Báo cáo khoa học: Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes ppt

Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes Jana Loeber 1 , Maike Claußen 1 , Olaf Jahn 2 and Tomas Pieler 1 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 Xenopus oocytes 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)byanti tag coimmunoprecipitation (MI:0007) l MINT-7994335: 42Sp50 (uniprotkb:P17506) physically interacts (MI:0915) with Vg1RBP (uniprotkb: O73932)byanti 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)bytandem affinity purification(MI:0676) l MINT-7994324: frgy2-b (uniprotkb:P45441) physically interacts (MI:0915) with Vg1RBP (uniprotkb: O73932)byanti tag coimmunoprecipitation (MI:0007) l MINT-7994345: 42Sp50 (uniprotkb:P17506 ) physically interacts (MI:0914)withstaufen (uniprotkb: Q5MNU4) and Vg1RBP (uniprotkb:O73932)byanti tag coimmunoprecipitation (MI:0007) l MINT-7994363: 42Sp50 (uniprotkb:P17506), Vg1RBP (uniprotkb:O73932), staufen (uniprotkb: Q5MNU4)and40LoVe (uniprotkb:Q6GM69) colocalize (MI:0403)bycosedimentation 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)bytandem 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. 4722 FEBS Journal 277 (2010) 4722–4731 ª 2010 The Authors Journal compilation ª 2010 FEBS Introduction During 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 J. Loeber et al. 42Sp50 interacts with the RNA localization complex FEBS Journal 277 (2010) 4722–4731 ª 2010 The Authors Journal compilation ª 2010 FEBS 4723 lysate 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-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 AB 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. 42Sp50 interacts with the RNA localization complex J. Loeber et al. 4724 FEBS Journal 277 (2010) 4722–4731 ª 2010 The Authors Journal compilation ª 2010 FEBS reduced 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. J. Loeber et al. 42Sp50 interacts with the RNA localization complex FEBS Journal 277 (2010) 4722–4731 ª 2010 The Authors Journal compilation ª 2010 FEBS 4725 42Sp50, 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. 42Sp50 interacts with the RNA localization complex J. Loeber et al. 4726 FEBS Journal 277 (2010) 4722–4731 ª 2010 The Authors Journal compilation ª 2010 FEBS family 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. J. Loeber et al. 42Sp50 interacts with the RNA localization complex FEBS Journal 277 (2010) 4722–4731 ª 2010 The Authors Journal compilation ª 2010 FEBS 4727 Because 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 NaHCO 3 , 0.82 mm MgSO 4 , 0.41 mm CaCl 2 , 0.66 mm KNO 3 )at18°C 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 18 °C 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 4 °C and precipitated with 15 lL Protein-A-sepharose (GE Healthcare, Milwaukee, WI, USA) for 1 h up to over- night at 4 °C. 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 lLof2· 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 4 °C 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 42Sp50 interacts with the RNA localization complex J. Loeber et al. 4728 FEBS Journal 277 (2010) 4722–4731 ª 2010 The Authors Journal compilation ª 2010 FEBS was performed in 6 mL of TEV digestion buffer using 150 U of AcTEV (Invitrogen, Karlsruhe, Germany) at 16 °C 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 16 °C. 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 NH 4 + 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 )DCT method [47] was used. The enrichment factor F was calculated as: F = E 1 · E 2 ⁄ E 3 (E 1 =2 )[CT (myc-IP) ) CT (total)] , E 2 =2 )[CT (myc-IP) ) CT (control ) IP)] , E 3 = 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. 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