Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA
* Author for correspondence (e-mail: kimberly_mowry{at}brown.edu)
Accepted 15 March 2004
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SUMMARY |
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Key words: RNA-binding protein, RNA localization, Polarity
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Introduction |
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RNA localization relies on cis-acting sequence elements within the RNA that
are recognized by trans-acting components of the localization machinery
(reviewed by Jansen, 2001).
These cis-elements generally reside within the 3' untranslated region
(UTR) of the localized message and are essential for proper transport;
examples are numerous, and include Xenopus Vg1 RNA
(Mowry and Melton, 1992
), and
bicoid (Macdonald and Struhl,
1988
; Macdonald and Kerr,
1997
; Macdonald and Kerr,
1998
) and oskar
(Kim-Ha et al., 1993
) mRNAs of
Drosophila. Variable in length, the localization elements (LEs)
mediate the interaction with transacting factors, resulting in formation of a
ribonucleoprotein (RNP) complex. RNA-binding proteins have been identified as
trans-acting localization factors in many animals, including Xenopus
and Drosophila. The Xenopus Vg1 LE interacts with at least
six different RNA-binding proteins, some of which have been shown to function
in vegetal RNA localization (Cote et al.,
1999
; Deshler et al.,
1998
; Deshler et al.,
1997
; Havin et al.,
1998
; Kroll et al.,
2002
; Mowry, 1996
;
Schwartz et al., 1992
;
Zhao et al., 2001
). In
Drosophila, genetic approaches have identified a number of
RNA-binding proteins required for localization of maternal mRNAs during
oogenesis. Examples include Staufen (St
Johnston et al., 1991
; St
Johnston et al., 1989
), Exuperantia
(Berleth et al., 1988
;
Macdonald et al., 1991
;
Marcey et al., 1991
), Swallow
(Berleth et al., 1988
;
Chao et al., 1991
;
Stephenson et al., 1988
),
Squid (Norvell et al., 1999
)
and Modulo (Arn et al., 2003
;
Perrin et al., 1999
), among
others. However, the many trans-factors identified are generally
non-homologous between vertebrates and invertebrates. This raises the issue of
whether the mechanistic strategies for RNA localization are shared among
organisms.
One factor that could mechanistically link RNA localization in vertebrates
and invertebrates is the double-stranded RNA (dsRNA)-binding protein Staufen.
Originally identified in flies, staufen mutants exhibited defects in
bicoid RNA localization (St
Johnston et al., 1989). Drosophila Staufen was found to
be localized to the posterior cortex with oskar mRNA in oocytes and
to the anterior cortex with bicoid mRNA in eggs, suggesting a role
for Staufen in localization of RNAs along the anteroposterior axis
(Ephrussi et al., 1991
;
Kim-Ha et al., 1991
;
St Johnston et al., 1991
).
Moreover, Drosophila Staufen was shown to be responsible for
asymmetric localization of prospero mRNA in neuroblasts
(Broadus et al., 1998
;
Li et al., 1997
;
Matsuzaki et al., 1998
;
Schuldt et al., 1998
;
Shen et al., 1998
). Since the
identification of Drosophila Staufen, several vertebrate homologs
have been found (Kiebler et al.,
1999
; Marion et al.,
1999
; Wickham et al.,
1999
). Nonetheless, the functions of known vertebrate Staufen
homologs are not yet clear. Mammalian Staufen has been shown to colocalize
with polyribosomes and rough endoplasmic reticulum in cultured cells and
hippocampal neurons (Kiebler et al.,
1999
; Marion et al.,
1999
; Wickham et al.,
1999
). Also, in hippocampal neurons, mammalian Staufen is
colocalized with RNA-containing granules in dendrites
(Mallardo et al., 2003
;
Tang et al., 2001
), suggesting
a role in RNA localization. However, interactions with specific localized RNAs
have not been uncovered, leaving open the question of whether this RNA-binding
protein may serve a conserved role in RNA localization.
To accomplish directional transport of localized RNAs within the cytoplasm,
roles for motor proteins have long been sought (reviewed by
Wilhelm and Vale, 1993), but
only recently have the identities of some of these come to light (reviewed by
Tekotte and Davis, 2002
). In
yeast, a myosin motor is responsible for the localization of ASH1 mRNA to
budding daughter cells (Bobola et al.,
1996
). During embryogenesis in flies, dynein has been shown to be
necessary for apical localization of pair-rule transcripts
(Bullock and Ish-Horowicz,
2001
; Wilkie and Davis,
2001
). In Drosophila oocytes, kinesin I has been
implicated in transport of oskar mRNA to the posterior during
oogenesis (Brendza et al.,
2000
), but this role may be indirect
(Cha et al., 2002
). In other
systems, including Xenopus, inhibition of RNA localization by
cytoskeletal disruption also provided clues towards involvement of molecular
motors (Knowles et al., 1996
;
Yisraeli et al., 1990
). While
motor proteins have been implicated in RNA sorting in vertebrate neurons
(Carson et al., 1997
), and
most recently in vegetal RNA localization in Xenopus
(Betley et al., 2004
), the
mechanisms by which specific motor proteins may be targeted to localized RNAs
is not at all clear.
We have identified a Xenopus homolog of Staufen (XStau) as a component of the RNA localization machinery in frog oocytes. We present evidence that XStau interacts with specific localized RNAs during oogenesis and is expressed in a spatial and temporal pattern consistent with a role in RNA localization. Importantly, we show that XStau is in an RNP complex that associates with a kinesin motor and that a mutant version of XStau blocks RNA localization in oocytes. These results suggest that XStau is an integral component of the machinery necessary to localize RNA to the vegetal cortex and that XStau may mediate interactions between localized RNAs and motor proteins that are crucial for transport.
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Materials and methods |
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To generate the GST-fusion clone pGEX-RBD2C, XStau sequence encoding dsRBD2 to the C terminus was PCR amplified using primers (forward 5'-ACGCGTCGACTCATGAAGCTGGGAAAGAAACCAATATAC-3' and reverse 5'-AAGGAAAAAGCGGCCGCGGCTGTGCTGTCCAGTGTTTGCCC-3') and cloned into pGEX4T-1 (Amersham Pharmacia) at SalI and NotI sites. FLAG-tagged XStau constructs were generated by PCR amplification of full-length XStau using primers RBD1-5' (forward 5'-GCAGATCTATGGACGTCACCATGTCTCAAGCT-3') and RBD5-3' (reverse 5'-AGCTCGAGGCTGTGCTGTCCAGTGTTTG-3'), and XStau234 using primers RBD2-5' (forward 5'-GCAGATCTATGCCTGTACGCGACAGCATCACG-3') and RBD4-3' (reverse 5'-AGCTCGAGGGCGGAGGGACTTTAAAACCTAG-3'), followed by ligation into pSP64TSNRLMCSFLAG at BglII and XhoI sites to generate pSP64TSNXStauFLAG and pSP64TSNXStau234FLAG.
Antibody production and immunoblotting
pGEX-RBD2C was transformed into E. coli strain BL21
CodonPlus(DE3)-RIL (Stratagene). Expression and purification of GST-XStau
peptide has been described previously
(Coligan et al., 1995), with
minor modifications. Lysis buffer contained 50 mM NaCl, 50 mM Tris pH 8.0, 5
mM EDTA, 0.1 µg/ml leupeptin, 0.1 µg/ml antipain, 0.1 µg/ml trypsin
inhibitor, 0.4 M Pefabloc and 200 µg/ml lysozyme. After sonication, lysed
cells were centrifuged at 40,000 g for 20 minutes, passed
through a 0.45 µm filter and loaded onto a glutathione column. Eluted
fusion protein was verified on 10% SDS PAGE, quantified using Bradford assay,
and concentrated using Centriplus YM-50 (Amicon). XStau-GST fusion peptides
(500 mg) were injected along with an equal volume of complete Freunds
adjuvant into New Zealand white rabbits to generate polyclonal anti-XStau
antibodies.
Immunoblotting was performed as described previously
(Denegre et al., 1997). XStau
antibodies were used at 1:2000; rpS6 antibodies (E-13, Santa Cruz
Biotechnology) at 1:800; dynein intermediate chain antibodies (DIC, Sigma) at
1:4000; tubulin antibodies (Sigma) at 1:2000; SUK4 antibodies
(Ingold et al., 1988
) at
1:1000; hnRNP I antibodies (Kress et al.,
2004
) at 1:2000; Vg1RBP antibodies
(Zhang et al., 1999
) at
1:30,000; and FLAG antibodies (Sigma) at 1:2000 dilutions in BLOTTO (250 mM
NaCl, 50 mM Tris pH 7.5, 5% dried milk, 0.1% Tween 20). Anti-rabbit secondary
was used at 1:160,000, anti-mouse secondary at 1:100,000, anti-goat secondary
at 1:8000 dilutions. All secondary antibodies used were peroxidase-conjugated
(Sigma) and detection was by enhanced chemiluminescence.
Oocytes and microinjection
Oocytes were obtained surgically from Xenopus laevis and
microinjection was performed as previously described
(Gautreau et al., 1997). VLE
RNA was transcribed from pSP73-340 (Mowry,
1996
) in reactions containing 0.5 mM ATP and CTP, 100 µM GTP, 1
mM diguanosine triphosphate, 450 µM UTP, 50 µM Alexa-546-14-UTP
(Molecular Probes), 1 µCi of 32P-UTP. Stage III albino oocytes
were injected with 150 pg of RNA and incubated at 18°C in OCM [50% L15
medium, 15 mM HEPES (pH 7.6), 1 mM glutamine and 1 µg/ml insulin] for 18-24
hours, fixed in MEMFA and either imaged directly, or subjected to
immunofluorescence as described below using anti-XStau and
Alexa-647-conjugated secondary antibody (Molecular Probes). For interference
assays, oocytes were injected with RNA transcribed from pSP64TSNXStauFLAG or
pSP64TSNXS-tau234FLAG using the mMessage mMachine kit (Ambion) and incubated
for 16 hours prior to VLE injection (as above).
Immunolocalization
Oocytes were defolliculated by treatment with collagenase as described
previously (Mowry, 1996) and
manually sorted into stages I-II, III-IV and V-VI
(Dumont, 1972
). After fixation
in MEMFA for 1 hour, whole-mount immunofluorescence was performed as described
previously (Sive et al.,
2000
). XStau antibodies were used at 1:250 dilution in PBT
(1xPBS, 2 mg/ml BSA and 0.1% Triton X-100) overnight at 4°C. Oocytes
were washed three times in PBT for 2-4 hours at room temperature. Goat
anti-rabbit Alexa-568- or -647-conjugated secondaries (Molecular Probes) were
added at 1:100 dilution in PBT overnight at 4°C. Oocytes were washed
extensively as above and dehydrated in methanol. Oocytes were cleared and
analyzed by confocal microscopy (Denegre
et al., 1997
). For colocalization analysis, oocytes were analyzed
on a Leica TCS SP2 inverted confocal microscope; excitation to detect
Alexa-546 labeled RNA was at 543 nm, with emission at 557-607 nm. To detect
XStau immunofluorescence with Alexa-647, excitation was at 633 nm and emission
was at 664-756 nm.
Subcellular fractionation
To prepare S10 lysates, total or staged oocytes were homogenized in an
equal volume of XB (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.5 mM DTT,
0.1 µg/ml leupeptin, 0.1 µg/ml antipain, 0.1 µg/ml trypsin inhibitor
and 0.4 mM Pefabloc). The homogenate was centrifuged twice for 10 minutes at
10,000 g to remove any insoluble material. The supernatant
(50-100 µl) (S10 lysate at 20 µg/µl protein) was applied to a 2
ml 5-40% sucrose gradient in XB with protease inhibitors. After centrifugation
for 2 hours at 237,000 g at 17°C, 100 µl fractions were
collected and either processed for immunoprecipitation (below) or precipitated
in 10% TCA and resolved by SDS-PAGE.
Immunoprecipitation
Protein G sepharose beads (2.5 mg, Amersham) were combined with 50 µg of
XStau or 30 µg of SUK4 antibody in a total volume of 500 µl of NET-150
[150 mM NaCl, 50 mM Tris (pH 8.0) and 0.05% NP-40] and mixed for 2 hours. The
beads were washed four times in NET-150 and mixed with S10 lysate. After
binding for 2 hours, the beads were washed four times in NET-150, and samples
were boiled in the presence of SDS buffer and resolved on SDS-PAGE as above.
For co-immunoprecipitation, NET-75 [75 mM NaCl, 50 mM Tris (pH 8.0) and 0.05%
NP-40] was used in place of NET-150. FLAG immunoprecipitations were preformed
as described previously (Kress et al.,
2004) using
FLAG-conjugated beads (Sigma).
RIP assay and RT-PCR
RNP immunoprecipitation (RIP) was performed as in Niranjanakumari et al.
(Niranjanakumari et al., 2002)
with modifications as follows: fractions from 5-40% sucrose gradients
containing the 20S Staufen complex were pooled and crosslinked in 0.1% to 0.2%
formaldehyde for 15 minutes at room temperature. Reactions were quenched in
0.25 M glycine and incubated for 15 minutes. Antibodies bound to beads were
mixed with 20 µg of competitor RNA for 30 minutes. XStau complexes were
immunoprecipitated for 1 hour and washed four times in NET-150. Crosslinking
was reversed by incubation at 70°C for 45 minutes. RT-PCR was performed
according to a previously published method
(LaBonne and Whitman, 1994
)
with minor modifications. Each sample was denatured for 10 minutes at
95°C, followed by 30 cycles (25 cycles for EF1
) of: 30 seconds at
95°C, 30 seconds at 55°C, and 30 seconds at 72°C. Primers used are
as follows: Vg1 (forward 5 '-CGATGACATCCACCCAACAC-3', reverse
5'-GAGGGTCACAGTCAGCAAGG-3'); VegT (forward
5'-CAAGTAAATGTGAGAAACCGTG-3', reverse
5'-CAAATACACACACATTTCCCGA-3')
(Zhang and King, 1996
); Xcat2
(forward 5'-GGCTGCGGGTTCTGCAGGAG-3', reverse
5'-GCCAGTCCCCCGAGGAGCCC-3'); Xwnt11 (forward
5'-ACAAAATGCAAGTGCCACGG-3', reverse
5'-TTGACAGCGTTCCACGATGG-3')
(Schroeder et al., 1999
); and
EF1
(forward 5'-CAGATTGGTGCTGGATATGC-3', reverse
5'-ACTGCCTTGATGACTCCTAG-3')
(Wilson and Melton, 1994
). The
accession number for the Xenopus Staufen sequence is AY342402.
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Results |
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XStau is associated with a kinesin motor
The presence of localized RNAs in the 20S XStau-containing complex
suggested that this RNP particle might be involved in RNA localization. To
investigate potential roles for motor proteins in late pathway vegetal
localization, we further defined the composition of the 20S XStau/Vg1 RNP by
immunoblotting gradient fractions for XStau, and for kinesin and dyein motor
proteins. As shown in Fig. 6A,
XStau (top) co-sediments with kinesin (middle), while dynein (bottom) migrates
elsewhere in the gradient. These results suggest a possible association
between Staufen and kinesin, and to investigate this interaction more
directly, we tested whether XStau and kinesin could be co-immunoprecipitated.
We first immunoprecipitated 20S XStau RNP-containing fractions with anti-XStau
and immunoblotted for kinesin (Fig.
6B, lane 2; top, SUK4). Indeed, a kinesin protein is
co-immunoprecipitated with XStau. Similarly, we immunoprecipitated with sea
urchin kinesin antibodies (SUK4), and immunoblotted with anti-XStau
(Fig. 6B, lane 4; bottom), and
found that XStau is also co-immunoprecipitated with kinesin. The SUK4
antibodies recognize conventional kinesin heavy chain
(Ingold et al., 1988;
Neighbors et al., 1988
;
Sawin et al., 1992
),
suggesting that the XStau-associated kinesin is kinesin I. These results
indicate that XStau is associated with a kinesin motor, which may provide a
mechanism for active transport of late pathway RNAs.
|
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Discussion |
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In this study, we have identified a Xenopus homolog of Staufen
(XStau) and provide evidence of a role in RNA localization during frog
oogenesis. We found that XStau is present in an RNP complex and associates
with localized RNAs and a kinesin motor. An interaction between Vg1 and XStau
is demonstrated by the findings that XStau is both colocalized with Vg1 RNA
and co-immunoprecipitated with Vg1 RNA (Figs
4,
5). Specificity of the
biochemical interaction is indicated as RNAs (Vg1 and VegT) transported by the
late pathway in Xenopus oocytes are co-immunoprecipitated with XStau,
while RNAs that localize through a different pathway (the METRO pathway) are
not (Fig. 5). The subcellular
distribution of XStau is also provocative; enrichment of XStau at the vegetal
cortex is coincident with the timing of late pathway RNA localization
(Fig. 3B). Strikingly, upon
injection of VLE RNA into stage III oocytes, XStau adopts a distribution that
is overlapping with the injected RNA undergoing localization to the vegetal
cortex. We interpret colocalization of XStau and VLE RNA as resulting from
recruitment of endogenous XStau to active localization complexes. More
importantly, we obtained functional evidence of a role for XStau in vegetal
RNA localization through in vivo interference experiments
(Fig. 7). We found that
expression of a dominant-negative (DN) version of XStau blocked VLE RNA
localization. The DN XStau contained dsRBDs 2-4
(Fig. 7A), and dsRBDs 3 and 4
are predicted to bind RNA most strongly
(Micklem et al., 2000). Thus,
the dominant effect of this protein may be due to binding of VLE RNA by DN
XStau, which may be defective in interaction with other components of the RNA
localization machinery. Indeed, we have found that XStau234 exhibits impaired
interaction with hnRNP I (Fig.
7F), a late pathway localization factor
(Cote et al., 1999
). Our
results provide evidence for interaction of XStau with late pathway RNAs both
in vitro and during localization in vivo, and reveal a role for XStau in
vegetal RNA localization in Xenopus oocytes.
Motor proteins have been implicated in transport of late pathway RNAs
because of the requirement for intact microtubules
(Yisraeli et al., 1990).
However, the nature of any such motors had remained elusive. Our results have
revealed that XStau is associated with kinesin I
(Fig. 6), and kinesin II has
recently been implicated with a role in Vg1 RNA localization
(Betley et al., 2004
).
Certainly, roles for kinesin I and kinesin II in vegetal RNA localization are
not mutually exclusive; each motor could have distinct functions in RNA
localization. Multiple kinesin-like proteins are expressed in Xenopus
(Boleti et al., 1996
;
Houliston et al., 1994
;
Le Bot et al., 1998
;
Tuma et al., 1998
;
Vernos et al., 1993
;
Vernos et al., 1995
), and we
have identified the XStau-associated kinesin as kinesin I based both on its
size (
120 kDa) and recognition by kinesin I-specific antibodies. The
antibodies we used to detect the XStau-associated kinesin, anti-sea urchin
SUK4, are specific for kinesin I (Ingold
et al., 1988
), and have been shown to recognize a kinesin I
doublet of
120 kDa in Xenopus extracts
(Neighbors et al., 1988
;
Sawin et al., 1992
). In
addition, a second kinesin I-specific antibody
(Romberg et al., 1998
)
recognized the XStau-associated kinesin as well (not shown). Based on these
findings, we propose that XStau interacts with kinesin I. In rat hippocampal
neurons, Staufen has been shown to co-sediment with kinesin I in gradients
(Mallardo et al., 2003
), and
kinesin I has also been linked to Staufen through genetic studies in
Drosophila. In the latter studies, mislocalization of Staufen protein
and oskar RNA resulted from deletion of kinesin heavy chain
(Brendza et al., 2000
). Our
biochemical results indicate that XStau and kinesin I interact within an RNP
complex that also contains vegetally localized RNAs. We propose that
interactions between Staufen and kinesin motors may be a conserved theme in
RNA localization pathways.
We have suggested that Staufen is a conserved feature in RNA localization;
paradoxically, however, the RNA targets appear rather unrelated. In flies,
Staufen has been suggested to interact with bicoid, oskar and
prospero RNAs (Broadus et al.,
1998; Ephrussi et al.,
1991
; Ferrandon et al.,
1994
; Kim-Ha et al.,
1991
; Li et al.,
1997
; Matsuzaki et al.,
1998
; Schuldt et al.,
1998
; Shen et al.,
1998
; St Johnston et al.,
1991
). In Xenopus, we have provided evidence of
association with the late pathway RNAs Vg1 and VegT. One cause for the range
of RNAs recognized by Staufen probably lies in the nature of the interaction
between dsRBDs and dsRNA, which is generally non-sequence specific
(St Johnston et al., 1992
).
Vg1 and VegT contain potentially double-stranded regions, but they are
specifically bound by XStau in vivo (Fig.
5B). So the question remains as to how Staufen could interact
specifically with disparate RNA targets. We propose that there are two classes
of RNA-binding factors involved in RNA localization. One class recognizes and
binds to RNA localization elements in a sequence-specific manner. Examples of
such factors in Xenopus include Vg1 RNA-binding proteins hnRNP I
(Cote et al., 1999
) and
Vg1RBP/vera (Deshler et al.,
1998
; Deshler et al.,
1997
; Havin et al.,
1998
; Schwartz et al.,
1992
). This class of factors may be cell-type specific and act to
establish a core ribonucleoprotein complex for transport. The other class of
factors, such as XStau, may act not at the level of sequence-specific RNA
recognition, but rather, recognize the core RNP complex and mediate the
interaction with the localization machinery. In such a model, some dsRBDs
would interact in a non-sequence specific manner with double-stranded regions
of RNA presented on the RNP, while other dsRBDs could interact with protein
components of the core RNP. Consistent with this idea, dsRBD2 and dsRBD5 of
Drosophila Staufen do not bind RNA in vitro, whereas dsRBD1, dsRBD3
and dsRBD4 bind dsRNA sequence nonspecifically
(Micklem et al., 2000
;
St Johnston et al., 1992
). We
have shown (Fig. 7) that
dominant-negative XStau234 is defective in interaction with hnRNP I,
suggesting that XStau dsRBD1 or dsRBD5 could potentially facilitate
interaction between XStau and hnRNP I. We suggest that this interaction is in
the context of an RNP, and we have previously shown that hnRNP I and
Vg1RBP/vera associate with Vg1 and VegT RNAs in the nucleus, prior to
recruitment of XStau to the cytoplasmic RNP
(Kress et al., 2004
). Our
observed biochemical interaction between XStau and kinesin
(Fig. 6) could further suggest
a role for XStau in motor recruitment, although this remains an issue for
future investigation. Thus, Staufen may represent a central component of the
RNA localization machinery, perhaps linking the localized RNP cargoes with the
motors that move them.
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ACKNOWLEDGMENTS |
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