Báo cáo khoa học: Annexin A2 binds to the localization signal in the 3¢ untranslated region of c-myc mRNA ppt

Annexin A2 binds to the localization signal in the 3¢ untranslated region of c-myc mRNA Ian Mickleburgh 1 , Brian Burtle 1 , Hanne Holla ˚ s 2 , Gill Campbell 3 , Zofia Chrzanowska-Lightowlers 4 , Anni Vedeler 2 and John Hesketh 1 1 School of Cell and Molecular Biosciences, University of Newcastle, UK 2 Department of Biomedicine, University of Bergen, Norway 3 Rowett Research Institute, Aberdeen, UK 4 School of Neurology, Neurobiology and Psychiatry, University of Newcastle, UK The delivery of newly synthesized proteins to their site of function is crucial for normal cell function. There is now evidence that in an increasing number of specific cases this not only involves targeting signals within proteins, but also signals in mRNAs resulting in their localization and translation in different cytoplasmic compartments [1–4]. Messenger RNA localization is observed during early development in Drosophila and Xenopus, in highly polarized neurones and glial cells [5–7], and in fibroblasts [8–10]. Such mRNA localiza- tion is dependent on cis-acting sequences almost exclu- sively found in the 3¢ untranslated regions (3¢UTRs) of the mRNAs concerned [7–14]. Using transfected cell lines expressing chimaeric gene constructs, 3¢UTR sequences have been found to be capable of targeting a reporter sequence to different cytoplasmic sites and to the cytoskeleton [8–13]. Complementary experiments have shown that removal of the appropriate 3¢UTR Keywords cytoskeleton; mRNA localization; RNA- binding protein; targeting; 3¢UTR Correspondence J. Hesketh, School of Cell and Molecular Biosciences, University of Newcastle, Newcastle-upon-Tyne NE1 7RU, UK Fax: +44 191 222 8684 Tel: +44 191 222 8744 E-mail: j.e.hesketh@ncl.ac.uk (Received 1 September 2004, revised 20 October 2004, accepted 15 November 2004) doi:10.1111/j.1742-4658.2004.04481.x Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis-acting sequences in the 3¢ untranslated regions (3¢UTRs) of the mRNAs concerned acting together with trans-act- ing proteins. The C-MYC transcription factor is a proto-oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c-myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3¢UTR. Here we show the specific binding of a trans-acting factor to the perinuclear localization ele- ment in the 3¢UTR of c-myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross-linking experiments showed that pro- teins in fibroblast extracts formed complexes with the region of c-myc 3¢UTR implicated in localization; a protein of  36 kDa exhibited specific, Ca 2+ -dependent binding. Binding was reduced by introduction of a muta- tion that abrogates localization. Using RNA-affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA–protein complex formed by cell extracts was further retarded by anti-(annexin A2). Purified annexin A2 bound to the same region of the c-myc 3¢UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c-myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel func- tional role for the RNA-binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton. Abbreviations DTT, dithiothreitol; MBP, myelin basic protein; MS, mass spectrometry; mRNP, messenger ribonucleoprotein; PVDF, poly(vinylidene fluoride); UTR, untranslated region. FEBS Journal 272 (2005) 413–421 ª 2004 FEBS 413 results in loss of, or altered, localization. For example, the transport and localization of both myelin basic protein (MBP) and b-actin mRNAs require a signal within the 3¢UTR [8,14]. RNA-containing particles are found colocalized with cytoskeletal components [9,14] and there is evidence that mRNAs are transported in RNA granules [15,16]. The detailed mechanisms of this spatial organization of the protein synthetic apparatus and mRNA localization by 3¢UTR signals are still poorly understood, particularly the nature of the pro- teins that bind to these localization signals. In fibroblasts, b-actin mRNA is transported to the cell periphery, whereas mRNAs encoding the tran- scription factors MYC and FOS are localized around the nucleus and are associated with the cytoskeleton [10,13]. In c-myc mRNA the localization signal lies in an 86-nucleotide region within the 3¢UTR and is abro- gated by a mutation in a conserved AUUUA [11]. The RNA-binding protein(s) involved in this retention of the c-myc mRNA on the cytoskeleton around the nuc- leus is not known but it is likely to be distinct from those with roles in the transport and peripheral local- ization of mRNAs such as b-actin and MBP. Here we describe the specific binding of a trans-acting factor to the region of the 3¢UTR of c-myc mRNA previously shown to contain the localization element, and iden- tify this protein as annexin A2. The multifunctional annexin A2 has previously been reported to have RNA-binding properties [17,18] and the data presented here indicate a functional role for such binding and provide evidence for a novel role of this protein in perinuclear localization of mRNA. Results The perinuclear localization element in the c-myc mRNA has previously been mapped to between nucleo- tides 194 and 280 of the 3¢UTR: the b-globin reporter is localized by nucleotides 194–440 (D3) and 194–280 (MW) from the wild-type c-myc 3¢UTR, but not by nucleotides 194–280 in which the AUUUA motif was mutated to AGGGA (MM) [11]. Protein binding to this localization signal was investigated by gel retardation and UV cross-linking assays using RNA transcripts that corresponded to these regions of the 3¢UTR. Gel retardation assays using D3 transcripts showed complex formation with increasing amounts of a S100 cell extract from Ltk – fibroblasts (Fig. 1A). Competitive experiments carried out using [ 32 P]UTP[aP]-labelled D3 transcripts and unlabelled MW transcripts (Fig. 1B) showed that the shorter 86-nucleotide transcripts competed effectively for protein binding to D3 tran- scripts. There was almost total inhibition of complex formation at 80-fold molar excess. In contrast, the presence of mutant MM transcripts, even at 80-fold molar excess, had little or no effect on protein A B Fig. 1. RNA–protein complex formation monitored by gel retardation assay. Complex formation was studied using [ 32 P]UTP[aP]-labelled D3 RNA (nucleotides 194–440 of c-myc 3¢ UTR) and S100 extract from Ltk – fibroblasts. (A) RNA was incubated with increasing amounts of S100 protein extract (1–5 lg). Complex formation is observed with 2 and 5 lg. (B) Complex formation was studied using [ 32 P]UTP[aP]-labelled D3 RNA (500 Bq) in the presence of 10–80-fold molar excess of unlabelled competitor RNA, either MW or MM transcripts as indicated. Annexin A2 binds to localization signal in c-myc mRNA I. Mickleburgh et al. 414 FEBS Journal 272 (2005) 413–421 ª 2004 FEBS binding to the D3 transcripts, showing that the MM transcripts did not compete for protein binding. These experiments indicate that one or more proteins capable of binding to nucleotides 194–440 of the c-myc 3¢UTR are present in cytoplasmic extracts of Ltk – fibroblasts and that this binding involves nucleotides 194–280 implicated in localization. Furthermore, the data indicate that the conserved AUUUA found necessary for localization [11] is necessary for full binding activity. The proteins binding to this region of the c-myc 3¢UTR were further investigated by UV cross-linking of RNA–protein complexes followed by SDS ⁄ PAGE. As shown in Fig. 2A, the D3 RNA exhibited binding to two major proteins and one other minor compo- nent. Comparison of the mobility of these proteins in SDS ⁄ PAGE with that of molecular mass standards indicated that the major proteins were of approximate molecular mass 36 and 50 kDa, with the minor protein of  90 kDa. The same pattern of binding was observed with a cytoskeletal fraction (from fibroblasts) that is known to be enriched in c-myc mRNA [9,19] but no binding was observed with cytosolic or endo- plasmic reticulum fractions (Fig. 2B). Binding of the proteins to nucleotides 194–280 was investigated by carrying out competition experiments in which the cell extract was incubated with both [ 32 P]UTP[aP]-labelled D3RNA and increasing amounts of unlabelled MW prior to cross-linking. As shown in Fig. 2(A), increas- ing amounts of unlabelled MW reduced the binding of RNA to the 36 kDa protein such that binding was reduced by 20-fold excess of competitor and was almost undetectable in the presence of 80-fold molar excess. In contrast, the 80-fold molar excess of unla- belled MW had comparatively little effect on binding to the other proteins. A similar excess of a nonspecific RNA, the c-myc coding region, had no effect on bind- ing (results not shown). These data indicate that the 36 kDa protein bound to the 194–280 region of the 3¢UTR. These observations were extended using shorter transcripts (nucleotides 205–280 of the c-myc 3¢UTR), RNase T 1 digestion and gel retardation. As shown in Fig. 3, these shorter transcripts also formed a complex with S100 extracts and complex formation was unaffected by an excess of homoribopolymer A and C (lanes 6, 7) but abolished by homoribopoly- mer G (lane 8) and to a lesser extent by homoribo- polymer U (lane 9); it was also Ca 2+ sensitive (compare lanes 2 and 4 with lanes 3 and 5). In addi- tion, formation of radiolabelled complex disappeared after incubation with unlabelled MW competitor (lane 10), but not after incubation with MM (lane 11). Again these data support the view that complex formation is due to binding of a protein to the 3¢UTR implicated in localization. Biotinylated RNA transcripts linked to streptavidin- coated magnetic beads were used to isolate proteins binding to the region of c-myc 3¢UTR implicated in localization. The beads were incubated with S100 B A Fig. 2. UV cross-linking analysis of proteins binding to c-myc 3¢UTR. (A) [ 32 P]UTP[aP]-labelled D3 (nucleotides 194–440 of c-myc 3¢UTR) RNA was incubated either with S100 extract alone or with a 2–80-fold molar excess of unlabelled competitor MW RNA (nucleo- tides 194–280 of c-myc 3¢UTR). After UV cross-linking, samples were subjected to SDS ⁄ PAGE and RNA–protein complexes were detected by the presence of radioactive bands. In the absence of competitor the D3 RNA formed a complex with two major proteins, one of  36 kDa (indicated by arrowhead) and one of  50 kDa, and a minor protein. The presence of competitor MW RNA reduced the complex formation between RNA and the 36 kDa protein but had no effect on complex formation with the larger protein. (B) [ 32 P]UTP[aP]-labelled D3 RNA was incubated with protein from a cytosolic (lane 1), cytoskeletal (lane 2) or membrane fraction (lane 3), or with S100 extract (lane 4), and after UV cross-linking, sam- ples were subjected to SDS ⁄ PAGE. Note that complex formation occurred with proteins, including a 36 kDa protein (arrow), in the cytoskeletal fraction. I. Mickleburgh et al. Annexin A2 binds to localization signal in c-myc mRNA FEBS Journal 272 (2005) 413–421 ª 2004 FEBS 415 extract from mouse Ltk – fibroblasts and after removal of excess extract and stringent washing, bound mater- ial was released and subjected to SDS ⁄ PAGE. In the absence of Ca 2+ the major protein bound to the RNA beads was of  50 kDa (Fig. 4A, lane 1). Following incubation in the presence of 1 mm Ca 2+ the major Fig. 3. RNA–protein complex formation with nucleotides 205–280 of the c-myc 3¢UTR. Gel retardation using [ 32 P]UTP[aP]-labelled D205 RNA (12 fmoles; nucleotides 205–280 of 3¢UTR) and 1 lg of protein from an Ltk – fibroblast S100 extract. RNase T 1 digestion was performed after the binding reaction. Lane 1 contains free probe and lanes 2–5 show retardation complex formed (arrowhead) with extract in a buffer either containing 40 m M or 120 mM NaCl and in the absence and presence of 1 mM CaCl 2 . The effects of competition with a 100-fold mass excess of homoribopolymers poly(A), poly(C), poly(G) and poly(U) are shown in lanes 6–9, respectively. Poly(G) and poly(U) dramatically reduce the calcium-dependent gel retardation complex. In lanes 10 and 11 a 160-fold molar excess of MW (nucleotides 194–280 of c-myc 3¢UTR) and MM (nucleotides 194–280 of c-myc 3¢UTR with AGGGA mutation), respectively, were used to compete with D205 RNA for protein binding. Note that MW competes much more effectively than MM. A B C Fig. 4. Isolation of proteins binding to nucleotides 205–280 of the c-myc 3¢UTR in the absence and presence of calcium. Proteins from an Ltk – S100 extract (1 mg) were incubated with biotinylated D205 RNA anchored to SA-PMP beads (see Experimental procedures) or to control SA-PMP beads with no RNA attached. Ten microlitres of unbound proteins (from binding solution after incubation) and half the volume of eluted proteins were separated by 12.5% (w ⁄ v) SDS ⁄ PAGE. (A) and (C) show gels stained with Coomassie Brilliant Blue and in (B) western blotting analysis was performed with monoclonal anti-(annexin A2) IgG at a 1 : 5000 dilution. In (A) and (B) the RNA-bound proteins and the unbound proteins (first wash) recovered in the absence of calcium are shown in lanes 1 and 2, respectively, and the RNA-bound and unbound proteins recovered in the presence of calcium are shown in lanes 3 and 4, respectively. In (C) Lane 1 shows proteins eluted from SA-PMP alone (no biotinylated RNA) in the presence of calcium compared with the eluate from D205 RNA-bound SA-PMP (lane 2). Black arrowheads indicate 36 kDa protein and white arrowhead points to 50 kDa protein. Annexin A2 binds to localization signal in c-myc mRNA I. Mickleburgh et al. 416 FEBS Journal 272 (2005) 413–421 ª 2004 FEBS protein bound was of  36 kDa (Fig. 4A, lane 3) and the 50 kDa band was less intense. Under these condi- tions the 36 kDa protein did not bind to control beads with no RNA (Fig. 4C). Because UV cross-linking indicates the specific bind- ing of a 36 kDa component to this region of the 3¢UTR and RNase T 1 digest ⁄ gel retardation shows Ca 2+ sensitivity of complex formation, our further analysis focused on the 36 kDa component of the pro- teins recovered in the eluates from the RNA beads. Western blotting (Fig. 4B) suggested that this major protein was annexin A2, an observation consistent with the observed Ca 2+ sensitivity of specific complex formation, the previously observed RNA-binding properties of annexin A2 [17,18] and the competition by poly(G) (Fig. 3; cf. [18]). The band corresponding to this major 36 kDa component was excised from the gel, digested with trypsin and subjected to MALDI- TOF ⁄ MS. Comparison of the digestion pattern with the available database confirmed that this protein band corresponds to mouse annexin A2. Taken together, the western blotting and MS data show that the 36 kDa protein that is present in mouse fibroblast extracts and which binds to nucleo- tides 205–280 of c-myc 3¢UTR is annexin A2. Gel retardation experiments with cell extracts in the pres- ence of anti-(annexin A2) showed that such antibod- ies caused increased retardation of the complex or ‘supershift’ (Fig. 5), but that this did not occur with a comparable concentration of a control IgG. Demonstration of this supershift with anti-annexin provides further evidence that annexin A2 is present in the complex formed by fibroblast extracts with the c-myc 3¢UTR transcripts. In further experiments, purified annexin A2 demon- strated the ability to bind to c-myc transcripts in vitro. The c-myc transcripts corresponding to either exon 3 (which contains both coding and 3¢UTR regions), the 3¢UTR or the region of the 5¢UTR containing the first 496 nucleotides, were labelled in vitro with [ 32 P]UTP[aP] and incubated with immobilized ann- exin A2 heterotetramer on nitrocellulose membranes. As shown in Fig. 6(A), transcripts of exon 3 or only the 3¢UTR bound to annexin A2, whereas the 1–496- nucleotide transcript of exon 1 did not interact. Fur- thermore, purified annexin A2 bound to labelled MW transcripts corresponding to the 194–280-mucleotide 3¢UTR region but markedly less (57%) to the mutant MM transcripts (Fig. 6B). There was essentially no binding of annexin A2 to control antisense transcripts (10% of binding to MW). These data indicate that, in vitro, annexin A2 binds to myc transcripts that con- tain 3¢UTR sequences. Discussion Using two independent methods, namely gel retarda- tion assays and UV cross-linking, the experiments presented here provide evidence for the existence of a protein of  36 kDa in fibroblast cell extracts that binds specifically to the region of the 3¢UTR previ- ously implicated in the localization of c-myc mRNA [11]. RNA-affinity experiments followed by MS iden- tified this protein as annexin A2, supershift assays showed annexin A2 to be present in the complexes formed by the fibroblast extracts and c-myc 3¢UTR transcripts, and in vitro experiments indicated that purified annexin A2 binds to this region of the c-myc 3¢UTR. In addition, assays with both cell extracts and purified annexin A2 indicated that the conserved Fig. 5. The effect of anti-(annexin A2) IgG on RNA–protein complex formation with nucleotides 205–280 of the c-myc 3¢UTR. Binding reactions were carried out using [ 32 P]UTP[aP]-labelled D205 RNA (12 fmoles; nucleotides 205–280 of 3 ¢UTR) and 2 lg of protein from an Ltk – fibroblast S100 extract in the presence of 120 mM NaCl and 1m M CaCl 2 . Following RNase T 1 digestion, 0.5 lg of antibodies was added and incubated with RNA-bound proteins w here indica- ted. Gel retardation was performed and complexes were separated for 4 h by native PAGE. Lanes 1 and 2 contain labelled D205 RNA in the absence and presence of Ltk – protein, respectively, with the RNP complex indicated with the white arrowhead. Anti-(annexin A2) IgG caused a supershift of the complex formed by Ltk – proteins (lane 3, black arrowhead). There was no apparent supershift by anti-biotin (lane 4). No complex is formed by anti-(annexin A2) IgG with D205 RNA in the absence of Ltk- proteins (lane 5). I. Mickleburgh et al. Annexin A2 binds to localization signal in c-myc mRNA FEBS Journal 272 (2005) 413–421 ª 2004 FEBS 417 AUUUA motif within this region of the 3¢UTR was necessary for full binding of the protein. These data correlate closely with earlier in situ hybridization data showing that not only is the 86-nucleotide region spanning nucleotides 194–280 in the c-myc 3¢UTR sufficient to target b-globin to the perinuclear cytoplasm and the cytoskeleton, but also that the AUUUA element is required for this targeting ability and for localization of c-myc mRNA [11]. Thus, the data suggest that annexin A2 is involved in the association of c-myc mRNA with the cytoskeleton and its localization. It has previously been shown that annexin A2 is recovered in a fraction released from the cell matrix by 130 mm KCl [17,18]. This fraction also contains cyto- skeletal components such as actin, messenger ribo- nucleoproteins (mRNPs) including polysomes, and specific mRNAs such as c-myc [9,17,19]. The observa- tion that the 36 kDa protein which binds to the local- ization signal in c-myc 3¢UTR is recovered in such a cytoskeletal fraction but not in the cytosolic or membrane fractions (Fig. 2B) is consistent both with the binding protein being annexin A2 and with previ- ous observations that c-myc mRNA is recovered in this fraction. Annexins are multifunctional proteins that can inter- act with both membranes and the cytoskeleton [20,21]. It has been proposed that these interactions and the resulting localization of annexins, including A2, can be modulated by post-translational modifications [21]. Annexin A2 interacts in a Ca 2+ -dependent manner with the two cytoskeletal proteins F-actin and non- erythroid spectrin [22]. Both the monomeric and tetra- meric forms of annexin A2 are able to associate with F-actin in the presence of Ca 2+ [20]. In addition, it has been suggested that annexin A2 is an integral member of mRNP complexes [17,21]. For example, UV cross- linking and immunoprecipitation of annexin A2 fol- lowed by phenol extraction revealed that annexin A2 was directly associated with small RNA sequences [21] that were most likely degraded mRNAs. Further stud- ies have shown that annexin A2 is present only in mRNPs associated with the cytoskeleton, either in the form of actively translating mRNPs in cytoskeleton- bound polysomes or inactive mRNPs [17]. Taken together with the ability of annexins to bind to F-actin, the observations that annexin A2 binds RNA and is an integral component of mRNP complexes [17,21] suggest that it may act as a linker between certain mRNAs and the actin filament system. However, it is likely that only a subfraction of annexin A2 has this function [21]. While this study was in progress it was observed that annexin A2 binds to c-myc mRNA [18]. Our data extend this observation by showing that the binding is to a specific region within the 3¢UTR implicated in mRNA localization and association with the cytoskele- ton, consistent with the finding that c- myc mRNA is translated on cytoskeleton-bound polysomes [19]. Few trans-acting factors involved in association of mRNAs with the cytoskeleton or in mRNA localiza- tion have been identified in mammalian cells. ZBP1 and hnRNPA2 have been implicated in b-actin and MBP mRNA localization [14,23], whereas HAX1 and eEF1c bind the region of the 3¢UTR of vimentin mRNA [24] implicated in localization [25]. The involve- ment of a different protein, annexin A2, in c-myc mRNA localization may reflect the different location of c-myc mRNA (perinuclear cytoplasm and cytoskele- ton) and ⁄ or different interactions with the cytoskeleton – with actin microfilaments in the case of c-myc and with intermediate filaments for vimentin. In conclusion, the data presented here indicate that annexin A2 binds to the 3¢UTR of c-myc mRNA and that the binding is to the defined section of the 3¢UTR B A Fig. 6. The binding of different c-myc transcripts to purified annexin A2. (A) Annexin A2 2 p11 2 heterotetramer (0.75, 1.5 and 3.0 lg) was immobilized on nitrocellulose membranes and the binding of 2 fmo- lÆmL )1 (5000 Bq) of uniformly [ 32 P]UTP[aP]-labelled c-myc tran- scripts was performed as described in Experimental procedures. Transcripts corresponded to nucleotides 1–496 of the 5¢UTR (lane 1), exon 3 (lane 2), and the 3¢UTR (lane 3). (B) 2 lg of annexin A2 2 p11 2 heterotetramer was incubated with 2 fmoles of MW (nucleotides 194–280 of c-myc 3¢UTR; lane 1), MM (nucleotides 194–280 of c-myc 3¢UTR with AGGA mutation; lane 2) or antisense MW (lane 3) transcripts. Incubation of MW with BSA was included as a negative control (lane 4). The binding was performed in solu- tion, 1 l gÆlL )1 yeast tRNA being present to prevent nonspecific RNA binding, and this was followed by UV cross-linking, RNase treatment and 10% (w ⁄ v) SDS ⁄ PAGE. Binding was visualized using a Canberra Packard Instant Imager. Migration position of annexin A2 is indicated by an arrowhead. Annexin A2 binds to localization signal in c-myc mRNA I. Mickleburgh et al. 418 FEBS Journal 272 (2005) 413–421 ª 2004 FEBS previously implicated in localization [11]. Our hypothe- sis is that the RNA-binding properties of annexin A2 have a novel functional role in perinuclear localization of mRNA and the association with the cytoskeleton. Further studies are in progress to investigate the role of other proteins in the perinuclear localization RNP complex. Experimental procedures Subcloning of fragments of the c-myc 3¢UTR and in vitro transcription Three sequences from the mouse c-myc 3¢UTR, namely bases 194–440 (D3), 194–280 containing a conserved AUUUA (MW), and 194–280 with a three-base change within the AUUUA sequence (MM) were transferred from vectors PM13 delta3, pSVc-myc1 and pSVc-mycSK ⁄ CL [11] into pBluescriptII SK (Stratagene, Amsterdam, the Netherlands) so as to maintain the RNA polymerase sites. Vector sequences 3¢ of the T7 promoter (including a tract of seven C residues) and bases 194–205 of the 3¢UTR sequence were removed from the MW construct by diges- tion with XhoI and KpnI to generate D205 which contains bases 205–280 of the 3¢UTR. Radiolabelled D3 transcripts were synthesized from linearized vectors using RNA Tran- scription Kit (Stratagene) and [ 32 P]UTP[aP] (800 CiÆ mmol )1 ). Templates for the transcription of MW, MM and D205 were generated by PCR with forward (5¢-TGAGCGC GCGTAATACG-3¢) and reverse (5¢-GCCCTATTTACAT GGAAAATTGG-3¢) primers and products purified using QIAquick columns (Qiagen, Crawley, Sussex, UK). Tran- scripts were labelled with [ 32 P]UTP[aP] (800 CiÆmmol )1 ) using a MAXIscript kit (Ambion, Austin, TX, USA), extracted with phenol ⁄ chloroform and precipitated with ethanol. Incorporation of radionucleotide into RNA was assessed by scintillation counting and unlabelled RNA was quantified spectrophotometrically; integrity was verified by denaturing gel electrophoresis. Transcripts corresponding to nucleotides 1–496 at the 5¢-end of c-myc mRNA were generated by in vitro transcription from the cDNA contain- ing exons 1 + 2 of the mouse c-myc gene in pBluescript SK. A 362 bp fragment encoding the 3¢UTR of mouse c-myc mRNA was synthesized by PCR from pBluescript containing the complete genomic mouse c-myc gene (a gift from T McDonnell, University of Texas, Houston, TX, USA) as template with forward (5¢-TACTGCAGACT GACCTAACTCGAGGAGG-3¢) and reverse (5¢-GCGGA ATTCTATGGTACATGTCTTAAAATC-3¢) primers con- taining a PstI site and an EcoRI site, respectively. This PCR product was inserted between the corresponding sites of the pGEM 3Zf + vector and used for in vitro transcrip- tion. All constructs were sequenced in both directions to confirm the orientation and sequences of the inserts. Cell extracts Ltk – fibroblasts were grown to  90% confluence in Dul- becco’s modified Eagle’s medium supplemented with 10% fetal calf serum and in a humidified atmosphere of 5% CO 2 at 37 ° C. S100 protein extracts were prepared following the method of Behar et al. [7] with modifications. Cells were resuspended in lysis buffer (130 mm NaCl, 5 mm MgCl 2 , 30 mm Tris ⁄ HCl pH 7.6, 2 mm dithiothreitol [DTT]) con- taining 0.5% (v ⁄ v) Nonidet P-40 and EDTA-free protease inhibitor cocktail (Roche, Lewes, East Sussex, UK), and lysed by passing them through a 21-gauge needle seven times. Large debris were removed by centrifugation at 5000 g for 10 min and the supernatant fluid was diluted with 3 vol. of 40 mm NaCl lysis buffer and centrifuged at 100 000 g for 1 h, 10% (v ⁄ v) glycerol was added to the supernatant fluid before freezing in aliquots in liquid nitro- gen. Cytosolic, cytoskeletal and membrane fractions were prepared using a sequential detergent ⁄ salt extraction proce- dure as described previously [9,26]. Cell pellets were resus- pended in 1 mL of buffer F (10 mm Tris, pH 7.6, 0.25 m sucrose, 25 mm KCl, 5 mm MgCl 2 , 0.5 mm CaCl 2 ) contain- ing 0.05% (v ⁄ v) Nonidet P-40, and after 10 min at 4 °C, the suspension was centrifuged at 1000 g for 5 min. The supernatant fluid (cytosolic fraction) was removed and after one wash in buffer F the pellet was resuspended in 1 mL of buffer F containing 130 mm KCl and 0.05% (v ⁄ v) Noni- det P-40, incubated for 10 min and centrifuged at 2000 g for 10 min. The supernatant fluid (cytoskeletal fraction) was removed, membrane components of the pellet solubi- lized by incubation in buffer F containing 130 mm KCl, 0.5% (v ⁄ v) Nonidet P-40 and 0.5% (w ⁄ v) deoxycholate for 10 min and the membrane fraction collected by centrifuga- tion at 3000 g for 10 min. Gel retardation and UV cross-linking assays Gel retardation reactions were carried out with 1–5 lg S100 extract and 500 Bq of 32 P-labelled RNA in binding buffer (10 mm Hepes, pH 7.6, 3 mm MgCl, 40 mm NaCl, 5% (w ⁄ v) glycerol, 1 mm DTT and 10 lg tRNA) in a total volume of 10 lLat22°C for 30 min. For competition experiments, labelled and unlabelled RNA were added simultaneously. Products were separated on 5% (w ⁄ v) non- denaturing polyacrylamide gels (60 : 1, 1· TBE at 20 VÆcm )1 for 3 h). For gel retardation analysis combined with T 1 treatment, digestion was carried out by adding 40 units of RNase T 1 to the binding reaction and incuba- tion continued for 5 min before the addition of 2 lLof 20% (w ⁄ v) Ficoll. For supershift assays, 0.5 lg mouse anti- (annexin A2) IgG or a control IgG (antibiotin) was added to binding reactions after RNase T 1 digestion and incuba- ted for 30 min at 4 °C prior to the addition of Ficoll. Complexes were separated by electrophoresis for 2 h at 20 VÆcm )1 through 5% (w ⁄ v) nondenaturing polyacrylamide I. Mickleburgh et al. Annexin A2 binds to localization signal in c-myc mRNA FEBS Journal 272 (2005) 413–421 ª 2004 FEBS 419 (79 : 1, 0.5· TBE) gels. Gels were dried and analysed by autoradiography. UV cross-linking reactions were carried out with 10–15 lg protein and 5000 Bq of 32 P-labelled RNA in 10 lL of binding buffer containing 100 mm NaCl and 125 ng tRNA. Heparin (5 mgÆmL )1 ) was then added and the incubation continued for 5 min. The reaction tubes were then placed on ice and irradiated in a Spectrolinker with 2 · 960 mJ to cross-link protein–RNA complexes. Unprotected RNA was then removed by incubation with 4 lg RNase A and 12 units RNase T 1 for 15 min at 37 °C, and the samples subjected to SDS ⁄ PAGE [27]. Protein isolation using paramagnetic beads A 0.6 mL aliquot suspension of prewashed MagneSphere streptavidin-coated paramagnetic particles (SA-PMP, Promega, Southampton, UK) was resuspended in 0.1 mL 0.5· NaCl ⁄ Cit containing 100 l g bovine serum albumin (BSA) and 100 lg yeast tRNA and incubated at  20 °C for 1 h with shaking. The suspension was washed twice with 0.3 mL 0.5· NaCl ⁄ Cit and then incubated with 20 lg biotinylated D205 transcripts (labelled with biotin-16-UTP [Roche] and produced by in vitro transcription) in 0.3 mL 0.5· NaCl ⁄ Cit for 10 min at room temperature. Unbound RNA was removed by washing twice with 0.3 mL 0.5· NaCl ⁄ Cit. SA-PMP with RNA bound were then incubated at 4 °C for 1 h with 1 mg cell extract in 40 mm NaCl lysis buffer containing 0.5 mgÆmL )1 yeast tRNA, 0.2 mgÆmL )1 BSA, and 800 unitsÆmL )1 RNasin (Promega), with or with- out 1 mm CaCl 2 , in a total volume of 0.5 mL. After 5 · 1 mL washes with 40 mm NaCl lysis buffer, proteins bound to the RNA were eluted by boiling in 45 mm Tris ⁄ HCl, pH 6.8, 10% (w ⁄ v) glycerol, 1% (w ⁄ v) SDS, 1% (v ⁄ v) 2-mercaptoethanol and 0.01% (w ⁄ v) bromophenol blue. Western blotting and mass spectrometry Proteins were separated by SDS ⁄ PAGE using a 12.5% (w ⁄ v) acrylamide separating gel. These were either stained with Coomassie Brilliant Blue or the proteins were trans- ferred to a poly(vinylidene fluoride) (PVDF) membrane by semidry electroblotting. Bands of interest from Coomassie- stained gels were excised for in-gel trypsin digestion followed by MALDI-TOF ⁄ MS (carried out by J Gray, Institute for Cell and Molecular Biosciences, University of Newcastle, UK). Proteins transferred to PVDF membranes were incubated with a monoclonal antibody to annex- in A2 (BD Transduction Laboratories, 1 : 5000 dilution) following the manufacturer’s instructions. After incuba- ting with anti-(mouse horseradish peroxidase-conjugated) serum (Sigma-Aldrich UK, Poole, Dorset, UK), the blot was developed using the POD chemiluminescence kit (Roche). Chemiluminescence was detected by exposure to Kodak X-Omat A2-5 film. RNA–annexin A2 binding assays The heterotetrameric annexin A2 2 p11 2 complex was puri- fied from pig intestinal epithelium [28]. mRNA filter bind- ing assays, using in vitro transcribed RNA and immobilized native annexin A2 tetramer, were carried out as described previously [17], except that 1 · Denhardt’s solution [0.02% (w ⁄ v) Ficoll, 0.02% (w ⁄ v) polyvinylpyrrolidone, 0.02% (w ⁄ v) BSA] was added to the RNA binding solution (10 mm triethanolamine, pH 7.4, 50 mm KCl, 1 mm DTT, 2mm MgSO 4 ,1mm CaCl 2 and 1 lgÆlL )1 of yeast tRNA), supplemented with 20 UÆmL )1 RNasin (Promega), to reduce the background. Membranes were incubated with 2 fmoleÆmL )1 of transcript for 20 min at room temperature, washed rapidly three times and then a further four times for 15 min in binding solution lacking yeast tRNA and Denhardt’s solution. The RNA binding was quantified and visualized using a Canberra Packard Instant Imager (Perkin Elmer, Pangbourne, UK). mRNA–annexin A2 binding assays in solution were performed as described by Kwon and Hecht [29]. Purified in vitro transcribed mouse c-myc RNA probes were heated at 72 °C for 3 min and cooled slowly to room temperature. mRNA (2 fmol; 5000 Bq) was incubated with the annexin A2 2 p11 2 complex in RNA binding solution, supplemented with 20 UÆmL )1 RNasin (Promega) containing 2.5% (w ⁄ v) Ficoll for 20 min in a final volume of 20 lL. After incubation, the RNA probes were covalently cross-linked to the proteins by exposure to UV light and RNase T 1 and A were then added for 30 min at 37 °C to digest unprotected RNA (see above). Nucleo- tide–protein complexes were separated from degraded mRNA probe by SDS ⁄ PAGE [27], the gels dried and mRNA–protein binding visualized using a Canberra Packard Instant Imager. 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Proc Natl Acad Sci USA 88, 3584–3588. I. Mickleburgh et al. Annexin A2 binds to localization signal in c-myc mRNA FEBS Journal 272 (2005) 413–421 ª 2004 FEBS 421 . that annexin A2 binds to the 3¢UTR of c-myc mRNA and that the binding is to the defined section of the 3¢UTR B A Fig. 6. The binding of different c-myc transcripts to purified annexin A2. (A) Annexin A2 2 p11 2 heterotetramer. that binding of annexin A2 to the localization signal in the c-myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel func- tional role for the. due to binding of a protein to the 3¢UTR implicated in localization. Biotinylated RNA transcripts linked to streptavidin- coated magnetic beads were used to isolate proteins binding to the region