
characteristic saltatory manner in both the plus- and minus-end direction
along the microtubular network. The average velocity of 0.12 µm/s is
similar to that observed for Staufen-containing granules
(Kohrmann et al., 1999
) and a
number of other RNA-containing granules, such as myelin basic protein
mRNA-containing granules (Ainger et al.,
1993
), suggesting that these RNPs move by a similar, but currently
unknown, molecular mechanism. In contrast to Vg1 mRNA and Vg1-RBP/Vera, which
have previously been reported to be localized via their
microtubule-independent binding to a subcompartment of the endoplasmic
reticulum in Xenopus oocytes
(Deshler et al., 1997
;
Etkin, 1997
;
Kloc and Etkin, 1998
;
Lee and Chen, 1988
), GFP-IMP1
does not appear to associate with the ER or other organelles in fibroblasts.
Moreover, endogenous IMP1 and IGF-II leader 3 mRNA from RD cells band at the
buoyant density of RNP particles in the Nycodenz gradient.
A subpopulation of GFP-IMP1 was anchored at nocodazole-insensitive sites in
the lamellipodia. Transport and anchoring are not necessarily linked
phenomena, as demonstrated by myelin basic protein mRNA, where one cis-element
appears to be necessary for transport and another mediates anchoring
(Ainger et al., 1997). Our data
are consistent with a model where IMP travels with its RNA cargo and becomes
anchored via a putative adaptor protein to filaments in the cortical region of
the lamellipodia, implying that the same cis-element mediates both events.
ß-actin mRNA localization in chicken embryo fibroblasts has previously
been reported to be disrupted by cytochalasin D, indicating that ß-actin
mRNA localization in these cells is connected to microfilaments
(Sundell and Singer, 1991
). In
the Xenopus oocyte, Vg1 mRNA is also anchored at the vegetal pole in
a microfilament-dependent manner (Yisraeli
et al., 1990
).
Deletion analysis of IMP1 revealed that the four KH domains, but not the
two RRMs, are sufficient for both intracellular granule formation and
trafficking of IMP1, as well as RNA binding in vitro. The maxi-KH domain is an
autonomously folded unit of about 70 amino acids with
ßßß
topology, which is present in a wide
variety of proteins, often in multiple copies, with the 15 repeats in vigilin
and its yeast homologue (Scp160) as an extreme. A common property among
members of the KH family is their RNA-binding ability, but the present study
strongly suggests that they are multifunctional motifs. Although it could be
argued that the regions between the four KH domains in the KH1-4 deletion
mutant could mediate the biological effects in terms of RNA trafficking, their
small sizes and non-conserved sequences make this unlikely. When the third KH
domain from the human Nova antigen was crystallized, it formed an
intermolecular tetramer (Lewis et al.,
1999
), suggesting that the KH domain may have an inherent ability
to oligomerize. The function of the two RRMs in the vertebrate branch of the
family is currently unclear. Although the two RRMs on their own cannot bind to
the high-affinity H19 RNA-target, they nevertheless contribute to a lower
Kd in the full-length molecule.
The three aspects of IMP1 localization RNA-binding, granule formation and anchoring in lamellipodia could not be separated in the present deletion analysis, suggesting that they are interdependent. A plausible explanation is that RNA-binding and granule formation are prerequisites for subsequent transport, possibly through a cumulative effect. This interpretation is supported by the narrow density distribution of IMP-RNP in the Nycodenz gradient, which implies that in spite of a broad range of particle sizes, the RNA-protein ratio in the particles is constant. Each granule must contain many GFP-IMP1 molecules (otherwise they would not appear as visible granules), and this high local concentration of IMP1 in a `burr'-like structure could lead to a much higher association rate of the granule to the localization apparatus than single IMP1 molecules would exhibit. The motifs needed for localization and granule formation of IMP1 as well as for efficient attachment of RNA cargo are situated within the conserved part of the protein, since both the recently characterised Drosophila IMP homologue and the apparent IMP homologue from C. elegans (accession number T23837) consist of four KH domains and no RRMs.
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Acknowledgments |
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