Results In an effort to identify novel proteins that are part of the vegetal RNA localization machinery in Xenopus oocytes, tandem affinity purification TAP-tagged ver-sions of the known v
Trang 1machinery in Xenopus laevis oocytes
Jana Loeber1, Maike Claußen1, Olaf Jahn2and Tomas Pieler1
1 Department of Developmental Biochemistry, Go¨ttingen Center for Molecular Biosciences, University of Go¨ttingen, Germany
2 Proteomics Group, Max-Planck-Institute of Experimental Medicine, Go¨ttingen, Germany
Keywords
EF1a; RNA localization; 42Sp50; Vg1RBP;
Xenopus laevis oocytes
Correspondence
T Pieler, Department of Developmental
Biochemistry, Go¨ttingen Center for
Molecular Biosciences, University of
Go¨ttingen, Justus-von-Liebig-Weg 11,
37077 Go¨ttingen, Germany
Fax: +49 551 3914614
Tel: +49 551 395683
E-mail: tpieler@gwdg.de
(Received 28 February 2010, revised 30
August 2010, accepted 9 September 2010)
doi:10.1111/j.1742-4658.2010.07878.x
Localization of a specific subset of maternal mRNAs to the vegetal cortex of Xenopusoocytes is important for the regulation of germ layer formation and germ cell development It is driven by vegetal localization complexes that are formed with the corresponding signal sequences in the untranslated regions
of the mRNAs and with a number of different so-called localization proteins
In the context of the present study, we incorporated tagged variants of the known localization protein Vg1RBP into vegetal localization complexes by means of oocyte microinjection Immunoprecipitation of the corresponding RNPs allowed for the identification of novel Vg1RBP-associated proteins, such as the embryonic poly(A) binding protein, the Y-box RNA-packaging protein 2B and the oocyte-specific version of the elongation factor 1a (42Sp50) Incorporation of 42Sp50 into localization RNPs could be con-firmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50 Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNAse-sensitive complexes, as well as to associ-ate specifically with several vegetally localizing mRNAs but not with nonlo-calized control RNAs Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between the nucleus and cytoplasm Taken together, these observations provide evidence for a novel function of 42Sp50
in the context of vegetal mRNA transport in Xenopus oocytes
Structured digital abstract
l MINT-7994313 : epab (uniprotkb: Q98SP8 ) physically interacts ( MI:0915 ) with Vg1RBP (uni-protkb: O73932 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7994335 : 42Sp50 (uniprotkb: P17506 ) physically interacts ( MI:0915 ) with Vg1RBP (uniprotkb: O73932 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7994166 : Vg1RBP (uniprotkb: O73932 ) physically interacts ( MI:0914 ) with Vg1RBP (uniprotkb: O73932 ), 42Sp50 (uniprotkb: P17506 ), frgy2-b (uniprotkb: P45441 ) and epab (uni-protkb: Q98SP8 ) by tandem affinity purification( MI:0676 )
l MINT-7994324 : frgy2-b (uniprotkb: P45441 ) physically interacts ( MI:0915 ) with Vg1RBP (uniprotkb: O73932 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7994345 : 42Sp50 (uniprotkb: P17506 ) physically interacts ( MI:0914 ) with staufen (uniprotkb: Q5MNU4 ) and Vg1RBP (uniprotkb: O73932 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7994363 : 42Sp50 (uniprotkb: P17506 ), Vg1RBP (uniprotkb: O73932 ), staufen (uniprotkb: Q5MNU4 ) and 40LoVe (uniprotkb: Q6GM69 ) colocalize ( MI:0403 ) by cosedimentation through density gradient ( MI:0029 )
l MINT-7994241 : Vg1RBP (uniprotkb: O73932 ) physically interacts ( MI:0914 ) with elrB (uni-protkb: Q91903 ), ElrA (uniprotkb: Q1JQ73 ), hnRNPI (uniprotkb: Q9PTS5 ), 40LoVe (uni-protkb: Q6GM69 ), staufen (uniprotkb: Q5MNU4 ) andVg1RBP (uniprotkb: O73932 ) by tandem affinity purification ( MI:0676 )
Abbreviations
EF1A, elongation factor 1a; ePAB, embryonic poly(A) binding protein; FRGY-2B, Y-box RNA-packaging protein 2B; LE, localization element;
OE, oocyte equivalent; TAP, tandem affinity purification; 42Sp50, oocyte-specific version of the elongation factor 1a.
Trang 2During oogenesis in Xenopus laevis, a group of
mater-nal transcripts becomes specifically localized to the
vegetal cortex By this means, an intracellular
asymme-try is created for subsequent use during early
embryo-nic development The respective vegetal mRNAs are
transported via two distinct routes [1] The early
(METRO-) pathway is activated in stage I–II oocytes;
RNAs associate with the mitochondrial cloud (also
referred to as the Balbiani body), a large conglomerate
enriched in mitochondria, endoplasmic reticulum
mem-branes and RNPs, also containing the germ plasm
Together with the mitochondrial cloud, the prospective
vegetal RNAs then migrate towards the vegetal cortex,
where they become anchored during stage III [1] The
association between the Balbiani body and the
mRNAs is considered to be established via a
diffu-sion–entrapment mechanism [2] and does not appear
to depend on the presence of intact microtubules [1];
however, a recent study provides evidence for a
facili-tating activity exerted by kinesin II [3] By contrast,
the late localization pathway can be efficiently blocked
when oocytes are treated with nocodazole, a
microtu-bule-depolymerizing drug [4] Although the late
locali-zing RNAs are excluded from the mitochondrial cloud
at the beginning of oogenesis, they accumulate between
the nucleus and vegetal pole during stage III and
sub-sequently translocate vegetally In stage IV oocytes,
the respective RNAs are found anchored at the cortex
of the entire vegetal hemisphere
All vegetally localizing RNAs contain regulatory
sequence elements in their untranslated regions, which
are necessary and sufficient for transport, and are
referred to as localization elements (LEs) In some
mRNAs, such as Vg1 or VegT, the number and
rela-tive positioning of short consensus sequence motifs
determines the localization efficiency [5–7] However,
the LEs of other localizing mRNAs, such as fatvg,
Xdead end, Xvelo1 and Xwnt11, contain only few or
none of these motifs, although they are still capable of
mediating vegetal transport [8–11] Therefore, the
sec-ondary structure of the LEs may as well be critical for
RNA localization, as is the case in Drosophila and
yeast [12–14]
LEs recruit trans-acting proteins, such as Vg1RBP,
hnRNPI⁄ PTB, Prrp, Staufen, 40LoVe and ElrA ⁄ B,
thereby forming the so-called localization complex or
‘locasome’ [5,15–20] Discrete RNP assembly steps
have been defined for Vg1 mRNA in the context of
the late vegetal RNA transport pathway In the
nucleus, Vg1 is recognized by the RNA-binding
pro-teins Vg1RBP and hnRNP I [7,21]; the export of this
complex into the cytoplasm is followed by a structural reorganization of the RNP Although, in the nucleus, the association between Vg1RBP and hnRNPI does not depend on the presence of RNA, the same interac-tion becomes RNA-dependent in the cytoplasm [21] Moreover, other proteins such as Staufen1 and Prrp join the complex in the cytoplasm [16–18] The trans-acting factors ElrA and 40LoVe can be detected in the nucleus as well as in the cytoplasm [20,22] However, it
is not yet clear at what stage of the transport process they join the localization complex Interestingly, Stau-fen has been found to interact with kinesin in X laevis oocytes, and might therefore provide the link between the localizing particle and a motor protein [18] The mature RNP eventually migrates to the vegetal cortex most likely along microtubules in a plus-end directed transport as the cargo of a kinesin motor [4,18,23,24]
At the vegetal cortex, the RNA molecule becomes anchored Cortical anchoring depends not only on the cytoskeletal network, but also on the presence of other localizing RNAs, such as Xlsirts and VegT [1,19,25] Although the RNP is assumed to dissociate at the cor-tex, trans-acting factors such as Prrp, ElrA, Staufen and 40LoVe remain enriched at the vegetal pole
in stage VI oocytes, when localization is finished [16,18–20]
To identify novel protein components of vegetal transport particles, we used over-expression of a tagged version of Vg1RBP to fish for novel binding partners that might play a role in RNA localization
We identified an oocyte-specific isoform of the protein translation elongation factor 1a (EF1A) as a novel Vg1RBP-interacting protein that is a specific compo-nent of vegetally localizing RNPs in Xenopus oocytes
Results
In an effort to identify novel proteins that are part of the vegetal RNA localization machinery in Xenopus oocytes, tandem affinity purification (TAP)-tagged ver-sions of the known vegetal localization factor Vg1RBP were expressed in stage III and IV X laevis oocytes by means of mRNA microinjection Two different tagged versions of Vg1RBP were employed: one carrying the TAP-tag at the N-terminus (N-TAP-Vg1RBP), and another one carrying it at the C-terminus (C-TAP-Vg1RBP) Lysates from microinjected oocytes were incubated with IgG sepharose beads and immobilized protein complexes containing TAP-Vg1RBP were eluted by proteolytic cleavage of Vg1RBP from the protein A moiety under native conditions; a control
Trang 3lysate from oocytes expressing the TAP-tag only
(TAP) was included Proteins contained in these
pre-parations were separated by SDS⁄ PAGE and
visua-lized by colloidal Coomassie staining (Fig 1A); the
arrays of proteins interacting with either
N-TAP-Vg1RBP or C-TAP-N-TAP-Vg1RBP were indistinguishable
from each other Proteins specifically interacting with
Vg1RBP were isolated and subjected to MS protein
identification Several of the protein species identified
by this means correspond to CBP-Vg1RBP (containing
the calmodulin binding peptide of the TAP-tag) or
endogenous Vg1RBP, as expected because of its
known ability to form homodimers, or degradation
products of the same protein Three novel proteins
could be identified that specifically co-purify with
Vg1RBP: embryonic poly(A)-binding protein (ePAB;
NCBI accession number: gi|13540314), Y-box protein
2B (FRGY-2B; NCBI accession number: gi|1175534)
and an oocyte-specific version of EF1A (42Sp50;
NCBI accession number: gi|416929)
The fact that other known localization factors, such
as Staufen or hnRNPI, were not identified, does not
necessarily imply that these proteins were not present
in the protein complex analyzed in the present study;
only proteins strongly stained by Coomassie were
reliably identified by MS analysis Different stoichio-metries of individual protein components, which assemble into one RNP with Vg1RBP, as well as struc-tural heterogeneity of RNPs containing Vg1RBP, may account for the fact that the expected interaction part-ners for Vg1RBP were not detected by Coomassie staining and MS analysis To confirm the presence of known localization factors, we analyzed TAP-Vg1RBP pulldown eluate by western blotting (Fig 1B) Staufen, 40LoVe, hnRNPI, ElrA and ElrB were found to co-purify with TAP-Vg1RBP, whereas GAPDH could not be detected This indicates that localization com-plexes were indeed isolated using the approach employed in the present study
The association between the novel proteins identified and Vg1RBP was verified by reverse co-immunoprecipi-tation For this purpose, Xenopus oocytes were micro-injected with synthetic mRNAs encoding myc-tagged versions of ePAB (myc-ePAB), FRGY-2B (myc-FRGY) and 42Sp50 (myc-42Sp50); complexes forming with these proteins were immunoprecipitated with a myc-specific antibody and co-precipitating Vg1RBP detected
by western blotting (Fig 2) It was found that endo-genous Vg1RBP is efficiently co-precipitated with ePAB
as well as FRGY-2B, and specifically, although with
Fig 1 Identification of Vg1RBP-interacting proteins (A) Oocyte extract prepared from uninjected stage III–IV oocytes and stage III–IV oocytes expressing either a TAP tag alone, N-terminally (N-TAP) or C-terminally (C-TAP) TAP-tagged Vg1RBP was incubated with IgG-sephar-ose beads and eluted by TEV protease cleavage of the TAP tag Eluted proteins were separated on 10% SDS ⁄ PAGE and visualized by colloi-dal Coomassie staining M, protein size marker The bands marked by blue lines were excised and analyzed by MS Putative Vg1RBP binding partners identified are ePAB, FRGY-2B and 42Sp50 Degradation products of Vg1RBP are marked with an asterisk (*) and proteins that could not be identified are labelled as n.i (B) Lysate from uninjected oocytes and oocytes expressing TAP or N-terminally TAP-tagged Vg1RBP was processed as above and analyzed by western blotting for the presence of known localization factors Input corresponds to 1%
of the material used in the pulldown experiment The presence of the TAP-Vg1RBP band in the anti-Staufen blot is a result of the strong binding of secondary anti-rabbit serum to the protein-A moiety of the TAP-tag Anti-CBP serum was used to show the expression of the TAP-tag alone.
Trang 4reduced efficiency, also with 42Sp50 These differences
in yield could reflect different protein stoichiometries in
the complexes that form After RNase-digestion,
Vg1RBP could no longer be detected in the
immunopre-cipitate, indicating that the interactions of these
differ-ent proteins are likely to be indirect, depending on the
presence of intact RNA as integral component of the
RNP
Association of 42Sp50 with mRNA has not been
reported previously If 42Sp50 is involved in vegetal
mRNA localization in Xenopus oocytes, as suggested
by its association with Vg1RBP, it would be expected
also to interact with other trans-acting localization
fac-tors, such as Staufen1 Co-immunoprecipitation
experi-ments from myc-42Sp50 programmed oocyte extract
do indeed reveal an RNA-dependent association of
42Sp50 with Staufen1 in addition to Vg1RBP (Fig 3)
Additional evidence for these proteins constituting
one RNP comes from density gradient centrifugation
analysis; fractionation of S16 total lysate from stage
III⁄ IV oocytes on a 5–60% glycerol gradient, followed
by western blotting, reveals 42Sp50 enrichment in high
density fractions together with Staufen1 and Vg1RBP
(Fig 4) These high density fractions contain large RNPs, with a size similar to 80S ribosomes; sensitivity
to RNAse treatment indicates that the RNA serves as
a scaffold for protein binding rather than protein–pro-tein interactions providing the driving force for the formation of these very large assemblies 42S tRNA storage particles that contain 42Sp50 do not co-migrate with 80S ribosomes and they are specific to stage I⁄ II oocytes [26] Taken together, these experi-mental observations provide strong evidence for 42Sp50 as being part of one large RNP complex together with other proteins known to serve functions
in vegetal RNA localization in Xenopus oocytes
If 42Sp50 was a trans-acting localization factor, it should be specifically associated with known vegetally localizing mRNA molecules To determine whether endogenous oocyte mRNAs are in a complex with
Fig 2 Vg1RBP interacts with ePAB, FRGY-2B and 42Sp50 in an RNase-dependent manner Stage III–IV oocytes were injected with RNA encoding either myc-tagged ePAB, FRGY or 42Sp50 Oocyte extract was prepared and subjected to immunoprecipitation with anti-myc serum in the presence or absence of RNase The precipitate was analyzed by SDS ⁄ PAGE and western blotting for the presence of Vg1RBP.
In a control immunoprecipitation with extract from uninjected oocytes, no Vg1RBP could be detected As input, 1% (for analysis with anti-Vg1RBP serum) or 20% (for analysis with anti-myc serum) of the total oocyte extract was loaded.
Fig 3 42Sp50 associates with Staufen1 in an RNase-dependent
manner Stage III oocytes were injected with Cap-RNA encoding
myc-42Sp50 and incubated overnight to allow protein expression.
Oocyte extract was used for co-immunoprecipitation using anti-myc
serum Precipitated proteins were analyzed by SDS ⁄ PAGE and
western blotting 42Sp50 interacts with both Vg1RBP and Staufen1
in the presence of intact RNA but not if cellular RNA is destroyed
by RNase digestion.
Fig 4 42Sp50 co-migrates with known components of the locali-zation complex in a glycerol gradient Extract from stage III–IV oocytes expressing myc-42Sp50 was fractionated on a 5–60% glycerol gradient either in the presence or absence of RNase Eleven fractions were collected and split into two aliquots to allow the detection of several proteins from the same gradient The fractions were subjected to SDS ⁄ PAGE, and western blot analyses were performed using specific antibodies against Vg1RBP, Staufen1 and 40LoVe or anti-myc serum to detect myc-42Sp50 Fractions 7–9 are enriched in the known localization factors Vg1RBP and Staufen1, and are therefore labelled as locasome.
Trang 542Sp50, we performed RNA co-immunoprecipitaion
experiments For this purpose, synthetic mRNA
encoding myc-tagged 42Sp50 was microinjected into
stage III and IV oocytes; S16 lysate was prepared after
24 h of incubation and used for an
immunoprecipita-tion with anti-myc The RNA was eluted from the
immunopellet and characterized for the presence of
dif-ferent mRNAs by quantitative real-time RT-PCR
cor-rected for the abundance of individual mRNA species
[20] It was found that of the three vegetally localizing
mRNAs tested, two, namely VegT and XNIF, are
sig-nificantly enriched in RNPs with 42Sp50 The
distribu-tion of only one of the vegetally enriched mRNAs,
namely Vg1, is similar to the nonlocalizing mRNAs
ornithine decarboxylase and lamin B1 (Fig 5) A
simi-lar result was obtained for RNP immunoprecipitation
with myc-tagged Vg1RBP (data not shown)
RNPs destined for vegetal localization assemble in
the oocyte nucleus and undergo at least one
remodel-ling step after export into the cytoplasm [7,21];
Vg1RBP has been shown to be part of the nuclear as
well as of the cytoplasmic vegetal-transport-RNP,
whereas Staufen1 joins the complex only in the
cyto-plasm [21] By means of immunolocalization on oocyte
sections, 42Sp50 was mainly detected in the cytoplasm
of stage I⁄ II oocytes [27,28] When nuclear and
cytoplasmic lysate from manually dissected stage III
oocytes were analyzed for the presence of myc-tagged
42Sp50 expressed by means of mRNA microinjection (Fig 6A), it was found that the majority of the protein
is similarly detected in the cytoplasmic fraction; how-ever, a small but perhaps significant amount of 42Sp50
is found in the nucleus The same blot was also probed with specific antibodies against Staufen1 and Vg1RBP;
as expected, Staufen1 was almost exclusively detected
in the cytoplasm; this was also the case for Vg1RBP The presence of 42Sp50 in nucleus and cytoplasm indicates that it might be a shuttling protein with a function in the nuclear export of localizing mRNAs
To determine whether 42Sp50 is capable of shuttling,
we injected the radioactively labelled in vitro-translated protein either into the nucleus or into the cytoplasm of
X laevis oocytes, isolated the nuclei manually at differ-ent time points after injection, and analyzed the nuclear and cytoplasmic fractions by SDS⁄ PAGE and phosphoimaging (Fig 6B) Although a minor fraction
of 42Sp50 is exported from the nucleus after 3 and 5 h (lanes 12 and 14), no import into the nucleus could be seen, even after 5 h of incubation (lane 7) Because export but not import can be detected, the export rate appears to be much higher than the import rate This would be in line with the steady-state distribution of the protein as described above (Fig 6A)
Discussion
mRNA transport to the vegetal cortex of Xenopus oocytes occurs in the context of large RNPs that can incorporate tagged variants of known localization pro-teins such as Vg1RBP expressed by means of oocyte microinjection Immunoprecipitation of such RNPs allowed for the identification of novel Vg1RBP-asso-ciated proteins such as ePAB, FRGY-2B and 42Sp50 Incorporation of 42Sp50 into localization RNPs could
be confirmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50 Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNase-sensitive complexes, as well as to associate specifically with several vegetally localizing mRNAs but not with nonlocalized control RNAs Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between nucleus and cytoplasm
ePAB, similar to the prototypical poly(A) binding protein, functions as a translational activator; however,
it is only expressed during Xenopus oogenesis and early embryogenesis [29,30] Interestingly, human prototypi-cal poly(A) binding protein, which is not only func-tionally, but also structurally closely related to ePAB, was reported to be in direct interaction with IMP1, similar to Vg1RBP, which is a member of the VICKZ
Fig 5 The 42Sp50 particle is specifically enriched in localizing
RNAs Extract from stage III–IV oocytes expressing myc-tagged
42Sp50 were subjected to immunoprecipitation using anti-myc
serum As a control, the same extract was used in a precipitation
without antibody Proteins and RNAs bound to the immunopellet
were eluted by incubation in 1% SDS RNA was isolated by
phenol ⁄ chloroform extraction Ten percent of the oocyte extract
was used for the isolation of total RNA using the same protocol.
RNA was reverse transcribed into cDNA and analyzed by
quantita-tive PCR Enrichment factors were calculated using the 2)DCT
method [47] as described in the Experimental procedures The
enrichment factor of each RNA was normalized to GAPDH, which
was set to one The mean of three independent experiments is
shown, with error bars indicating the standard deviation ODC,
ornithine decarboxylase.
Trang 6family of RNA binding proteins [31–33] Together with
the findings obtained in the present study, interaction
of VICKZ and poly(A) binding proteins thus appears
to define a conserved feature of mRNPs forming in
dif-ferent biological systems The function of these mRNPs
is obviously not solely related to RNA transport
FRGY-2B is a germ cell specific RNA packaging
protein that stabilizes stored mRNAs and prevents
their translation [34,35] Tanaka et al [36] have
identi-fied proteins associated with FRGY2 in Xenopus
oocytes Interestingly, and in full agreement with data
reported in the present study, both Vg1RBP and ePAB
were among the proteins identified It is not known
why proteins that function as translational repressors,
such as FRGY2, or as translational activators, such as
ePAB, should be part of one and the same complex;
on the basis of the experimental results obtained in the
present study, we cannot exclude the possibility that
ePAB and FRGY2 are indeed part of different RNPs
together with Vg1RBP and⁄ or 42Sp50 Translational
repression would contribute to localized protein
expression after RNA localization has occured It is
not known whether the function of FRGY2 might be
dominant over the one exerted by ePAB and⁄ or how
translational repression would eventually be relieved
Although ePAB and FRGY-2B thus appear to
inter-act with a broad spectrum of mRNAs, 42Sp50 was
ori-ginally identified as one of two proteins that are found
in association with tRNA molecules in the 42S storage particles 42Sp50 is specifically expressed in previtello-genic phases of oogenesis (stage I and II); in structure, the protein is closely related to EF1A and it also exhi-bits aminoacyl tRNA transfer activity [27,37] The finding that 42Sp50 is part of vegetal RNA localization complexes in Xenopus oocytes relates to the more recent demonstration that EF1A is required for the intracellular localization and cortical anchoring of b-actin mRNA in chicken embryonic fibroblasts [38]; furthermore, EF1A was reported previously to interact with components of the cytoskeleton such as actin [39]
On the basis of these observations, it was therefore proposed that the EF1A-actin complex serves as a scaffold for b-actin mRNA anchoring [38] A similar notion might hold true for 42Sp50 in the context of vegetal mRNA localization in Xenopus oocytes; because we classified 42Sp50 as a shuttling protein, it might join the localization complex in the nucleus and mediate anchoring to cortical actin upon arrival of the RNP at the vegetal pole of the oocyte In the context
of these localizing RNPs, but also as integral part of the 42S storage particle, 42Sp50 might exert an addi-tional function for RNA export from the nucleus; however, direct experimental evidence in support of such a notion is currently not available
A
B
Fig 6 Intracellular localization of 42Sp50 in X laevis oocytes (A) 42Sp50 is present in the cytoplasm and in the nucleus The nuclei from stage III oocytes expressing myc-42Sp50 were manually isolated Nuclear (1–10 OE) and cytoplasmic fractions (0.5 and 1 OE) were analyzed
by SDS⁄ PAGE and western blotting using anti-myc serum As a control, the membrane was probed with anti-Vg1RBP and anti-Staufen1 sera Approximately 5% of myc-42Sp50 can be detected in the nucleus, whereas endogenous Vg1RBP or Staufen is not visible in the nuclear fraction (B) 42Sp50 shuttles between nucleus and cytoplasm Myc-42Sp50 was expressed in vitro in rabbit reticulocyte lysate, and radiolabelled with [ 35 S]methionin The reticulocyte lysate was injected either into the nucleus or the cytoplasm of stage VI oocytes After incubation for 0, 3 and 5 h, respectively, the nuclei and cytoplasms of 15 oocytes per timepoint were manually separated Myc-42Sp50 was recovered from the cytoplasmic and nuclear fractions by immunoprecipitation using anti-myc serum, separated by SDS ⁄ PAGE and analyzed
by phosphoimaging As a positive control, ribosomal protein L5 was co-injected Although 42Sp50 is exported from the nucleus, nuclear import cannot be detected.
Trang 7Because 42Sp50 was originally identified as a tRNA
specific RNA-binding protein and the experiments
con-ducted in the present study had revealed that it is in a
large complex with several different vegetally localizing
mRNAs and localization proteins such as Staufen1
and Vg1RBP, we also tested for direct binding of
42Sp50 to the LEs of different vegetal mRNAs For
this purpose, lysate from stage III and IV oocytes
expressing myc-tagged 42Sp50 was employed for UV
crosslinking experiments; however, no direct
interac-tion of 42Sp50 with the different LEs could be
demon-strated (data not shown) These negative results
indicate that 42Sp50 is either not in direct contact with
the LEs or that it associates with a different region of
the corresponding mRNAs To address the function of
42Sp50 in the process of vegetal mRNA localization
more directly, we aimed to generate dominant negative
effects by microinjection of various deletion mutants
of 42Sp50 into stage III⁄ IV oocytes However, reporter
RNA localization was found not to be affected in
these experiments (data not shown); because another
assay for the putative dominant negative activity of
the 42Sp50 deletion constructs is unavailable, the
inter-pretation of these observations remains elusive
Experimental procedures
Plasmids
The expression plasmids used in the TAP technique
(pCS2+-N-TAP-Vg1RBP and pCS2+-C-TAP-Vg1RBP)
were generated by inserting the coding sequence of the
TAP tag derived from pZome-1-N or pZome-1-C
(Euro-scarf, Heidelberg, Germany) into the BamHI and EcoRI
sites of the pCS2+ plasmid The full length coding region
of Vg1RBP (allele D, obtained from J Yisraeli) [40] was
ligated into the EcoRI site of the resulting pCS+-N-TAP
and pCS2+-C-TAP vectors The full length coding regions
of Xenopus 42Sp50 and ePAB were cloned from EST clones
of the NIBB X laevis project into the EcoRI and XbaI
sites of pCS2+-MT [41] The full length coding region of
FRGY-2B was amplified from oocyte cDNA using the
primers FRGY-2B_F: 5¢-GGAATTCCATGAGTGAGGC
GGAACC-3¢ and FRGY-2B_R: 5¢-GGTCTAGACAGCG
ACTGAGTTCATTCTG-3¢ and ligated into the EcoRI and
XbaI sites of pCS2+-MT
Oocyte microinjection and cultivation
Oocytes were obtained surgically from X laevis females,
defolliculated in 2.5 mgÆmL)1 Blendzyme 3 (Roche,
Mannheim, Germany) and stages III–IV were sorted
according to size [42] Cap-RNA was prepared from
pCS2+-TAP-Vg1RBP or pCS2+-myc-42Sp50, which were linearized with NotI using the mMessage mMachine kit (Ambion, Austin, TX, USA) and purified using the RNeasy kit (Qiagen, Hilden, Germany) Oocytes were injected with
15 nL of RNA (250–500 ngÆlL)1) each and incubated in
1· NaCl ⁄ Mes (10 mm Hepes, pH 7.4), 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mm MgSO4, 0.41 mm CaCl2, 0.66 mm KNO3) at 18C for 20–24 h Oocytes were har-vested in batches of 100, shock-frozen in liquid nitrogen and stored at)80 C until further use
For import and export assays, stage VI oocytes were injected with radioactively-labelled proteins produced in reticulocyte lysate programmed with pCS2+-myc-42Sp50
or pCS2+myc-L5 [43] and incubated in 1· NaCl ⁄ Mes at
18C for up to 6 h After incubation, nuclei were manually isolated with forceps The red colour of the reticulocyte lysate served as an indicator for successful nuclear injection Nucleus and cytoplasm of 15 oocytes per time point were homogenized in 500 lL of NET-2 buffer (50 mm Tris⁄ HCl,
pH 7.4, 150 mm NaCl, 0.05% Nonidet P-40), supplemented with protease inhibitors (Roche) and incubated with pro-tein-G-sepharose coupled anti-myc sera for 1 h at room temperature The immunoprecipitates were washed three times with NET-2, separated by SDS⁄ PAGE and analyzed
by phosphoimaging
Oocyte extract preparation and immuno-precipitation
Oocytes were lysed in IPP145 buffer (50 mm Tris⁄ HCl, pH 8.0), 145 mm NaCl, 0.05% NP-40, 5% glycerol, 1 mm phe-nylmethanesulfonyl fluoride, Proteinase Inhibitors (Roche)
in diethylpyrocarbonate-treated water) at 5 lL per oocyte equivalent (OE) After centrifugation at 16 000 g, yolk pro-teins were removed from the supernatant (S16) by Freon extraction (DuPont, Wilmington, DE, USA) The extract
of 100 OE was incubated with 1 lL of anti-myc serum for 1–
2 h at 4C and precipitated with 15 lL Protein-A-sepharose (GE Healthcare, Milwaukee, WI, USA) for 1 h up to over-night at 4C If indicated, 5 lL of RNase A (10 mgÆmL)1) was added together with the antibody Immunopellets were washed with ice-cold IPP145, mixed with 30 lL of 2· SDS loading dye and analyzed by western blotting
IgG affinity chromatography For the large scale IgG pulldown, 1000 oocytes (approxi-mately 9 mg of total protein) were used The oocytes were lysed in 5 lL of IPP145 per OE and Freon-extracted S16 lysate was prepared Three hundred microlitres of IgG-sepharose was added and incubated at 4C overnight The IgG-pellet was then washed in ice-cold IPP145 and subsequently in TEV digestion buffer (50 mm Tris, pH 7.5,
50 mm NaCl, 0.1% NP40, 5% glycerol) The TEV digest
Trang 8was performed in 6 mL of TEV digestion buffer using
150 U of AcTEV (Invitrogen, Karlsruhe, Germany) at
16C for 4 h To elute the proteins from the IgG beads,
the NaCl concentration was increased to 150 mm and the
mixture was incubated for another 2 h at 16C The
volume of the eluate was reduced by ultracentrifugation
through Vivaspin columns (Sartorius, Go¨ttingen,
Ger-many), the proteins were precipitated with trichloroethane,
and visualized on an 8–16% SDS gel by colloidal
Coomas-sie staining Protein bands were picked manually and
pro-cessed for MS protein identification For the western blot
analysis of the IgG purified complexes, 100 oocytes
expres-sing either TAP tag alone or TAP-Vg1RBP, as well as
uninjected oocytes, were used, in accordance with the
affi-nity purification protocol described above
SDS/PAGE, western blot analysis and colloidal
Coomassie staining
For western blot analysis, proteins were separated by 10%
SDS⁄ PAGE and electroblotted onto nitrocellulose
membrane Proteins were detected using antibodies against
myc-tag (9E10; Sigma, St Louis, MO, USA), Vg1RBP
[J Yisraeli (University of Cambridge, UK)], Staufen1
[N Standart (Hebrew University, Jerusalem, Israel)],
40LoVe [I Mattaj (EMBL, Heidelberg, Germany)],
anti-hnRNPI (4E11; Antibodies-online,
http://www.antibodies-online.com), HuR (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), Calmodulin-binding peptide (Upstate
Biotech-nology, Lake Placid, NY, USA) and GAPDH (Abcam,
Cambridge, MA, USA)
For staining with colloidal Coomassie, the gel was fixed
in 10% acetic acid and 40% ethanol for 1 h, washed twice
in distilled water and incubated in freshly prepared staining
solution overnight The staining solution was prepared as a
stock containing 0.1% (w⁄ v) Coomassie Brilliant Blue
G250, 2% (w⁄ v) ortho-phosphoric acid and 10% (w ⁄ v)
ammonium sulfate Four parts of this stock were mixed
with one part methanol and used immediately
Identification of proteins by MS
Manually excised gel plugs were subjected to an automated
platform for the identification of gel-separated proteins
[44] as described in recent large-scale proteome studies
[45,46] An Ultraflex MALDI-TOF-mass spectrometer
(Bruker Daltonics, Bremen, Germany) was used to acquire
both peptide mass fingerprint and fragment ion spectra,
resulting in confident protein identifications based on
sequence information and peptide mass Database searches
in the NCBI nonredundant primary sequence database
restricted to the taxonomy X laevis were performed using
the mascot, version 2.0 (Matrix Science, Boston, MA,
USA) with the parameter settings described previously
[45,46] All datasets were researched without taxonomy
restriction to account for potential matches to sequences from Xenopus tropicalis The minimal requirement for accepting a protein as identified was at least one peptide sequence match above homology threshold in coincidence with at least four peptide masses assigned in the peptide mass fingerprint
Co-immunoprecipitation and RT-PCR analysis
In total, 200 myc-42Sp50 expressing stage III–IV oocytes were lysed in 800 lL of IPP145 S16 lysate was prepared Three hundred microlitres of S16 were used for immuno-precipitation with anti-myc serum As a control, the same amount S16 was processed in parallel without antibody Precipitated proteins and RNAs were eluted by short incu-bation in IPP145 containing 1% SDS and 5 lgÆmL)1 glyco-gen RNAs were isolated by phenol⁄ chloroform extraction and NH4+ acetate⁄ ethanol precipitation The RNA pellet was washed in 80% ethanol, dried and resuspended in
20 lL of diethylpyrocarbonate-treated water Thirty micro-litres of S16 were used for the isolation of total RNA employing the same protocol
In total, 1.5 lL of precipitated RNA or 0.3 lL (2%) of total RNA were reverse-transcribed in a 10 lL reaction Some 2.5 lL of cDNA were analyzed by quantitative PCR
in a 25 lL reaction using the iQ SYBR Green Supermix and the iCycler system (Bio-Rad, Munich, Germany) The primers used for the amplification have been described pre-viously [20]
To determine the specific enrichment of the RNAs ana-lyzed, the 2)DCTmethod [47] was used The enrichment factor
F was calculated as: F = E1· E2⁄ E3 (E1= 2)[CT (myc-IP) )
CT (total)]
, E2= 2)[CT (myc-IP) ) CT (control ) IP)], E3=
2)[CT (control ) IP) ) CT (total)]) as reported previously [20] The enrichment factor for each RNA was normalized to GAPDH, which was set to 1
Acknowledgements
The authors would like to thank J Yisraeli (Hebrew University, Jerusalem, Israel) for providing the a-Vg1RBP antibody; N Standart (University of Cambridge, UK) for providing the a-XStaufen 1 antibody; K Czaplinski and I W Mattaj (EMBL, Heidelberg, Germany) for providing the a-40LoVe antibody; and Andreas Nolte for help with the DNA sequencing This work was supported by funds from the Deutsche Forschungsgemeinschaft to T.P
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