ABSTRACT
In investigating the composition of stored (maternal) mRNP particles in
Xenopus
oocytes, attention has focussed primarily on the phosphoproteins pp60/56, which
are Y-box proteins involved in a general packaging of mRNA. We now identify a third, abundant, integral component of stored
mRNP particles, Xp54, which belongs to the family of DEAD-box RNA helicases. Xp54 was first detected by its ability to
photocrosslink ATP. Subsequent sequence analysis identifies Xp54 as a member of
a helicase subfamily which includes: human p54, encoded at a chromosomal
breakpoint in the B-cell lymphoma cell line, RC-K8;
Drosophila
ME31B, encoded by a maternally-expressed gene, and
Saccharomyces pombe
Ste13, cloned by complementation of the sterility mutant
ste13
.
Expression studies reveal that the gene encoding Xp54 is transcribed maximally
at early oogenesis: no transcripts are detected in adult tissues, other than
ovary. Using a monospecific antibody raised against native Xp54, its presence
in mRNP particles is confirmed by immunoblotting fractions bound to oligo(dT)-cellulose and separated by rate sedimentation and buoyant density. On
isolating Xp54 from mRNP particles, it is shown to possess an ATP-dependent RNA helicase activity. Possible functions of Xp54 are discussed
in relation to the assembly and utilization of mRNP particles.
For many years, it has been apparent that pools of non-translating, non-polysomal or `free' mRNP particles exist in the cytoplasm of
eukaryotic cells, being particularly prominent in the oocytes of many
organisms, including
Xenopus
(
1
). Oogenesis in
Xenopus
occurs over several months, during which time each oocyte accumulates a vast
pool of ~10
12
ribosomes and 2 * 10
11
stored mRNP complexes, eventually brought together for translation at oocyte
maturation, fertilization and early embryogenesis (
2
). During the rapid cell divisions which follow fertilization, transcription is
severely limited (
3
), and only after the mid-blastula transition is zygotic transcription initiated (
4
). Thus, the embryo relies on the pool of stored, maternal mRNP to drive the
early stages of its development.
The mRNP particles isolated from
Xenopus
oocytes contain a set of at least four abundant proteins with apparent
molecular masses on SDS-PAGE in the range of 50-60 kDa (
5
). The two proteins of largest apparent mass, pp60/mRNP4 and pp56/mRNP3, have
been characterized as `masking' proteins, because they are tightly bound to
mRNA (
6
), and are able to repress translation
in vitro
(
7
), an activity regulated via their phosphorylation by an mRNP-associated protein kinase (
8
,
9
). More recently, the masking proteins have been identified as belonging to the
family of Y-box proteins (
10
-
12
), a family with dual roles in transcription regulation and mRNA packaging (
13
). However, the other abundant mRNP proteins have remained unidentified.
In this report we describe a 54 kDa protein, which is an abundant component of
mRNP particles. It turns out to belong to the family of DEAD-box RNA helicases, proteins which can regulate RNA secondary structure in
translation initiation, splicing and ribosome biogenesis (
14
,
15
). DEAD-box proteins contain a set of conserved motifs necessary for RNA helicase
activity, including the NTPase `A' (AXXGXXGKT) and `B' (DEAD) motifs, involved
in ATP binding and hydrolysis. Within this increasingly large group of
proteins, there are distinct subfamilies.
Xenopus
Xp54 belongs to a subfamily which includes: human p54, found at a chromosomal
breakpoint in the cell line RC-K8, derived from a diffuse large B-cell lymphoma (
16
) and now described as a putative proto-oncogene (
17
); a mouse equivalent (
18
);
Drosophila
ME31B, maternally-expressed in oocytes and nurse cells (
19
);
Schizosaccharomyces pombe
Ste13 cloned by functional complementation of the sterility mutant
ste13
(
20
) and
Saccharomyces cerevisiae
DHH1 (
21
). We describe the structure of Xp54, its developmental expression, and its
presence and activity in mRNP particles. With reference to what is known about
its putative homologues p54, ME31B and Ste13, we suggest possible functions for
Xp54.
Oocyte stages of
Xenopus laevis
are listed according to Dumont (
22
), and developmental stages according to Nieuwkoop and Faber (
23
). XTC cells were cultured as described previously (
24
).
Ovary containing only previtellogenic (stage I) oocytes was dissected from an immature female
X.laevis
. cDNA was synthesized from poly(A)
+
RNA extracted from glycerol gradient fractions enriched for polysomes (
9
). Double-stranded cDNA was cloned directionally into the bacteriophage [lambda] expression vector Uni-ZapXR (Stratagene). Two libraries of 0.8 * 10
5
clones (XlaIp1) and 3.7 * 10
5
clones (XlaIp2) were constructed and each was amplified to give 10
9
p.f.u./ml. For screening purposes equal volumes from each library were mixed.
Poly(A)
+
RNP particles were isolated from clarified homogenates of previtellogenic ovary by affinity chromatography using oligo(dT)- cellulose (Pharmacia) as described previously (
25
). Binding was performed in a solution containing: 0.3 M KCl; 2 mM MgCl
2
; 2 mM dithiothreitol; 0.5% Nonidet P-40; 10 mM Tris-HCl, pH 7.5. After extensive washing with binding buffer, mRNP
particles were eluted in 5 mM Tris-HCl pH 7.5.
Differential elution of proteins could be achieved by increasing the salt
concentation in the binding buffer. Whereas the mRNA-masking proteins, pp60 (also known as FRGY2a) and pp56 (FRGY2b) remain
stably bound to the column in the presence of 2 M NaCl, other mRNP-associated proteins could be eluted over the range of 0.4-1 M NaCl.
Proteins were also separated into two classes on the basis of thermostability.
On heating the isolated poly(A)
+
RNA fraction to 80oC for 10 min, followed by cooling to 0oC and centrifugation at 13 000 r.p.m. for 5 min in a microcentrifuge, the two masking
proteins, pp56/60, remain in the supernatant, whereas the other proteins can be
recovered from the pellet (
26
). Pellet fractions were raised in 80% formic acid and cyanogen bromide was
added in the ratio 2 mg CNBr/1 mg of protein. The reaction proceeded at room
temperature for 20 h in the dark. Polypeptide fragments were resolved by SDS-PAGE on 20% gels. Conditions for fragmentation and separation were
determined using mRNP particles labelled with
125
iodine by the Iodigen (Pierce) reaction. Polypeptides were detected by staining gel transfers and individual bands were excised and partially sequenced using a gas-phase automated sequencer.
To test for ATP binding, mRNP particles were photocrosslinked to [[alpha]-
32
P]ATP. [[alpha]-
32
P]ATP (2 [mu]l) (Amersham International, 3000 Ci/mmol) was added to 100 ml containing ~30 mg of poly(A)
+
mRNP adjusted to 20 mM Tris-HCl, pH 7.5 and 2 mM MgCl
2
. Samples were photocrosslinked on ice for 20 min with UV-light at 254 nm, 600 J/m
2
/s. After photocrosslinking, samples were digested with RNase and analysed by
SDS-PAGE and autoradiography.
To test for phosphorylation by the mRNP-associated protein kinase activity (
9
), poly(A)
+
mRNP were adjusted to 20 mM Tris-HCl, pH 7.5 and 2 mM MgCl
2
, as above. [[gamma]-
32
P]ATP (2 [mu]l) (Amersham International, 3000 Ci/mmol) was added to each sample.
Reactions proceeded for 30 min at 37oC, then samples were digested with RNase and analysed by SDS-PAGE and autoradiography.
RNA was extracted from pools of: 100 oocytes (stages I and II); 50 oocytes
(stages III and IV); 25 oocytes (stages V and VI); 25 embryos (stages 4, 8, 12,
18, 27 and 42), as described previously (
27
). Samples equivalent to two oocytes or embryos were denatured and separated on denaturing (formaldehyde) gels, using polyadenylated RNA standards as size markers. After vacuum transfer on to nylon membrane
(BioRad, GT), the RNA was crosslinked by UV-irradiation. Radiolabelled antisense riboprobes were synthesized as run-off transcripts from an
Eco
RI-
Xho
I subclone of Xp54 in pBlueScript (Stratagene), using T7 RNA polymerase (Pharmacia) in the presence of [[alpha]-
32P
]CTP (400 Ci/mmol, Amersham). Hybridization of the probe to transfers was
carried out at 65oC in a solution containing 1% dried skimmed milk, 1* SSC and 2% SDS.The transfers were then washed in decreasing salt concentrations to
a final step of 0.1* SSC at 80oC, dried and exposed in contact with X-ray film (Agfa).
Antisera were raised in rabbits against an electrophoretically-purified protein of 54 kDa isolated from oocyte mRNP particles. IgG
fractions were obtained by binding to, and elution from, protein A-Sepharose (Pharmacia). Affinity-purified antibodies were prepared by binding IgG to nitrocellulose
membrane saturated with extracts of bacterially-expressed pGEX 4T/Xp54 fusion proteins. Samples equivalent to two oocytes
or embryos, or to 10
5
cells or to 10 [mu]g of fusion protein, were separated by SDS-PAGE, transferred to nitrocellulose and reacted with anti-p54 at a dilution of 1/3000. Detection was with peroxidase-conjugated anti-rabbit IgG (Sigma) reacted with diaminobenzidine/ hydrogen peroxide.
Samples containing ~150 [mu]g of poly(A)
+
mRNP were loaded on to gradients of 10-25% glycerol made up in gradient buffer: 100 mM KCl; 2 mM MgCl
2
; 1 mM DTT; 10 mM Tris-HCl, pH 7.5. Centrifugation was at 18 000 r.p.m. for 14 h at 0oC, using a Beckman SW28 rotor.
Gradients of 20-60% Nycodenz in the buffer: 2 mM MgCl
2
; 1 mM EDTA; 20 mM Tris-HCl, pH 7.5; were prepared according to the manufacturer's specifications
(Nyegaard & Co., Oslo, Norway). Samples containing ~150 [mu]g of poly(A)
+
mRNP in 0.5 ml buffer were loaded on to 10 ml gradients, which were centrifuged
at 36 000 r.p.m. for 18 h at 0oC using a Beckman SW55Ti rotor. Twenty-one 0.5 ml fractions were collected for analysis. Densities were determined by optical refractometry using the equation: density [rho] (g/cm
3
) = 3.242[eta] - 3.323 where [eta] is the refractive index.
The vector pGEM-T (Promega) was used to synthesize complementary RNA transcripts from the T7 and an SP6 promoters. In order to generate
transcripts of suitable lengths part of the polylinker region was deleted by
cutting at the
Nsi
I and
Pst
I restriction sites, which were then ligated. T7 strand: the plasmid was
linearized at the
Spe
I site, generating a 60 base transcript: 5'-GGGCGAATTGGGCC
To generate the double-stranded probe: 1 [mu]g of the labelled strand, specific activity 2.5 [mu]Ci/[mu]g RNA, was mixed with 2 [mu]g of cold strand, in a volume of 100 [mu]l of DEPC-treated dH
2
O. The mixture was denatured at 95oC for 5 min, then adjusted to 200 mM NaCl; 2 mM EDTA; 10 mM Tris-HCl, pH 7.5, and incubated at 60oC for 1 h for hybridization.
Double-stranded probes were also made (as detailed above) using sense and
antisense transcripts from a subclone representing the 3' end of an oocyte-specific [beta]-tubulin gene. The annealed strands should generate
compex stuctures containing regions of intrastrand duplex within the 280 nucleotide length of complementary sequence (
28
). These structures also present 5' single-stranded ends.
Any detectable RNA helicase activity unwinds and separates the strands, of which
only one is radioactively labelled. Hence the activity is measured as a
mobility shift towards the single-stranded form. As a control, the probe was denatured in 1 M glyoxal + 50%
DMSO (dimethylsulfoxide) in 10 mM sodium phosphate buffer and placed at 50oC for 1 h. Helicase assays were performed in 50 [mu]l reactions in helicase buffer: 20 mM Tris-HCl, pH 7.5; 80 mM KCl; 1 mM MgCl
2
; 1.5 mM DTT; with ATP (at 0-1 mM). Double-stranded probe (0.1 [mu]Ci per reaction) was included and protein samples (ranging from
2 to 500 ng protein) were tested. Reactions proceeded at 20oC for 20 min, after which 10 [mu]l of gel loading buffer was added (50% glycerol, 0.25% bromophenol
blue, 0.25% xylene cyanol, in DEPC-treated dH
2
O) before loading the reactions on an acrylamide gel. The gel contained 6%
acrylamide (sequencing grade) in TAE buffer (0.04 M Tris-acetate, 1 mM EDTA) and 10% glycerol. The gel was run for ~2 h at 200 V, fixed for 20 min in 10% acetic acid, dried and set up
for autoradiography.
PCR primers were designed around two of the sequenced peptides: (M)GWEKPSPIQ and (M)RQEHRNRVFH. Based on the alignment with human p54, the peptide sequence MGIFE
A cDNA clone containing an insert of 2.5 kb was selected from the cDNA library
and sequenced (EMBL accession number X92421). The length of the sequenced insert is 2491 bp, containing an open reading frame of 1445 bp extending from positions 204 to 1649. The
proposed initiation codon occurs in a sequence context conforming to the
eukaryotic consensus and to start sites for other proteins expressed in
Xenopus
oocytes. A polyadenylation site (AUUAAA) occurs 34 bp upstream from the 3'-terminal poly(A). The 3' UTR contains AU-rich stretches and three AUUUA motifs which could
represent instability elements (
31
). The predicted molecular mass of the encoded protein is 54.1 kDa, matching the size of the ATP-crosslinked protein already described (Fig.
1
B). This protein is referred to as Xp54 (
Xenopus
p54).
An alignment with the DEAD-box RNA helicase family (not shown) confirms that Xp54 is a member of a
subfamily that includes human and mouse p54,
Drosophila
ME31B,
S.pombe
Ste13 and
S.cerevisiae
DHH1. The extent of conservation from yeast to man is on the whole impressive,
and in particular,
Xenopus
Xp54 is 94% identical to human p54 at the amino-acid level. Among the five members of this subfamily, the overall amino
acid sequence identity is 54%, and the overall level of similarity is at least
76% when conservative substitutions are taken into account. Figure
2
shows the alignment of Xp54 with its putative homologues.
The expression pattern of Xp54 mRNA was examined through oogenesis, early
development and in adult tissues (Fig.
3
). Hybridization of radiolabelled antisense riboprobes to transfers of poly(A)
+
RNA from ovary identifies a transcript of 2.6 kb, but no transcript larger than
this, even on high level loading of the gel (Fig.
3
A). This observation compares well with the cloned cDNA, which is 2491 bp long
and was derived from previtellogenic mRNA. Although a minor signal at 1.5 kb is also apparent, this would barely
contain the coding region alone (1445 bp) and its identity remains unknown.
GST (glutathione
S
-transferase) fusion proteins were prepared by subcloning Xp54 into pGEX-4T (Pharmacia) vectors. A polyclonal antiserum, raised against a
band-excised mRNP protein of 54 kDa, recognized three out of four Xp54-GST fusion proteins (Fig.
4
A). Specific reactions were obtained with fusions containing the middle portion
and the carboxyl end of Xp54:
Pvu
II-
Pvu
II;
Dra
I-
Dra
I;
Xho
I-
Xho
I. The antiserum did not recognize the
Pvu
II-
Xho
I fusion protein which encodes 14.3 kDa of Xp54 near the amino end, including
the `A' motif involved in ATP-binding. This antiserum is referred to as anti-p54 and was affinity-purified by binding an IgG fraction to nitrocellulose
saturated with bacterial extract containing the
Pvu
II-
Pvu
II fusion protein.
Anti-p54 was used to track expression of the Xp54 protein through oogenesis and
early embryogenesis (Fig.
4
B). The level of Xp54, on a per oocyte/embryo basis, is at a maximum at Dumont
stage I and remains fairly constant through to the end of oogenesis and, after
fertilization, up to blastula. From blastula, through gastrula and neurula,
there is a substantial decline in the level of Xp54. This pattern of expression
is similar to that of the Y-box mRNA-packaging proteins, pp60/56 (
32
), and therefore correlates well with the relative abundance of maternal mRNP
particles. However, one difference is that whereas pp60/56 are not detected
much after blastula (stage 8), low levels of the immunostained 54 kDa band
remain detectable through to at least the free-feeding tadpole (embryonic stage 42).
Although all evidence indicates that the 2.6 kb transcript which encodes
Xenopus
p54 is exclusively maternal, immunoblotting with anti-p54 shows that a 54 kDa protein is expressed in
Xenopus
culture cell lines (XTC, from metamorphosing tadpoles; XP, from adult kidney),
indicating that a somatic transcript exists to direct the synthesis of a very
similar protein. A 54 kDa protein is also detected with anti-p54 in rat ovary extracts (Fig.
4
B) and it is reasonable to suppose that similar proteins are expressed across
vertebrate species.
That Xp54 is a major cytoplasmic protein of small oocytes is seen directly by
immunostaining ovarian sections with anti-p54. The fluorescent image reveals a widespread distribution of
particulate structures in the cytoplasm (Fig.
5
A). Although no immunostaining is detected in the nucleoplasm, nuclear structures, including nucleoli and
chromatin (seen more clearly in chromosome preparations, not shown), give a
significant reaction, particularly in the smaller (stage I) oocytes. The
cellular distribution of Xp54, then, appears to be similar to that of pp60/56,
except for the more intense staining of nucleoli with anti-p54.
In order to determine to what extent Xp54 is associated with particles
sedimenting at a rate expected of mRNPs, clarified homogenates from
previtellogenic oocytes were centrifuged through 15-40% glycerol gradients. It has been shown previously that mRNP particles
sediment at 30-100S (
5
,
9
). On analysing gradient fractions by SDS-PAGE and immunoblotting, it is seen that a 54 kDa protein, which reacts
specifically with anti-p54, mostly sediments between 42S and 80S marker peaks (Fig.
6
A). The observed distribution is similar to that of the mRNP packaging proteins
pp60/56 using anti-FRGY2 (not shown). The coincidence of pp56/60 and Xp54 suggests that both
packaging proteins and RNA helicase are bound to the same population of mRNA
molecules. From the presence of some Xp54 near the top of the gradients, it is
possible that in early oocytes excess RNA helicase occurs in RNA-free protein complexes, as has been described for pp60/56 and other mRNP
proteins (
9
,
33
).
On binding poly(A)
+
mRNP to oligo(dT)-cellulose, a subset of proteins, including pp56/60, remains bound to the
resin at high salt concentration (2 M NaCl). These proteins are eluted under
conditions which release the poly(A)
+
RNA (no salt or 60% formamide;
5
,
25
,
28
). However, before eluting the salt-stable mRNP, individual proteins can be washed off the column by
increasing salt concentration (Fig.
7
A). At 0.4 M NaCl, a 54 kDa protein is eluted, whereas at 0.6 and 1.0 M NaCl, a
much wider range of mRNP proteins elute (Fig.
7
B). Selected fractions were immunoblotted with anti-p54 and anti-FRGY2. The immunoblots confirm that most of pp60/56 is only released
in the final no salt elutions, although small amounts can be detected from the 0.6 M eluate onwards, but not in the 0.4 M eluate (not shown). In contrast, Xp54 is
enriched in the 0.4 M eluate, but is also present in higher salt elutions up to
1.5 M NaCl (Fig.
7
C). Thus Xp54 appears to be bound to the mRNP through charge interactions;
whether it is bound directly to mRNA, or to other mRNP proteins, is not known.
The RNA helicase Xp54 is an abundant and integral component of stored (non-translating) mRNP in
Xenopus
oocytes. Xp54 was first identified through peptide sequencing and ATP
crosslinking, and then cloned by PCR using degenerate primers. A partially
purified fraction containing Xp54 was shown to possess an ATP-dependent RNA helicase activity. Xp54 belongs to the family of DEAD-box proteins, and includes the conserved motifs that are the
hallmark of that family. More specifically, Xp54 belongs to a subfamily that
includes human p54, to which it is 94% identical, mouse p54,
Drosophila
ME31B,
S.pombe
Ste13 and
S.cerevisiae
DHH1. Information in the literature describing these putative homologues can
now be related to Xp54.
The first member of this subfamily to be cloned was
Drosophila
ME31B
, a maternally expressed gene (
19
). It is expressed in germ-line cells, including the 15 nurse cells and oocytes, but not the
surrounding somatic cells.
ME31B
mRNA is detectable only in the early embryo, 0-2 h after fertilization. The
S.pombe
gene
STE13
was cloned by functional complementation of a sterility mutant (
20
). Prior information indicated that
STE13
is essential for nitrogen-starvation-induced G
1
arrest, leading to the initiation of sexual development (
35
). The yeast protein is therefore required for the progression of meiosis. Yeast
mutants were successfully complemented with a cDNA construct encoding
Drosophila
ME31B, but not by one encoding Vasa, a well characterized maternally-expressed RNA helicase, consistent with ME31B being the functional homologue of Ste13. Maekawa
et al.
(
20
) suggested that Ste13 may have a role in the translational control of meiotic
mRNAs.
Human p54 was first described by Dan and Yunis (
16
). These authors were investigating the chromosomal translocation
t(11;14)(q23;q32) found in the cell line RC-K8 derived from a diffuse, large B-cell lymphoma. A putative RNA helicase, p54, 75% identical to
Drosophila
ME31B, was found at this breakpoint. The expression of its mRNA was detected at
high levels, in a variety of tissues, in the form of a 6.7 kb transcript. More
recently, Akao
et al.
(
17
) have used an anti-p54 antiserum to detect the 54 kDa protein in different tissues.
Significantly, they detect moderate amounts of p54 in the neuroblastoma cell
line IMR-32 and glioblastoma cell line T98G, and higher levels in the
rhabdomyosarcoma cell line RMS-YM and lung cancer cell lines LU99A and LU99B. These cell lines are
derived from tissues in which the same authors do not detect p54, leading to
the suggestion that p54 is a candidate proto-oncogene.
As in the case of
Drosophila
ME31B (
19
), we were unable to detect Xp54 mRNA in adult tissues. Highest levels of Xp54
mRNA are seen in early oogenesis, when the demand for mRNP proteins is highest.
However, Xp54 protein is detected on Western blots at least until the free-feeding tadpole stage, and is detected in the tadpole cell line XTC and
the adult kidney cell line XP. It is therefore likely that the cDNA described
in this paper is a germ cell-specific transcript, and that a somatic transcript directs the synthesis
of a similar protein. The presence of other germ-cell specific transcripts in
Xenopus
oocytes is well documented (
32
).
What, then, is the specific function of Xp54? The
Xenopus
oocyte can be considered to be in a mode of suspended cell proliferation. Until
oocyte maturation, the oocyte is stalled in the first meiotic prophase. During
this time, the lampbrush chromosomes are highly active in transcribing genes
whose products will drive early development. For example, c-
mos
is transcribed from early oogenesis onwards, but its mRNA is not translated
until oocyte maturation (
36
). Another example is c-
myc
mRNA which accumulates from early oogenesis and is stored for translation
during early embryonic development (
37
,
38
). Two possibilities arise. The first is that Xp54, as an integral component of
stored mRNP, is required for the efficient translational recruitment of stored
mRNAs. More specifically, Xp54 might unwind duplex structures in the 5' UTR during translation initiation (mimicking the role of eIF4A), or may
even unwind duplexes in the coding region and 3' UTR that might impede elongation. The key property would be the ability
to enhance translation at a time when large quantities of product are required.
Akao
et al.
(
17
) have detected p54 on the rough ER, consistent with a role in translation and
our own immunostaining studies on XTC cells lead to a similar conclusion. An
alignment of 45 DEAD- and DEXH-box RNA helicases representing all known subfamilies (not shown),
suggests that the Xp54 subfamily is more closely related to eIF4A than to any
other helicase, again suggesting that Xp54 has a role in translation
initiation.
An alternative function might relate to the formation of stored mRNP particles,
a process which appears to be initiated in the nucleus (
13
). In this scenario, Xp54 would unwind RNA sequences to facilitate protein binding, notably of the mRNA-packaging Y-box proteins, FRGY2a/b, which show a marked preference for single-stranded RNA (
25
). Whereas immunostaining of cell sections (Fig.
5
) indicates that Xp54 can be detected in the nucleus, an exclusively nuclear
function would not explain the high levels of Xp54 which are maintained in
cytoplasmic mRNP. Like the Y-box proteins, which package the mRNA, Xp54 levels decline on progression
from rapid embryonic cell cleavage to mid-blastula, when stored maternal mRNP particles are mostly used up and
zygotic transcription takes over (
4
).
In considering what might regulate the onset of Xp54 helicase activity, two
aspects are worth mentioning. (i) It has been shown previously that mRNP
particles in oocytes carry an associated casein kinase II (CK2) activity (
9
) and that phosphorylation of FRGY2a/b stabilizes its binding to mRNA (
28
,
30
). Xp54 also has multiple potential CK2 phosphorylation sites: four, out of a total of five, being located near the C-terminus. However, it is significant that Xp54, in comparison with FRGY2a/b, is inefficiently phosphorylated both
in vitro
and
in vivo
in immature oocytes (
9
). Perhaps the turnover of phosphates in Xp54 is much slower, or else the CK2
sites are blocked during the assembly of mRNP particles, only becoming
accessible for phosphorylation at some later stage. Modification at these sites, whether by phosphorylation or dephosphorylation, might act as a trigger for helicase activation at an appropriate stage of development. (ii) Full helicase activity
in vivo
may require the assistance of an additional protein factor, much in the same
way that eIF4A requires eIF4B (
39
). Such a cofactor might only be available at certain times of development,
and/or might only bind the mRNP complex in response to an appropriate change in
the phosphorylation status of Xp54. It is possible that a cofactor is present in the column fraction used in this report to demonstrate helicase activity, since Xp54 was not purified.
The combination of Xp54, Y-box proteins and CK2 with maternal mRNA should provide a useful model to
explore the functioning of stored mRNP particles. Findings would be relevent,
not only to germ cells (
32
) and early development, but also to proliferating somatic cells in which high
levels of Y-box proteins are detected (
40
) and to somatic cells in which an association of Y-box proteins with stored mRNA has been demonstrated (
41
).
We thank Dr Graham Kemp (St Andrews) for kind assistance in peptide
sequencing.This work was supported by a grant from The Wellcome Trust.
*To whom correspondence should be addressed. Tel: +44 1334 463583; Fax: +44 1334
463600; Email: js15@st-and.ac.uk
+
Present address: MRC Human Genetics Unit, Western General Hospital, Edinburgh
EH4 2XU, UK
REFERENCES
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