1 Department of Biochemistry, University of Montreal, Montreal, H3C 3J7,
Canada
3 Centre de Recherches en Sciences Neurologiques, University of Montreal,
Montreal, H3C 3J7, Canada
2 Max-Planck-Institute for Developmental Biology, Tübingen, Germany
* These authors contributed equally to this work
Author for correspondence (e-mail:
luc.desgroseillers{at}umontreal.ca
)
Accepted 5 June 2002
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Summary |
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Key words: Staufen, Ribonucleoparticle, Ribosome, RNA-binding protein, RNA transport
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Introduction |
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RNA transport is thought to be initiated by the recognition of cis-acting
RNA motifs by RNA-binding protein(s) and their assembly into ribonucleoprotein
(RNP) complexes. RNPs are then recruited and transported on the cytoskeleton
and anchored to their final destination. To restrict RNA translation to the
appropriate time and place, the mechanisms of mRNA transport must be tightly
coupled to those of translation: translation of transported RNAs has to be
repressed during transport, and repression has to be removed after arrival at
the proper location and/or following signaling events. Evidence also supports
a model in which the delivery of new mRNAs to the dendrite occurs in motile
structures called RNA granules. Neuronal RNA granules were described as large
clusters of ribosomes, RNAs, some translation factors and proteins and can be
observed in living neurons using the RNA-binding dye SYTO14
(Knowles et al., 1996). They
were suggested to be translationally incompetent and to represent reservoirs
of silent RNA, which upon cell stimulation release RNAs for local translation
on polyribosomes (Krichevsky and Kosik,
2001
).
We and others have cloned and characterized Stau1 in mammals
(Marión et al., 1999;
Wickham et al., 1999
;
Monshausen et al., 2001
), a
protein similar to the Drosophila Staufen protein
(St Johnston et al., 1991
).
Staufen is a dsRNA-binding protein involved in mRNA localization events in
Drosophila oogenesis and neurogenesis
(St Johnston, 1995
). In
mammals, Stau1 is ubiquitously expressed and localizes to the rough
endoplasmic reticulum (RER) in vivo and cosediments with polyribosomes. In
mammalian neurons, Stau1 was detected as particles in the soma and dendrites
but not in axons (Kiebler et al.,
1999
; Monshausen et al.,
2001
). A Stau1/GFP fusion protein colocalized with SYTO14-labeled
granules and moved in dendrites at approximately the same speed as
RNA-containing granules (Köhrmann et
al., 1999
), showing that Stau1 is a component of ribonucleoprotein
complexes. Immunoelectron microscopic analyses confirmed that Stau1 is present
in RNA granules in neurons (Krichevsky and
Kosik, 2001
). These observations, along with the evolutionary
conservation of the protein structure, strongly support the involvement of
Stau1 in RNA transport in mammalian cells.
Recently, a second Staufen homologue (Stau2) located on human
chromosome 8 has been reported on the basis of genomic and EST analyses
(Buchner et al., 1999). In
cultured neurons, the amount of Stau2 in dendrites seems to correlate with
that of RNA, suggesting that, like Stau1, Stau2 plays an important role in the
delivery of RNA to dendrites (Tang et al.,
2001
). Here, we report the identification of two additional Stau2
isoforms generated by differential splicing and the cellular and molecular
characterization of these proteins. Since Stau2 is mainly expressed in the
brain, our studies concentrate on neurons. We show that Stau2 has many
conserved features in common with Stau1. However, they also differ in other
aspects that are likely to fulfil different though complementary roles in
neurons.
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Materials and Methods |
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RNA analysis
For northern blotting, mouse tissues were dissected and total RNA
immediately prepared using Trizol (Life Technology, NY). Poly(A)+
RNA was isolated with the Oligo(dT)-cellulose matrix. Poly(A)+ RNAs
(4 µg) were loaded on a denaturing formaldehyde agarose gel and transferred
to a nylon Hybond N+ membrane. Hybridization was carried out with the
ExpressHyb solution (Clontech, Palo Alto, CA, USA) as proposed by the
supplier. Three [32P]DNA probes were used: a fragment coding for
the C-terminus of Stau2 (BamHI site to the Stop codon); a fragment
coding for the C-terminus of Stau1 (EcoRI to the Stop codon); and a
fragment coding for the entire open reading frame of mouse actin.
RT-PCR assays were performed with total RNA isolated from different tissues using the RNA PCR kit (Applied Biosystems, Foster City, CA). To determine the relative abundance of the Stau262 and Stau259 transcripts in a tissue, we amplified isolated RNA using RT-PCR in a single reaction tube with the sense 5'-CGCAGTTTTGTGGAGCTGTGAGGG-3' and antisense 5'-CCATGTCTGCTCGCCAAGACTCAG-3' oligonucleotides. These primers flank the alternatively spliced exon and therefore amplified both transcripts. Different amounts of starting RNA (250 to 1000 ng) and PCR cycles (94°C for 30 seconds, 60°C for 30 seconds) were compared to ensure that the amplification was below the level of PCR saturation and that the ratio of the two bands did not vary with the number of cycles used.
Northwestern assay
To construct the maltose-binding protein (MBP)-Stau259 fusion
protein (MBP-FL), the full-length GT10 cDNA insert was first cloned
into Bluescript SK (Stratagene, LA Jolla, CA) and then digested with
SalI and SacI for cloning in the corresponding sites of the
pMalC vector (New England Biolabs, Mississauga, ON, Canada). To construct
GST-1 to GST-5, DNA fragments coding for different Stau2 domains were PCR
amplified with the Vent DNA polymerase (New England Biolabs, Mississauga, ON,
Canada), cloned into Bluescript SK at the EcoRV site and subcloned
into pGEX-T (Amersham Pharmacia, Baie d'Urfé, QC, Canada) using the
SalI and NotI restriction sites. To construct the GST-1
fusion protein, we used the sense
5'-TTCTCTCCAAGATAAAATGGCAAACCC-3' and antisense
5'-AGACTTTTCTGGAATTGGCTCAATCTG-3' primers. Similarly, GST-2,
GST-3, GST-4 and GST-5 fusion proteins were constructed with
5'-GAGGGATACGGAAGTTTGATC-3' and
5'-AGACTTTTCTGGAATTGGCTCAATCTG-3';
5'-ACAGATTGAGCCAATTCCAGAAAAGTCT-3' and
5'-GTTTTGGCTTCTCTACCACAGG-3';
5'-TCCTGTGGTAGAGAAGCCAAAAC-3' and
5'-GAGCGGATCCTGAAGACTGGTG-3', and with
5'-CACCAGTCTTCAGGATCCGCTC-3' and
5'-ATTCGTTTCCTAGAACACAGACACC-3' oligonucleotides, respectively.
Northwestern assays were performed as previously described
(Wickham et al., 1999
) using
[32P]bicoid 3'UTR RNA as probe.
Production of Stau2-specific antibodies
Monoclonals
A Stau2-GST fusion protein encoding the C-terminal portion of Stau2
(Thr379 to Stop) was expressed in BL21 pLysS DE3 (Invitrogen,
Burlington, ON, Canada) and affinity purified on a Glutathione Sepharose
matrix (Amersham Pharmacia, Baie d'Urfé, QC, Canada). The C-terminal
Stau2 peptide was eluted from the column by cleavage with thrombin. Mice were
immunized by multiple injections of 10 µg of antigen per injection, and
spleens from Stau2-positive mice were isolated, and monoclonal antibodies
(1C6) were prepared as described previously
(Crine et al., 1985).
Polyclonals
Alternatively, the same E. coli overexpressed and purified
Stau2-GST antigen (non thrombin-cleaved) was used for injecting mice
intraperitoneally four times with 50 µg of antigen per injection. Samples
of blood were then taken through the tail vein and antisera prepared as
described previously (Harlow and Lane,
1988): diluted 1:1 in glycerol and stored at -20°C. Polyclonal
antibodies were also prepared by injecting rabbits with 40 µg of the
thrombin-cleaved antigen per injection, as described previously
(Wickham et al., 1999
). All
these immune sera were tested for their specificity. First, no Stau2-specific
signal was obtained with the pre-immune serum isolated from the corresponding
mouse or rabbit. Second, pre-incubation of the antibodies with the Stau2-GST
fusion protein antigen coupled to sepharose abolished the Stau2-specific
signal. Finally, the mouse antibodies recognize Stau2 but not Stau1 when
overexpressed in BHK21 cells (data not shown).
Western blot assay
Western blotting was carried out as previously described
(Wickham et al., 1999). Human
autoimmune P serum (a generous gift of M. Reichlin, Oklahoma Medical Research
Foundation, Oklahoma, USA) and rabbit anti-L7 antiserum (a generous gift of A.
Ziemiecki, University of Berne, Berne, Switzerland) were used to detect
ribosomes. Monoclonal anti-calnexin (Stressgen, Victoria, BC, Canada),
anti-
-tubulin (ICN, Irvine, CA) and AE-4 (Santa Cruz Biotechnology,
Santa Cruz, CA) antibodies were used to detect endoplasmic reticulum,
-tubulin and histone H1, respectively.
Primary cultures of neurons
For the biochemistry experiments, cortical neurons were isolated and
cultured as described before (Shaw et al.,
1985; Bassell et al.,
1998
) with the following modifications. Briefly, cerebral cortex
was dissected from embryonic day 17-18 Sprague Dawley rats and digested with
0.25% trypsin. Neurons were plated at high density (80x106
per 150 mm plate) on poly-L-lysine- (10 µg/ml, overnight) and
laminin-coated (2 ng/ml for 4 hours) 150 mm tissue culture dishes. After
neurons had attached to the substrate (4 hours) in MEM+10% horse serum,
cultures were allowed to differentiate in glutamate-free MEM with N2
supplements, including transferrin (100 µg/ml), insulin (5 µg/ml),
progesterone (20 nM), putrescine (100 µM) and selenium dioxide (30 nM). In
addition, extra glucose (600 mg/l), sodium pyruvate (1 mM), ovalbumin (0.1%)
and 5% FBS were added to the media. Cells were allowed to achieve polarity for
5 days prior to extraction. For IF, primary hippocampal neurons derived from
E17 rat embryos were cultured at low density according to the standard
protocol (Goslin and Banker,
1991
) as described previously
(Kiebler et al., 1999
). B27
supplements (Life Technologies, Karlsruhe, Germany) was used instead of the N2
supplement.
Immunocytochemistry
Immunocytochemistry was carried out essentially as described previously
(Kiebler et al., 1999). The
following primary antibodies (incubation overnight at 4°C) were used:
rabbit anti-calnexin antibodies (dilution 1:400; a generous gift of A.
Helenius, ETH, Zurich, Switzerland), mouse monoclonal anti-
-tubulin
antibodies (dilution 10,000; Sigma, Munich, Germany), rabbit anti-Staufen1
antibodies (dilution 1:400, a generous gift of J. Ortin, Madrid, Spain)
(Kiebler et al., 1999
;
Marión et al., 1999
)
and mouse anti-Staufen2 antibodies (dilution 1:400 from the glycerol stock,
see above). The rabbit anti-Stau1 antibodies (Juan Ortín, Madrid)
recognize Stau1 but not Stau2 (data not shown). As secondary antibodies,
Texas-Red-conjugated affinipure goat anti-rabbit IgG Fab fragment (dilution
1:800; Dianova, Hamburg, Germany), Alexa Fluor 488 goat anti-rabbit IgG (H+L)
conjugate (dilution 1:500; Molecular Probes, Leiden, Holland),
biotin-conjugated sheep anti-mouse IgG (dilution 1:500; Roche Diagnostics,
Mannheim, Germany), FITC-conjugated streptavidine (dilution 1:2,000; Amersham
Pharmacia, Freiburg, Germany), Cy3-conjugated affinipure goat anti-mouse IgG
(H+L) (dilution 1:800; Dianova, Hamburg, Germany) and Cy2-conjugated
affinipure goat anti-mouse IgG (H+L) (dilution 1:800; Dianova, Hamburg,
Germany) were used. Fluorescence and confocal microscopy were performed
essentially as described previously
(Kiebler et al., 1999
).
Neurons were also stained with monoclonal anti-MAP2 (dilution 1,000; Sigma,
Munich, Germany) and anti-tau-1 (dilution 10,000; Chemicon International,
Temecula, CA) antibodies, two markers of dendrites
(Caceres et al., 1984
) and
axons (Binder et al., 1985
),
respectively. In the case of Fig.
6B, the Stau2 immunofluorescence signal was amplified using the
biotin-streptavidine cascade.
|
Cytoplasmic extract preparation and crude cell fractionation
Cytoplasmic extracts were prepared from high-density cultures of cortical
neurons. Neurons were washed in cold PBS (pH 7.5), then in isotonic buffer
(110 mM KOAc, 2 mM MgOAC, 1 mM DTT, 10 mM HEPES, pH 7.5) and recovered with a
rubber policeman in hypotonic buffer (10 mM KOAc, 2 mM MgOAc, 1 mM DTT, 5 mM
HEPES pH 7.5) supplemented with 1 U/ml RNase inhibitors and EDTA-free COMPLETE
protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada). Cells were
broken by two sets of 20 strokes in a 23-gauge syringe followed by
centrifugation at 1,500 g for 10 minutes. Supernatants were adjusted
to 100 mM KCl and allowed to stand on ice for 30 minutes. The S100-P100
fractionation was performed by centrifugation at 100,000 g for 1 hour
in the Sorvall SW50. 1Ti rotor as described previously
(Siomi et al., 1996).
Treatments of the cytoplasmic extracts before generating the S100/P100
fractions include 25 mM EDTA; 300 U/ml Micrococcal nuclease for 15 minutes
prior to addition of 5 mM EGTA; 0.5% Nonidet P40; 0.5 M KCl. Incubations were
carried out on ice for at least 30 minutes.
Sucrose gradient analysis
We used a 10-step discontinuous gradient ranging from 20 to 60% sucrose and
containing 100 mM KCl, 10 mM KOAc, 2 mM MgOAc, 1 mM DTT and 5 mM HEPES, pH 7.5
as described previously (Luo et al.,
2002). Gradients were centrifuged at 175,000 g in a
SW41Ti rotor for 3 hours and fractions of 0.8 ml were collected. Proteins were
recovered by acetone precipitation and analyzed by western blotting. Images
were obtained on a BioRad Fluor-S MAX Multi-Imager.
For ribosome analysis, cytoplasmic extracts were left on ice for 30 minutes and centrifuged on a continuous 10 to 40% sucrose gradient containing 100 mM KCl, 10 mM KOAc, 25 mM EDTA, 1 mM DTT and 5 mM HEPES pH 7.5 for 4 hours at 250,000 g in a SW41 rotor. Fractions of 0.8 ml were recovered, and the RNA sedimentation profile was monitored with a spectrophotometer set at 254 nm. Proteins were recovered and analyzed as described above.
Co-immunoprecipitation
Neurons were washed in isotonic buffer, and cytoplasmic extracts were
prepared as described above. Immunoprecipitation was carried out with either
the anti-ribosomal P protein human antiserum as previously described
(Siomi et al., 1996) or with
the rabbit polyclonal anti-Stau2 antibodies. Immunoprecipitated proteins were
resolved by SDS-PAGE, transferred to nitrocellulose membrane and revealed by
western blotting. Immunoprecipitated RNAs were purified, separated on a
formaldehyde agarose gel, transferred to nylon membranes and hybridized with a
[32P] 18S ribosomal RNA.
Ribosome pull down on 1.5 M sucrose cushion
Cortical neurons were cultured for 4 days at high density. Neuronal
cultures were either serum starved for 4 hours or starved for 4 hours and
refed with 5% FBS for 2 hours before lysis. Untreated cells were refed with
fresh 5% FBS two hours before cytoplasmic extraction. Other cultures were
treated with either cycloheximide (100 µg/ml) or puromycin (100 µg/ml)
for 30 minutes, or with pactamycin (2 µg/ml) or rapamycin (20 ng/ml) for 1
hour before cytoplasmic extraction. Neurons were then recovered, and
cytoplasmic extracts were prepared in 0.5 ml KCl and Nonidet P40, which were
adjusted to 0.11 M and 0.5%, respectively. Extracts were left on ice for 30
minutes and then placed on a 1 ml sucrose cushion (1.5 M) containing 110 mM
KOAc, 2 mM MgOAc, 1 mM DTT, 0.05% Nonidet P40 and 10 mM HEPESK pH 7.4. Tubes
were centrifuged at 300,000 g for 3 hours in a Beckman TLA 100.3
microfuge. After centrifugation, the pellets (ribosome enriched fraction) were
resuspended in 1 vol Laemmli sample loading buffer. Supernatant/cushions were
recovered, the proteins TCA-precipitated and recovered in 1 vol Laemmli sample
loading buffer. Proteins were resolved on a 8.5% SDS-polyacrylamide gel and
analyzed by western blotting. All experiments were performed in duplicate.
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Results |
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The predicted structure of Stau2 is similar to that of the
Drosophila Staufen based on the existence of the different RBDs. In
contrast to mammalian Stau1, Stau2 contains the corresponding
Drosophila dsRBD1. Whereas Stau262 has a full-length
dsRBD1 domain, Stau259 contains only the C-terminal half of the
domain (Fig. 1C). Sequence
analysis also revealed the presence of domains that correspond to the
Drosophila split domain (dsRBD2) and the major (dsRBD3) and minor
(dsRBD4) RNA-binding domains. A region of low sequence identity with the
microtubule-binding domain of MAP1B is also present downstream of dsRBD4, as
observed before in mammalian Stau1
(Wickham et al., 1999).
Finally, only the C-terminal part of a putative RBD5 is found in a
rearranged form in Stau2 (Fig.
1B).
Stau2 is mainly expressed in brain
Northern blot analysis with RNAs isolated from mouse tissues revealed that
Stau2 is mainly expressed in brain and heart
(Fig. 2B). Longer exposure of
the blot allowed us to detect transcripts in the kidney, testis and ovary
(data not shown). For comparison, a Stau1-specific probe hybridized to RNA in
most tissues (Fig. 2B), as
observed before in humans and rats
(Marión et al., 1999;
Wickham et al., 1999
;
Monshausen et al., 2001
).
Therefore, there are clear tissue-specific differences in the expression of
the two genes. RT-PCR amplification of brain RNA with primers located on each
side of the differentially spliced exon of 131 nucleotides demonstrated that
the two transcripts are expressed in the brain. Consistent with the western
blots, transcript T2 encoding Stau259 is more abundant than T1
(Fig. 2C). Varying
concentrations of brain RNA and multiple numbers of cycles assured that PCR
amplification was kept in non-saturable conditions.
Mapping of functional domains in vitro
To identify the functional RNA-binding domain(s) in Stau2, we used a
Northwestern assay as previously described for Stau1
(Wickham et al., 1999). A
full-length Stau259-MBP fusion protein was first produced in
bacteria and tested in the dsRNA-binding assay. This protein bound
[32P]bicoid 3'UTR mRNA
(Fig. 3A). None of the
overexpressed MBP, BSA or the bacterial proteins in the extracts bound the
probe, demonstrating the specificity of the RNA-binding assay. We then fused
individual domains to GST (Fig.
3C), expressed them in bacteria and tested their capacity to bind
the probe (Fig. 3A). In these
conditions, GST-3 strongly bound the probe, whereas GST-4 bound the probe only
very weakly. By contrast, GST-1, GST-2 and GST-5 did not bind at all.
Fig. 3B shows that equivalent
amounts of the fused proteins were loaded. These results demonstrate that
dsRBD3 is the major dsRNA-binding determinant, as determined previously for
the other Staufen homologues.
|
Stau2 localizes in the somatodendritic domain of neurons but not in
axons
We next determined the subcellular distribution of Stau2 in mature primary
hippocampal neurons in culture (16 DIV), using a polyclonal antiserum. This
antiserum is specific for Stau2 and does not recognize Stau1. Rat Stau2
localized in both the soma and neurites where it appeared as granules
(Fig. 4A). In neurites,
Stau2-containing particles appeared to be aligned on individual tracks.
Consistently, our results show a significant colocalization of aligned Stau2
particles with -tubulin (Fig.
4B), strongly suggesting that Stau2 is associated with polarized
microtubular tracks. Stau2 immunoreactivity was found in the same compartment
as MAP2 (Fig. 5A), clearly
demonstrating that it is present in dendrites. By contrast, Stau2 was absent
from axons as it did not colocalize with Tau
(Fig. 5B), a marker of
axons.
|
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Stau1 and Stau2 are components of distinct particles in
dendrites
The distribution pattern of Stau2 is similar to that of Stau1, which was
predominantly found in large granules both in the periphery of the cell body
as well as in dendrites of mature hippocampal neurons
(Kiebler et al., 1999).
Therefore, we tested whether Stau1 and Stau2 colocalize in the same granules.
We performed double-immunofluorescence microscopy of the same hippocampal
neurons using specific Stau1 and Stau2 antibodies that do not cross-react with
each other (data not shown). From these experiments it became clear that Stau2
is significantly more abundant in distal dendrites than Stau1
(Fig. 6A). At higher
magnification in distal dendrites, the vast majority of both proteins did not
colocalize (Fig. 6B),
demonstrating that they are components of distinct particles.
Stau2 isoforms are found in the P100 fraction of cytoplasmic
extracts
To better study the subcellular distribution of the Stau2 isoforms, we
first used a crude cytoplasmic and nuclear fractionation approach starting
from high-density primary cultures of rat cortical neurons. Western analysis
of the fractions with the monoclonal antibody 1C6 indicated that
Stau262, Stau259 and Stau252 were all present
in the cytoplasmic fraction (Fig.
7A).
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We then fractionated the cytoplasmic extracts into S100/P100 fractions by high-speed centrifugation. Under these conditions, soluble proteins are found in the supernatant (S100), whereas membrane-bound, heavy-complex-associated proteins and organelles sediment in the pellet (P100). As shown in Fig. 7B, the three Stau2 isoforms were consistently found in the P100 fraction, as do calnexin (CNX), a marker of the RER and L7a, a ribosomal protein. This result showed that Stau2 isoforms are associated with dense particles/organelles. By contrast, tubulin (Tub) was found mainly in the S100 fraction. Pre-treatment of the cytoplasmic extracts with non-ionic detergent (Nonidet P40) before centrifugation did not abrogate the sedimentation of Stau2 proteins in P100, showing that solubilization of the membranes did not affect Stau2 association with dense particles/organelles. As a control, calnexin was now shifted to the S100. Similarly, pre-treatment with EDTA did not displace Stau2 from the dense particles. By contrast, treatment with 0.5 M KCl released roughly 50% of Stau2 in the S100 fraction. These observations demonstrate that all Stau2 isoforms are non-covalently associated with stable, EDTA- and detergent-resistant high-density particles/organelles. Treatment with a high concentration of RNase only released very low amounts of Stau2 isoforms in the S100 fraction, suggesting that their association with high-density particles may not be mediated mainly by RNA intermediates.
Stau2 splice isoforms are found in several complexes
To better characterize these Stau2-containing particles, we next
fractionated cytoplasmic extracts on a 20-60% sucrose density gradient.
Fractions were recovered and analyzed by western blotting with an anti-Stau2
monoclonal antibody, anti-calnexin and anti-L7a. Interestingly, more than 70%
of both Stau259 and Stau252 co-fractionated with the
major peak of ribosomes (Fig.
8A). Significant amounts (about 30%) of the two proteins were also
found in fractions of higher density, which also contained low amounts of
ribosomes. These proteins are not associated with membranes since detergent
treatment of the cytoplasmic extract prior to sedimentation did not abolish
the signal (data not shown). By contrast, Stau262 was found only in
the lightest fractions of the gradient. These results demonstrate that
Stau259 and Stau252 have the same distribution, that
they are components of at least two complexes and that Stau262 has
a distinct distribution compared to the two shorter isoforms.
|
To study this possible interaction between Stau259/Stau252 and the ribosomes, we first separated cytoplasmic extracts using a sharper sucrose gradient (10 to 40%) and a longer centrifugation time in the presence of EDTA to separate ribosome subunits. In these conditions, Stau259 and Stau252 still cofractioned with ribosome subunits and were found in the same fractions as both the 40S and 60S subunits (Fig. 8B). Second, we immunoprecipitated ribosomes with a human anti-ribosomal protein antiserum and detected Stau2 by western blotting with the anti-Stau2 antibody. A band corresponding to Stau259 was visible on the blot (Fig. 9A). The presence of a weak band in the immunoprecipitate is consistent with an association of Stau2 with only a small fraction of the total pool of ribosomes in the cells. Unfortunately, Stau252 comigrates with the remaining antibodies and therefore cannot be detected on the blot. A Stau259-specific band was not visible when a normal human serum was used for immunoprecipitation. Similarly, when Stau2 was immunoprecipitated with an anti-Stau2 antibody, the P ribosomal protein (Fig. 9B) and 18S rRNA (Fig. 9C) were detected in the immunoprecipitate. No signal was found when immunoprecipitation was done with the preimmune serum. All these results are consistent with an interaction of two Stau2 isoforms with ribosomes, pointing to a very new role for Stau2 in translation.
|
Stau259/Stau252 association with ribosomes is
independent of translation
Finally, we tested whether the translational activity of ribosomes
modulates the association of Stau2 with ribosomes. Neurons were treated with
pactamycine (an inhibitor of translation initiation). rapamycin (an inhibitor
of the FRAP/TOR signaling pathway) or puromycine or cycloheximide (two
inhibitors of translation elongation). In other experiments, cells were first
incubated in a serum-free medium, and half of the cultures were re-exposed to
normal serum concentrations. Cytoplasmic extracts were fractionated through a
1.5 M sucrose cushion in the presence of detergent. Supernatants and
ribosome-enriched pellets were analyzed for the presence of Stau2. In
untreated cells, Stau259 and Stau252 were found in the
pellet as expected. In treated cells, they were also associated with the
pellets and not released in the supernatant (data not shown). These results
strongly suggest that translation does not influence association of both
Stau259 and Stau252 with ribosomes.
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Discussion |
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Mouse Stau2 also shares high amino-acid sequence identity with the
paralogous protein Stau1. As observed previously with Stau1
(Kiebler et al., 1999;
Köhrmann et al., 1999
),
Stau2 is found in the somatodendritic compartment of neurons and colocalizes
with microtubules in dendrites. Consistently, drugs that disrupt microtubule
organization impair both Stau1 (Kiebler et
al., 1999
; Köhrmann et
al., 1999
) and Stau2 (Tang et
al., 2001
) distribution in dendrites. Nevertheless, double
immunofluorescence microscopy demonstrates that Stau1 and Stau2 are components
of distinct particles. This conclusion is further supported by experiments
with co-immunoprecipitation (T.F.D. and L.DG., unpublished) and biochemical
cell fractionation (A.D. and M.A.K., upublished). All these results suggest
that Stau1 and Stau2 may have similar and/or complementary function(s) in
neurons but that they are components of distinct population of complexes.
Evolutionary conserved features of Stau2
Conservation of the overall structure and the primary sequence of
corresponding dsRBDs among Staufen homologues in different species suggests
that these domains have kept their unique functional features throughout
evolution. In all species, dsRBD3 is the major RNA-binding domain
(St Johnston et al., 1992;
Micklem et al., 2000
). By
contrast, dsRBD2 and dsRBD5 do not bind to RNA in vitro. Nevertheless there
are requirements for dsRBD2 for localization of oskar RNA at the
posterior pole and dsRBD5 for the derepression of translation of
oskar mRNA once localized at the posterior pole
(Micklem et al., 2000
). dsRBD5
is also involved in bicoid and prospero mRNA localization in
oocytes and neuroblasts, respectively
(Micklem et al., 2000
). Most
probably, dsRBD5 establishes protein-protein interaction as it was shown to
bind to Miranda in neuroblasts (Schuldt et
al., 1998
). The rearrangement/absence of dsRBD5 in the mouse Stau2
isoforms may be used to modulate these functions. The role of dsRBD1 and
dsRBD4 is still unknown. In contrast to Drosophila dsRBD1, mouse
Stau2 dsRBD1 does not bind to dsRNA in our in vitro conditions. Whether it
binds to RNA in vivo is still an open question. Interestingly, whereas Stau1
lacks dsRBD1, Stau2 lacks a bona fide dsRBD5, suggesting that during evolution
some functions of Drosophila Staufen were split between the two
paralogues.
Stau2-containing particles
In the cell fractionation assay, Stau2 isoforms are consistently found in
the P100 fraction, demonstrating that they are components of rather large
complexes. These Stau2-containing complexes share common properties. First,
they seem remarkably stable. They are resistant to EDTA and to relatively high
concentration of KCl. Second RNAse treatment only releases very small amounts
of Stau2 isoforms in the supernatant, in contrast to the poly(A)-binding
protein (PABP), which is completely released in the supernatant (data not
shown). Third, Stau2 association with complexes appears to be independent of
translation. Drugs that inhibit translation or serum starvation do not affect
these associations.
Our results with sucrose gradients further demonstrate that Stau2 isoforms
are distributed in several complexes. Stau262 fractionates in
fractions of low density. These fractions also stain for calnexin but are
devoid of ribosomes. This calnexin-positive material has been observed in
neurons (Villa et al., 1992)
but not in other cell lines such as COS7, HEK293 or HeLa and may represent the
smooth ER elements that extend into both axons and dendrites
(Krijnse-Locker et al., 1995
).
However, the comparative behaviors of Stau262 and calnexin in the
S100/P100 fractionation experiments suggest that Stau262 may not be
physically linked to these vesicles.
Stau259 and Stau252 have a more complex subcellular
distribution than Stau262. They are found in at least two complexes
of different density across the sucrose gradient. The first complex is found
in fractions of high density. These complexes are not associated with the RER
since they are not solubilized with detergent. Whether they are associated
with ribosomes and/or represent large clusters of ribosomes, as those recently
observed in neurons and shown to contain Stau1
(Krichevski and Kosik, 2001)
do, need to be resolved. The second complex is associated with ribosomes.
Sucrose gradients, co-immunoprecipitation and ribosome pull-down assays are
all consistent with this observation. However, it is possible that this small
population of Stau2-containing ribosomes is also bound to the RER in vivo.
Indeed, confocal microscopy shows colocalization of Stau2 and calnexin in vivo
(data not shown). Nevertheless, the harsh lysis conditions used in our
experiments establish that the primary binding site of Stau259 and
Stau252 is likely to be the ribosomes and not the membranes
themselves.
Molecular characterization of Staufen's functions in Drosophila
demonstrated that both Staufen-RNA and Staufen-protein interactions occur
during localization (Ferrandon et al.,
1994; Li et al.,
1997
; Schuldt et al.,
1998
; Micklem et al.,
2000
). The heterogeneity of Stau2- and Stau1-containing
particles/organelles suggests that they are part of a dynamic and multi-step
process that modulates the composition of RNA/protein complexes along the
pathway. This is essential for proper localization and/or translational
regulation of mRNAs. We continue to be challenged to determine the subcellular
localization and the roles of each isoform in neurons in relation to these
complex processes. Since new tools to address these problems are now
available, we can anticipate answers to these questions.
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Acknowledgments |
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References |
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