Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
Author for correspondence (e-mail:
huber.1{at}nd.edu)
Accepted 18 September 2002
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SUMMARY |
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Key words: FUSE-binding protein, KSRP, Prrp, RNA localization, Vg1 mRNA, VgRBP71, ZBP2, Xenopus
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INTRODUCTION |
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It appears that the majority of localized mRNAs in a variety of cells from
yeast to neurons are organized in large particles
(Bertrand et al., 1998;
Ferrandon et al., 1994
;
Hoek et al., 1998
;
Ross et al., 1997
;
Schroeder and Yost, 1996
;
Shen et al., 1998
;
Wilhelm et al., 2000
). This
observation suggests that several trans-acting factors are required
to support this process, which usually requires concomitant translational
repression of the mRNA, at least until localization is complete. For example,
a minimum of five different proteins are required for the localization of
bicoid mRNA in Drosophila oocytes and an equal number of
trans-acting factors are involved in the localization of
Ash1 mRNA in yeast (Mohr and
Richter, 2001
). Six different polypeptides can be crosslinked to
the localization element of Vg1 mRNA upon UV irradiation, suggesting that a
multi-component complex also mediates transport of this RNA
(Mowry, 1996
). Three proteins
that bind to the VLE have been identified. The first, alternatively called Vg1
RBP or Vera, is a Xenopus homolog of chicken zipcode-binding protein,
which binds to and determines localization of ß-actin mRNA in fibroblasts
(Deshler et al., 1998
;
Havin et al., 1998
). Vg1
RBP/Vera appears to be necessary for the association of Vg1 mRNA with
microtubules (Elisha et al.,
1995
) and is itself associated with a subcompartment of the
endoplasmic reticulum (Deshler et al.,
1997
). A second factor, which is a homolog of hnRNP I, binds to
and colocalizes with Vg1 mRNA (Cote et al.,
1999
). Mutations in the VLE that prevent this interaction abolish
localization of the RNA. A proline-rich protein, dubbed Prrp, binds to Vg1
mRNA and is concentrated in the vegetal cortex of stage III/IV oocytes
(Zhao et al., 2001
). It also
interacts with the actin-associated protein profilin. Prrp can bind to other
mRNAs (e.g., VegT, An1, An3) that use the late pathways for localization in
Xenopus oocytes, but does not bind to mRNAs that use the early
pathway. The exact functions of these three factors have not been
determined.
Identification of all the components of the localization machinery is a
necessary step towards understanding how a particular mRNA is delivered to its
proper intracellular destination. We have screened a Xenopus oocyte
expression library for proteins that can bind to the localization element of
Vg1 mRNA and have identified the Xenopus homolog of a protein that
has been implicated in an extraordinary number of activities. Human far
upstream element (FUSE) binding protein, FBP, is a transcriptional activator
of the Myc gene (Duncan et al.,
1994). Two paralogs, FBP2 and FBP3, have been subsequently
identified, establishing the existence of a family of putative transcription
factors that differ substantially at the nucleotide level, but show
appreciable similarity in amino acid sequence
(Davis-Smyth et al., 1996
).
Although FBP was first identified as a single-stranded DNA binding protein, it
contains four K-homology (KH) RNA-binding domains, and members of the FBP
family have now been found in a variety of complexes that regulate alternative
splicing, stability and even editing of mRNA
(Grossman et al., 1998
;
Irwin et al., 1997
;
Lellek et al., 2000
;
Min et al., 1997
). Of
particular significance, an FBP2 homolog binds to the zipcode element of
ß-actin mRNA and affects its localization in fibroblasts and neurons
(Gu et al., 2002
).
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MATERIALS AND METHODS |
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Plasmids and nucleic acids
The plasmid pGEX-4T-2-c4 carries the complete coding sequence of VgRBP71
inserted into the BamHI and EcoRI restriction sites of the
vector. This plasmid was used for the expression of VgRBP71 as a glutathione
S-transferase fusion protein. A DNA fragment generated by PCR
amplification, which encodes the first 449 amino acids of VgRBP71, was
inserted into the NdeI and XhoI sites of pET-23b for
expression of the truncated polypeptide with a C-terminal polyhistidine tag.
The plasmid vectors used in the yeast two-hybrid assays were purchased from
Clontech. The 32P-labeled probe for the northern blot assay was
prepared from the SalI-NotI restriction fragment of a
VgRBP71 cDNA clone (pGEMEX-4) using the DECAprime II Random Priming
DNA Labeling Kit (Ambion). Plasmids containing the localization elements of
VegT mRNA (nt 1849-2294) (Kwon et al.,
2002
) and Xcat-2 mRNA (nt 393-748)
(Zhou and King, 1996
) or the
entire 3'UTR of An1 mRNA (nt 2268-2789) were constructed using RT-PCR
reactions. Total oocyte RNA was isolated from mixed staged oocytes using
RNAwiz (Ambion). First strand DNA synthesis used a combination of random
hexamers (Promega) and T16 (Roche) primers along with Thermoscript
reverse transcriptase (Invitrogen). The resulting cDNA was amplified with
specific primers and Taq DNA polymerase. The PCR products were ligated into
pCR2.1-TOPO (Invitrogen). The An1 fragment was subsequently subcloned into the
BamHI and XhoI sites of pBSKSII(+) (Stratagene). RNA was
synthesized by run-off transcription using phage T7 RNA polymerase and
plasmids linearized with either BamHI (VegT and Xcat-2) or
XhoI (An1).
Antibody
Antiserum was prepared against the GST-VgRBP71 fusion protein and antibody
purified by affinity chromatography. A polypeptide containing the first 449
amino acids of VgRBP71 (10 mg) was coupled to a HiTrap column (Amersham
Pharmacia); serum (10 ml) was applied to the column and bound antibody eluted
as described previously (Harlow and Lane,
1988). The affinity-purified antibody showed very little
cross-reaction with proteins in extract prepared from oocytes. Most
importantly, there is no cross-reaction with the Xenopus homolog of
FBP, which has a predicted molecular mass of 64.8 kDa. Anti-hemagglutinin
antibody was purchased from Clonetech.
RNA-binding assays
In vivo assays were performed essentially as described before
(Zhao et al., 2001), except
that the VgRBP71 RNP complexes were immunoprecipitated from oocyte homogenate
using affinity-purified, polyclonal antibody prepared against VgRBP71. In
vitro RNA-binding assays (10 µl) were in buffer containing 40 mM HEPES (pH
7.5), 100 mM KCl, 1 mM MgCl2, 1 mM DTT, 5% glycerol, 0.2 mg/ml
yeast tRNA, 0.2 mg/ml heparin and 4 U RNasin (Promega). Each reaction
contained 1 nM internally radiolabeled VLE RNA and the indicated amount of
unlabeled competitor RNA. VgRBP71 and RNA was incubated for 2 hours on ice and
then separated by electrophoresis on 7% polyacrylamide (40:1,
acrylamide/bisacrylamide) gels at 200 V at ambient temperature.
Confocal microscopy
Albino oocytes were treated with type II collagenase (1 mg/ml) in
OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM
Na2HPO4, 5 mM HEPES, pH 7.8) (Opresko, 1991) for 1 hour
at 28°C, and rinsed in Modified Barth's Solution-High Salt (MBSH: 110 mM
NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM NaHCO3, 0.5 mM
Na2HPO4, 15 mM Tris-HCl, pH 7.6). The oocytes were fixed
for at least two hours in MEMFA (2 mM EGTA, 1 mM MgSO4, 3.7%
formaldehyde, 0.1 M MOPS, pH 7.4)
(Hemmati-Brivanlou et al.,
1990) and rinsed with four changes of TBS (137 mM NaCl, 2.7 mM
KCl, 25 mM Tris-HCl, pH 7.4). The oocytes were then equilibrated in TBSN (TBS
+ 0.2% NP-40) for 5 minutes. The equilibration buffer was removed and the
oocytes were incubated in 0.5 ml TBSN containing 2% BSA and 10 µl rabbit
anti-VgRBP71 antibody for 24 hours at room temperature with gentle shaking.
Control oocytes were processed in the same fashion in the absence of the
primary antibody. Following the incubation with antibody, oocytes were washed
in TBS for 24 hours; the wash buffer was changed every 6-8 hours. The oocytes
were then stained for 24 hours in 0.5 ml TBSN containing 2% BSA and 5 µl
goat anti-rabbit antibody conjugated to Alexa Fluor 568 (Molecular Probes).
Unbound secondary antibody was removed by washing the oocytes at room
temperature with gentle shaking for a total of 24 hours in TBS with the buffer
changed every 6-8 hours. The oocytes were then dehydrated using several
changes of methanol, cleared with benzyl benzoate:benzyl alcohol (2:1, v/v)
and analyzed by confocal microscopy (Dent
et al., 1989
). Oocytes to be bisected were collected, washed,
fixed and rinsed as described above. Individual oocytes were placed on a glass
slide and sliced in half with a #10 scalpel using a single quick motion. A
drop of buffer was then added to the oocyte halves, which were then
transferred to a 24-well plate containing TBS. The samples were rinsed to
remove debris from the sectioning and then processed in the same fashion as
whole oocytes. A BioRad MRC 1024 scanning confocal system attached to a Nikon
Diaphot 200 inverted microscope was used to collect images of stained
oocytes.
Yeast two-hybrid assays
Matchmaker Two-Hybrid System 3 was used to screen a Xenopus oocyte
cDNA library cloned into the vector pACT2 (Clontech). The bait plasmids
contained the complete, the N-terminal (amino acids 1-251) or the C-terminal
(amino acids 242-360) coding sequences of Prrp inserted into the vector,
pGBKT7. The complete coding sequence of VgRBP71, the N-terminal region
encompassing the four KH domains (amino acids 1-449) or the C-terminal domain
containing the tyrosine-rich repeats (amino acids 450-672) were inserted into
the prey vector, pGADT7. Protein expression was verified in each case by
western blot assays using the Myc or HA epitope encoded in the bait or prey
vector, respectively. The bait plasmid carrying Prrp exhibited no autonomous
activation of reporter genes on its own in the host strain (AH109). However,
this plasmid does slow the growth of these cells, so bait and prey plasmids
were transformed together. The transformation mixtures were spread on plates
containing synthetic drop-out (SD) medium lacking adenine, histidine, leucine
and tryptophan and kept at 30°C for 4 days. In the case of the library
screen, colonies were transferred onto SD medium lacking leucine and
tryptophan in order to eliminate multiple prey plasmids within any given
colony. After this second round of selection, colonies were spotted onto SD
plates for a colony lift assay to measure ß-galactosidase activity
(Breeden and Nasmyth, 1985).
Colony lift assays were performed in triplicate, in order to estimate the
strength of the interaction between individual pairs of proteins. Prey
plasmids identified in the screen were initially classified based upon
restriction enzyme digestions. Representative clones from these groups were
sequenced, and from this latter group several were chosen and used as probes
in southern blot assays. Several of the positives clones identified by
southern hybridization were confirmed by DNA sequencing.
Coimmunoprecipitation assays
VgRBP71 carrying an HA epitope (pGADT7-KSRP) and Prrp carrying a
Myc epitope (pGBKT7-Prrp) were synthesized using the TnT T7 Coupled
Reticulocyte Lysate System (Promega). The reactions were run at 30°C for
90 minutes and used directly for immunoprecipitation reactions. Aliquots of
each protein synthesis reaction (15 µl) were added to 150 µl of NET-2
buffer (150 mM NaCl, 0.05% NP-40, 50 mM Tris-HCl, pH 7.4) containing protein-A
Sepharose coated with rabbit anti-HA antibody (Clontech). Negative controls
lacked either VgRBP71 or anti-HA antibody. The effect of RNA on the
VgRBP71:Prrp interaction was tested by treating aliquots of the two proteins
with 1 µl RNase A (10 mg/ml) for 10 minutes at 37°C prior to incubation
with the antibody coated protein-A Sepharose beads. The protein mixtures were
incubated with the beads for 1.5 hours at 4°C with end-to-end rotation.
The beads were collected by brief centrifugation, washed three times with 200
µl NET-2 buffer containing 1 mM PMSF and 1 µg/ml pepstatin. The samples
were suspended in SDS/dye solution, placed at 100°C for 5 minutes, and
separated on a 10% SDS polyacrylamide gel. The proteins were transferred to a
nitrocellulose filter that was exposed to X-ray film. For
co-immunoprecipitation assays from oocyte extract, [35S]-labeled
Prrp was synthesized from pGBKT7-Prrp in reticulocyte lysate. Approximately
100 stage III/IV oocytes were injected with 25 nl of reticulocyte lysate and
incubated overnight in MBSH solution at 18°C. Oocytes were homogenized in
NET-2 buffer and cleared in a microcentrifuge. Equal amounts of lysate were
incubated with protein A-Sepharose beads carrying affinity-purified VgRBP71
antibody or with protein A-Sepharose beads alone for 2 hours at 4°C with
gentle rotation. The supernatant was removed and the beads were washed four
times with NET-2 buffer. The samples were processed as described for the in
vitro assays.
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RESULTS |
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FBP was first identified as a single-stranded DNA-binding protein that
binds to an upstream control element (FUSE) of the human MYC gene
(Duncan et al., 1994). The
central region of the protein has four KH domains, which are capable of
binding to either RNA or single-stranded DNA. The C-terminal region of FBP2
has four repeats of a tyrosine-rich motif that can function independently as a
transcriptional activation domain
(Davis-Smyth et al., 1996
).
There are at least three members of the FBP family that exhibit appreciable
similarity in amino acid sequence, but that differ substantially at the
nucleotide level due to differences in codon usage
(Davis-Smyth et al., 1996
).
FBP2 has been independently identified in a human neural cell line as a
splicing regulatory factor called KSRP, which is a component of a multiprotein
complex that forms on an intronic splicing enhancer in Src pre-mRNA
(Min et al., 1997). KSRP has a
segment of 58 amino acids beginning at residue 9 that is not present in the
Xenopus sequence. Whether this is a true difference between the human
and Xenopus FBP2 genes or is a consequence of alternative splicing
needs to be established, as the presence or absence of this sequence could
determine the specialized functions of these proteins. The 58 amino acid
sequence specific to KSRP was not found in a search of the Xenopus
EST database. The complete sequence of human FBP2 has not been reported, so it
is not known whether this N-terminal segment is present in this form of the
transcription factor. Because of this difference between the Xenopus
protein and KSRP, we refer to the former as VgRBP71 (Vg1 RNA-binding protein
71) in accordance with the convention used to designate other proteins that
bind to the VLE.
Gu et al. (Gu et al., 2002)
recently reported that a chicken homolog of FBP2/KSRP binds to the
localization element (the zipcode) of ß-actin mRNA in fibroblasts and
neurons. Similar to the case with VgRBP71, the only substantial differences
between the chicken protein, called zipcode binding protein 2 (ZBP2), and KSRP
occur in the N-terminal regions. Whereas VgRBP71 is missing a 58 amino acid
segment relative to KSRP, ZBP2 has a 47 amino acid insertion
(Fig. 1). The amino acid
sequences of VgRBP71 and ZBP2 have 73% identity and 78% similarity.
A search of the sequence databases using BLAST2 revealed that a rat
protein, which binds to the localization element in the mRNA encoding
microtubule-associated protein 2 (MAP2), is also a homolog of FBP2. MARTA1,
along with a second protein, binds to a targeting element in the 3'UTR
of MAP2 mRNA that mediates localization to dendritic regions in primary
neurons (Rehbein et al.,
2000). The rat sequence has 97% and 78% identity with the human
and Xenopus sequences, respectively. The important conclusion to be
drawn from these observations is that the cell-type specific localization of
mRNAs encoding unrelated proteins, nonetheless, uses some conserved factors
common to this process.
The remaining single clone isolated in the screen of the library is 81% identical to human FBP, the founding member of this family. The nucleotide sequence of the Xenopus FBP homolog identified in this work can be found at DDBJ/EMBL/GenBank accession number AF533514. Whereas the overall amino acid identity of Xenopus FBP and FBP2 is only 57%, the sequences within the four KH domains of the two proteins average about 70% identity. This may explain why a clone of the former was identified in the VLE-binding screen: FBP and FBP2 possibly share some overlapping RNA-binding specificity.
VgRBP71 binds to the VLE with high affinity and specificity
The goal of the screen was to identify proteins that bind to the
localization element of Vg1 mRNA. The affinity and specificity of binding to
VLE RNA was determined using mobility shift assays with VgRBP71 expressed as a
fusion protein with glutathione S-transferase. The dissociation constant for
the VgRBP71-VLE complex was estimated from the concentration of protein needed
to reach half-saturation in binding titration assays (data not shown). The
apparent Kd in optimized buffer conditions is 5 nM.
The specificity of binding was tested by incubating a constant amount of
VgRBP71 and radiolabeled VLE RNA with increasing concentrations of competing,
unlabeled VLE RNA (Fig. 2,
lanes 2-6) or a 355 nucleotide non-cognate RNA (lanes 7-10) transcribed from
the plasmid pBSIISK. VLE RNA competes efficiently against itself in this
assay; whereas, in the presence of a 100-fold excess of non-cognate RNA,
55% of the VgRBP71-VLE complex still remains intact. These assays
establish that the interaction is specific and that binding affinity is
comparable with other RNA-binding proteins that have KH domains
(Buckanovich and Darnell, 1997
;
Chkheidze et al., 1999
;
Lin et al., 1997
).
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Temporal expression of VgRBP71
There are two temporally distinct pathways for the localization of vegetal
RNAs in Xenopus oocytes
(Forristall et al., 1995;
Kloc and Etkin, 1995
). The
early pathway operates during stages I and II of oogenesis; whereas, the late
pathway, used by Vg1 mRNA, is active during stages III and IV. Oocytes were
separated into three groups according to the Dumont stages
(Dumont, 1972
) and the levels
of mRNA encoding VgRBP71 measured by northern hybridization
(Fig. 3A). The amount of
VgRBP71 mRNA in stage I to stage II oocytes is low; however, there is a
substantial increase at stage III to stage IV that is maintained for the
remainder of oogenesis. We observed a similar increase in the level of Prrp
mRNA at stage III (Zhao et al.,
2001
). The single transcript seen in the northern blot is
approximately 2700 nucleotides, which matches the length expected from
sequencing and cRACE determinations.
|
The levels of VgRBP71 protein were measured in western blot assays and they
mirror those of the mRNA (Fig.
3B). There is very little VgRBP71 detected prior to stage III, at
which time the amount of protein increases substantially and then remains
fairly constant. Thus, there is a correlation between the temporal expression
of VgRBP71 and the time at which localization of Vg1 mRNA takes place.
Expression of VgRBP71 continues beyond oogenesis. There is another increase in
the level of the protein upon oocyte maturation, after which it remains
constant within the developing embryo. This observation is consistent with the
assignment of other functions to orthologs of this protein that are not
limited to oocytes, such as roles in alternative splicing in neurons
(Min et al., 1997), as well as
mRNA localization in fibroblasts and neurons
(Gu et al., 2002
). It is
important to note that the antibody used in these studies was affinity
purified and showed no detectable cross-reaction with Xenopus
FBP.
VgRBP71 is associated with other localized mRNAs
We tested for the in vivo association of VgRBP71 with Vg1 mRNA and other
localized mRNAs using an immunoprecipitation assay. Oocytes were manually
disrupted and whole cell extract incubated with anti-VgRBP71 antibody
immobilized on protein A-Sepharose. RNA specifically retained on these beads
after multiple washes was detected by an RT-PCR assay using gene-specific
primers (Fig. 4). A standard
for each RNA was generated by amplifying cDNA prepared from total oocyte RNA
(lanes marked PCR control). In no case did protein A-Sepharose beads alone
yield a PCR product (antibody). However, beads adsorbed with pre-immune
serum in some cases did give a small amount of product that, nonetheless,
could be distinguished from a specific signal.
|
These assays detected an association of VgRBP71 with Vg1 mRNA as well as
with VegT mRNA, which also uses the late pathway for localization.
Interestingly, there is also a distinct PCR product for Xcat2 mRNA.
This mRNA is normally localized through the early pathway; however, when it is
injected into stage IV oocytes, it can be localized through the late pathway
(Zhou and King, 1996). This
latter observation indicates that Xcat2 mRNA can associate with at
least one or more trans-acting factors of the late pathway, one of
which is apparently VgRBP71. Xwnt11 mRNA, which is also localized to the
vegetal pole by the early pathway, gave an extremely weak signal in this
assay. Because the levels of VgRBP71 are low during the operation of the early
pathway, it seems unlikely that it would be found with Xwnt11 mRNA.
The latter result brings up an important caveat concerning this assay. It
cannot be assumed that immunoprecipitation in this case is quantitative.
Recovery of individual complexes is likely to be influenced by several
factors, including subcellular location, association with other cellular
structures and accessibility of epitopes in any given complex to the
antibody.
VgRBP71 also binds to two mRNAs (An1 and An3) that are localized to the animal hemisphere. Thus, the possibility arises that VgRBP71 is an RNA-binding protein that is generally associated with mRNA in Xenopus oocytes. To address this point, we tested for binding of the protein to mRNAs encoding actin and transcription factor IIIA (Fig. 4) and to ribosomal protein L5 and profilin (data not shown), which are not known to be localized. These mRNAs are abundant in Xenopus oocytes, making it likely that we could detect any association with VgRBP71. There was no PCR product generated for these four mRNAs. Together, these results indicate that VgRBP71 is associated with localized mRNAs in Xenopus oocytes, but that it is not specific to a particular directional (vegetal/animal) pathway.
The apparent association of VgRBP71 with several different localized mRNAs
in these assays presents the question of whether this factor binds directly to
all of them or whether, in some cases, it is due to protein-protein
interactions within a larger RNP complex. The only other cis-acting
elements, which control localization through the late pathway, that have been
identified are for VegT (Bubunenko et al.,
2002; Kwon et al.,
2002
) and Xcat2 (Zhou and
King, 1996
) mRNAs. We synthesized RNAs containing these segments
as well as the entire 3'UTR of An1 mRNA (579 nt) and used them for in
vitro binding assays. In order to compare directly the binding of each of
these RNAs to VgRBP71, we once again used competition assays
(Fig. 5). The competition
strength of the VegT and An1 localization elements are very similar to the
VLE, establishing that VgRBP71 binds directly to these RNAs with high
affinity. The apparent affinity of VgRBP71 for Xcat2 is somewhat less than its
affinity for the other RNAs, but is distinctly greater than the nonspecific
RNA control. This result may reflect the fact that Xcat2 is normally
transported through the early pathway, but can use the late pathway when
injected into stage III oocytes. It is possible that the interaction of
VgRBP71 with Xcat2 mRNA in vivo may be facilitated by other factors that bind
to the localization element of the latter. Although we find that the binding
of VgRBP71 to these other localization elements and to VLE RNA is mutually
exclusive, this does not mean that the complexes are identical or even
similar. Alternative combinations of KH domains within the protein could
mediate interactions with the different RNAs. Moreover, these results do not
exclude the possibility that an interaction with other proteins helps to
recruit VgRBP71 to these mRNAs in vivo.
|
Intracellular location of VgRBP71 during oogenesis
The nuclear function of human FBP is well documented
(Davis-Smyth et al., 1996;
Liu et al., 2001
) and the
protein appears to have three distinct nuclear localization elements
(He et al., 2000
), which are
also present in VgRBP71. All of the FBP proteins contain four KH domains, an
RNA-binding motif that was first identified in hnRNP K
(Siomi et al., 1993
). The
latter belongs to a family of hnRNP proteins that shuttle between the nucleus
and cytoplasm. Human hnRNP K contains a bipartite-basic nuclear localization
signal (NLS) as well as a second domain, termed KNS, that mediates
bi-directional movement of the protein across the nuclear envelope
(Michael et al., 1997
).
VgRBP71, however, does not contain a KNS domain or other known shuttling
sequences such as M9 (Michael et al.,
1995
) or HNS (Fan and Steitz,
1998
). Additionally, there is no identifiable nuclear export
sequence (NES) in the protein.
Confocal fluorescence microscopy was used to examine the intracellular
distribution of VgRBP71 during oogenesis and, most importantly, to determine
whether any amount of the protein is located in the cytoplasm, consistent with
its binding to localized mRNAs (Fig.
6). In accord with the northern
(Fig. 3A) and western
(Fig. 3B) assays, there is
little VgRBP71 detected in stage I and II oocytes. The protein that is present
in early stage oocytes is positioned evenly around the cell cortex and
uniformly through the nucleus. The marked increase of VgRBP71 at stage III is
most conspicuous in the nucleus; however, there is a concomitant increase in
the cytoplasm as well. Although still enriched in the cortex, VgRBP71 is found
in appreciable amounts throughout the cytoplasm of stage III oocytes. Although
much of the protein remains nuclear at stage IV, the cytoplasmic distribution
has changed somewhat. There is an enrichment of the protein in the animal
hemisphere in the region between the nucleus and the cortex. In order to
improve the immunochemical staining, a stage IV oocyte was bisected along the
animal-vegetal axis prior to incubation with the primary antibody
(Fig. 6, IV*).
Although the nucleus is lost during this procedure, the manipulation improves
the staining of the oocyte and yields considerably greater detail. The
distinct cortical enrichment is still observed, but a punctate cytoplasmic
distribution is more apparent. By stages V and VI, cytoplasmic VgRBP71 is
concentrated at the periphery of the oocyte with a modest excess in the animal
hemisphere relative to the vegetal. The amount of protein in the nucleus
remains high. A western blot assay with manually dissected, staged oocytes has
confirmed the nucleocytoplasmic distribution of VgRBP71 during oogenesis
(results not shown). The confocal images establish that a significant amount
of VgRBP71 resides in the cytoplasm, consistent with its ability to bind to
localized mRNAs. Correspondingly, about 10% of total cellular ZBP2 is located
in the cytoplasm of fibroblasts and neurons
(Gu et al., 2002).
|
The cytoplasmic distribution of VgRBP71 is different from the other three
VLE-binding proteins that have been characterized so far. Vg1 RBP/Vera,
VgRBP60/hnRNP I, and Prrp all exhibit a clear co-localization with Vg1 mRNA at
the vegetal cortex in stage IV oocytes, although VgRBP60 also shows diffuse
staining in the animal hemisphere (Cote et
al., 1999). VgRBP71, however, ultimately accumulates in a band
around the entire periphery of the oocyte. This disposition is in accordance
with the ability of the protein to bind to mRNAs that are localized to either
hemisphere of the oocyte, further supporting the idea that VgRBP71 does not
confer directional specificity. In fact, these results suggest that it
functions in some general aspect of localization, which could include
associated activities such as translational control or stabilization of
localized mRNAs, rather than transport itself.
VgRBP71 interacts with Prrp
The first protein identified in the screen of the expression library with
VLE RNA was a novel proline-rich hnRNP protein called Prrp
(Zhao et al., 2001). This
protein co-localizes with Vg1 mRNA in stage IV oocytes and can bind to other
mRNAs that use the late pathway for localization (e.g. VegT). The proline-rich
domain of Prrp interacts with profilin, an actin-associated protein that
appears to regulate microfilament assembly
(Schlüter et al., 1997
),
suggesting that the protein may play a role in anchoring Vg1 mRNA to the
vegetal cortex. We have used Prrp as the bait in a yeast two-hybrid screen of
a Xenopus oocyte library in an effort to identify other components of
the RNP complex that forms on the VLE or factors that otherwise associate with
this complex. A screen of 3.5x106 colonies yielded 59 that
exhibited ß-galactosidase activity. Of these positive clones, at least 15
encode FBP2 (VgRBP71) and three encode FBP. Thus, two proteins, Prrp and
VgRBP71, identified independently by their ability to bind to the localization
element of Vg1 mRNA, appear to interact with each other. Neither Vg1 RBP/Vera
nor VgRBP60 were found in this screen. However, Prrp itself was recovered
three times, suggesting that it may form dimers or other higher order
structures that are typical of many hnRNP proteins
(Kim et al., 2000
). Most of
the remaining positive clones contain unique sequences (based upon restriction
enzyme digestions and southern blot analysis) that are either not found in the
sequence databases or that we have not yet sequenced.
The two-hybrid assay was also used to determine which domains in Prrp and VgRBP71 mediate their association. The results reveal that two segments in each protein are involved in this interaction. The N-terminal half of Prrp (amino acids 1-251) contains two RNP domains that interact with the N-terminal segment of VgRBP71 (amino acids 1-449) that extends through the four KH domains of that protein (Fig. 7A, lane 6). In addition, the C-terminal of Prrp (amino acids 242-360), which contains several polyproline repeats, and the C-terminal of VgRBP71 (amino acids 450-672), also interact (Fig. 7A, lane 9). The other combinations of bait and prey were negative.
|
We also tested the association of Prrp with VgRBP71 using immunoprecipitation assays (Fig. 7B). VgRBP71, carrying a hemagglutinin (HA) epitope, and Prrp were expressed individually in rabbit reticulocyte lysate; Prrp was labeled with [35S]. Aliquots of both proteins were mixed together and then incubated with protein A-Sepharose beads coated with anti-HA antibody. The precipitate was analyzed by SDS gel electrophoresis followed by autoradiography. The immunoprecipitation of VgRBP71 also brings down Prrp confirming an interaction between the two (lane 1). Prrp was not recovered with protein A-Sepharose beads that had not been coated with antibody (lane 2) nor was it recovered in the absence of VgRBP71 (lane 3). Because both Prrp and VgRBP71 bind to the VLE, there is some possibility that their apparent association is mediated by RNA and is not direct. This seemed unlikely, as the C-terminal segments of the two proteins, which lack RNA-binding domains, give a positive signal in the two-hybrid assay. Nonetheless, this possibility was tested in the co-immunoprecipitation assays. Samples of Prrp and VgRBP71 were treated with RNase A prior to mixing together. This treatment had no effect on the recovery of Prrp with VgRBP71 (results not shown).
An immunoprecipitation assay was also used to determine whether these two proteins interact in oocytes (Fig. 7C). Immunoprecipitation of VgRBP71 from oocyte homogenate followed by a western blot using Prrp antiserum was inconclusive because of interference from the first (VgRBP71-specific) antibody. In order to circumvent this problem, stage III/IV oocytes were injected with [35S] labeled Prrp and kept overnight. VgRBP71 was retrieved from extract prepared from these oocytes using affinity-purified antibody bound to protein A-Sepharose. The immunoprecipitate was analyzed by electrophoresis on an SDS polyacrylamide gel followed by autoradiography. Prrp co-precipitated from oocyte extract with VgRBP71 (Fig. 7C, lane 3), demonstrating an association of these two factors in vivo.
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DISCUSSION |
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There is only circumstantial information on the function of these proteins.
Vg1 RBP/Vera is required for the association of Vg1 mRNA with microtubules
(Elisha et al., 1995), which
may either be direct or mediated through a specialized region of the
endoplasmic reticulum (Deshler et al.,
1997
). The ability of Prrp to bind to profilin suggests that this
protein may be involved in the actin-dependent anchoring of Vg1 mRNA in the
vegetal cortex (Zhao et al.,
2001
). A complete description of Vg1 mRNA localization will
require the identification of all the factors that assemble on the VLE. By
extension, identification of the binding partners of these proteins should
provide clues to their individual functions. Thus, the indications that Vg1
RBP/Vera is microtubule associated and Prrp is microfilament associated
suggest roles in translocation and anchoring, respectively.
We have identified a member of the FBP family of proteins, specifically a
homolog of FBP2, that binds to the VLE. This protein is a general factor that
can associate with mRNAs localized to either the animal or vegetal hemispheres
of the oocyte. Interestingly, the sequence of the DNA-binding site for FBP,
found upstream of the human MYC gene, is present near the 3'
end of the VLE (nt 243-269); however, this sequence does not occur in the
3'UTR of other localized mRNAs to which VgRBP71 binds. ZBP2, which is a
chicken homolog of VgRBP71, binds to the 54 nt zipcode element in ß-actin
mRNA (Gu et al., 2002). A
comparison of the sequence of the zipcode with the VLE did not reveal any
appreciable alignment. The binding of VgRBP71 to its cognate sites, then, may
be structure rather than sequence-dependent. It is also possible that
different RNAs utilize different KH domains in VgRBP71 and, thus, present
different identity elements for recognition by the protein. For example, the
binding of human FBP to FUSE DNA is determined primarily by KH domains 3 and 4
(Braddock et al., 2002
;
Duncan et al., 1994
).
What constitutes a functional localization signal in the mRNAs that are
sorted in Xenopus oocytes is not yet apparent. The localization
element of VegT mRNA, which also uses the late pathway for movement to the
vegetal cortex, has recently been delineated by two groups
(Bubunenko et al., 2002;
Kwon et al., 2002
). Despite
little apparent conservation in primary sequence or predicted secondary
structure, the localization signals of Vg1 and VegT mRNAs seem to have a
similar functional organization and bind the same set of proteins
(Bubunenko et al., 2002
). The
3'UTR of Vg1 mRNA contains four different repeated sequence elements
(Deshler et al., 1997
);
repeats of one of these sequences, the 5-nt E2 element, also occur in the
3'UTR of VegT mRNA (Kwon et al.,
2002
). The E2 sequence is the recognition element used by Vera/Vg1
RBP and these repeats are necessary for the localization of Vg1 and VegT mRNAs
(Bubunenko et al., 2002
;
Kwon et al., 2002
). A second
sequence element, VM1, which is the binding site for hnRNP I
(Cote et al., 1999
), may also
be needed for efficient localization of these mRNAs to the vegetal cortex
(Bubunenko et al., 2002
). The
sequences required for translocation and anchoring of Xcat2 mRNA to
the vegetal cortex through the late pathway are within the first 150 nt and
the last 120 nt of the 3'UTR. The former sequence has a single E2 and
two VM1 motifs; otherwise, the 3'UTR of Vg1 and Xcat-2 mRNAs exhibit no
segments of notable sequence identity. Surprisingly, the 3'UTR of An1
contains three repeats of the short E2 element and nine variants of the VM1
motif. Bubunenko et al. (Bubunenko et al.,
2002
) have suggested that localization elements may not be defined
so much by nucleotide sequence or secondary structure, but rather the
strategic placement of multiple protein-binding motifs that assemble a common
group of trans-acting factors. This scenario has the potential to
account for the binding of the same set of proteins to localization signals
that have very little sequence similarity. This model also implies that
protein-protein interactions also play an important role in the assembly of
the RNP complexes that form on the localization element. Thus, simple
comparative sequence analysis may not immediately reveal the localization
signals used by the multiple trans-acting factors that participate in
the localization process. In accordance with these ideas, the in vitro binding
assays demonstrate that VgRBP71 can bind directly to several different
localization signals. In the case of Xcat2, other factors may
strengthen the association of VgRBP71 with this RNA when it anomalously uses
the late pathway.
Members of the FBP family have been implicated in an extraordinary array of
disparate activities. The founding member was first identified as a
transcriptional activator of Myc
(Duncan et al., 1994) and
subsequent members, FBP2 and FBP3, also contain tyrosine-rich C-terminal
domains that are potent activation domains
(Davis-Smyth et al., 1996
).
KSRP/FBP2 is part of a complex of proteins that binds to an element that
regulates the tissue-specific splicing of Src mRNA
(Min et al., 1997
).
Interestingly, another component of this splicing complex is polypyrimidine
tract binding protein (PTB), a homolog of hnRNP I
(Markovtsov et al., 2000
).
Thus, two proteins that bind to the Vg1 VLE are found in this splicing
complex. Although there is no evidence for a direct interaction between hnRNP
I and KSRP, binding of the latter to the splicing enhancer element required
hnRNP H and hnRNP I. Similarly, a multi-protein complex that regulates the
alternative splicing of ß-tropomyosin also contains PTB/hnRNP I and FBP
(Kaminski and Jackson, 1998
).
These observations raise the question whether some components of nuclear hnRNP
complexes involved in RNA processing can, in some cases, have a distinct
second role in the cytoplasm. The localization of fushi tarazu mRNA
in Drosophila embryos requires prior exposure of the transcripts to
nuclear components, which lead to the proposal that cytoplasmic localization
uses hnRNP proteins that become associated with the mRNA in the nucleus
(Lall et al., 1999
). It is
possible to imagine that these hnRNP complexes are dynamic with their
composition changing as the mRNA is processed, exported from the nucleus,
translocated and positioned in the cytoplasm, yet with some subset of proteins
remaining associated with the RNA through several, if not all, steps.
Members of the FBP family have been identified in other nuclear and
cytoplasmic processes. An FBP homolog binds to an element in the 3'UTR
of the mRNA encoding the neuronal phosphoprotein, GAP43; this sequence
controls the stability of the RNA (Irwin
et al., 1997). Once again, homologs of FBP and hnRNP I are
functionally connected. However, in this case, binding of the two proteins to
the stability element appears to be mutually exclusive, suggesting that they
have counteracting activities. Perhaps the most surprising discovery is that a
FBP2/KSRP ortholog is a component of the enzyme complex that edits
apolipoprotein B mRNA (Lellek et al.,
2000
). It is not obvious how these various activities are related
or whether they need to be. However, sequencing of peptides derived from
purified human DNA helicase V revealed that it is FBP; notably, this protein
preparation also worked as an RNA helicase
(Vindigni et al., 2001
). If
FBP or any of its homologs are truly RNA helicases, it may be that the protein
is involved in the assembly or remodeling of RNP complexes on mRNA. This might
then explain the widespread nucleocytoplasmic distribution of this protein, at
least in Xenopus oocytes.
A search of the sequence databases revealed that VgRBP71 is also a homolog
of rat MARTA1, which is one of two trans-acting factors that bind to
a localization element in the 3'UTR of rat MAP2 mRNA, which directs it
to dendritic compartments (Rehbein et al.,
2000). As is the case with Xenopus VgRBP71, MARTA1 is
found in the cytoplasm and the nucleus; additionally, the dissociation
constant of the MARTA1:MAP2 mRNA complex is the same as that determined here
for the VgRBP71:VLE complex. These similarities imply that VgRBP71
participates in some general aspect of mRNA localization and may account for
its association with several different localized mRNAs in Xenopus
oocytes, irrespective of their final intracellular destination. This idea is
reinforced by the recent identification of ZBP2, which mediates localization
of ß-actin mRNA to the leading edge of embryonic fibroblasts and the
growth cones of developing neurons (Gu et
al., 2002
). The two identified factors that bind to the zipcode
element, ZBP1 and ZBP2, are orthologs of two of the proteins that bind to the
VLE, Vg1 RBP/Vera and VgRBP71, respectively. Thus, there is an extraordinary
conservation of proteins involved in RNA localization that extends across
different cell types and species.
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ACKNOWLEDGMENTS |
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Footnotes |
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