Báo cáo khoa học: Two splicing isoforms of the Y-box protein ctYB-1 appear on the same mRNA molecule

Two splicing isoforms of the Y-box protein ctYB-1 appear on the same mRNA molecule Dmitry Nashchekin, Sergej Masich, Teresa Soop, Alexander Kukalev, Elizaveta Kovrigina, Oxana Nashchekina and Bertil Daneholt

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden

Keywords mRNA transport; mRNP; protein isoforms; YB-1; Y-box proteins

Correspondence B. Daneholt, Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden Fax: +46 8 313529 Tel: +46 8 524 87370 E-mail: bertil.daneholt@ki.se

(Received 31 August 2006, revised 2 November 2006, accepted 7 November 2006)

doi:10.1111/j.1742-4658.2006.05576.x

Y-box proteins constitute an evolutionarily conserved family of DNA- and RNA-binding proteins involved in the regulation of transcription and translation. In the dipteran Chironomus tentans, a homologue to the verte- brate Y-box protein YB-1 was recently characterized and designated ctYB-1. It is transferred from nucleus to cytoplasm bound to mRNA and is likely to affect translation. It appears in two size variants, p40 and p50. We fur- ther analysed the two size variants and their interaction with mRNA. Southern blot analysis, in situ hybridization and RT-PCR analysis sugges- ted that there is just one YB-1 gene, and that the two size variants repre- sent splicing isoforms. In a C. tentans epithelial cell line, only p40 is present, whereas both variants appear together in eight tissues from fourth- instar larvae, although in somewhat different proportions. Furthermore, the appearance of the two isoforms was studied in relation to a specific 35– 40 kb mRNA transcript in the salivary glands, the Balbiani ring mRNA. Because of their exceptional size, Balbiani ring messenger ribonucleoprotein particles in nucleoplasm and Balbiani ring polysomes in cytoplasm could be identified and selectively studied. We were able to establish that both isoforms are associated with both nuclear and cytoplasmic Balbiani ring mRNA. In addition, a p50-specific antibody coimmunoprecipitated p40 from Balbiani ring polysomes, suggesting that the two splicing isoforms are located along the same Balbiani ring mRNA molecule. The functional sig- nificance of the two isoforms is being discussed.

they are abundant

(masked) messenger

Y-box binding (YB) proteins constitute a family of evolutionarily conserved, multifunctional and nucleic acid-binding proteins [1–3]. They obtained their name from the early observation that one member of the family, the human YB-1, bound to the Y-box sequence in the promoter region of MHC class II genes [4]. Fur- ther investigations revealed that several YB proteins are single-stranded, DNA-binding transcriptional fac- tors involved in the activation and repression of many genes [5].

Many YB proteins bind to mRNA and affect the translation of mRNA. These proteins become associ- ated with mRNA cotranscriptionally and accompany it

from the gene to the cytoplasm [6,7]. It was noted in translationally early on that repressed ribonucleoprotein (mRNP) particles in germ cells of amphibians and ver- tebrates [8–11]. Later on, it was shown that the YB proteins are required for male and female fertility in mammals [12]. YB-1 is a major component of cyto- [13]. Verte- plasmic mRNPs in somatic cells as well brate YB-1 is required for optimal translation: it is essential for initiation of mRNA translation in vitro [14] and protects mRNA from 5¢-end degradation [15]. However, at high concentrations YB-1 inhibits the initiation of protein synthesis [16–18]. YB proteins are

Abbreviations BR, Balbiani ring; CSD, cold shock domain; mRNP, messenger ribonucleoprotein; YB, Y-box binding.

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supposed to exert their effect on translation by modify- ing the mRNP structure making mRNA more or less available for translation [10,13].

and p50 [6]. The variants share the first 258 amino acids, although the C-termini differ; the p40-specific terminus comprises six amino acids and the p50-specific terminus comprises 59 amino acids. It has previously been shown that ctYB-1 binds mRNA cotransciprio- nally and accompanies it from the gene to polysomes [6]. Here, we further study the appearance of the two variants in the salivary gland cell, in particular in rela- tion to the BR mRNA. First, we demonstrate that there is only one YB-1 gene, suggesting that the two size variants are splicing isoforms. We also show that the variants are expressed in all larval tissues studied, although at different proportions. Both variants are associated with BR mRNA in the nucleus and cyto- plasm, and they appear together along the same BR mRNA transcript.

YB proteins consist of three domains, a middle, highly conserved, 80 amino acid, cold shock domain (CSD) surrounded by more variable N- and C-terminal domains. Remarkably, the vertebrate CSD is > 45% identical to bacterial cold shock proteins [19]. The CSD is a five-stranded b-barrel containing RNP1- and RNP2-like consensus motifs that recognize both DNA and RNA [2,20,21]. The N-terminal domain is rich in alanine and proline. In vertebrates and some inverte- the C-terminal domain contains alternating brates, clusters of basic and acidic amino acids [6,9]. The C-terminal tail of YB proteins contributes to the bind- ing of DNA and RNA and also mediates protein– protein interactions [2,22].

Results

The two ctYB-1 variants are encoded by a single copy gene

The N- and C-termini of YB proteins can vary con- siderably, not only among species, but also within a given species. More than one gene could be present, but alternative splicing could also contribute to the in mouse somatic cells at variability. For example, least two different YB proteins are expressed, YB-1 ⁄ MSY1 and DBPA ⁄ MSY3. Moreover, MSY3 appears as two splicing variants MSY3S and MSY3L [23]. In germ cells, the situation is even more complicated. Here, all three somatic proteins are expressed as well as two isoforms of the germ-cell-specific MSY2 [11]. Thus, a total of five YB protein variants are expressed in germ cells and three in somatic cells [23].

To examine whether the two ctYB-1 size variants are generated from one or two genes, we performed South- ern blot analysis and in situ hybridization (Fig. 1). For Southern blot analysis, C. tentans genomic DNA was digested with restriction enzymes (Not, EcoRI or Hind III). Digested DNA was fractionated in an 0.8% agarose gel and hybridized with a ctYB-1 cDNA probe. Single bands were observed with Not and HindIII, which do not cut the ctYB-1 cDNA (Fig. 1A, lanes 1 and 3). Two bands were detected with EcoRI, which cleaves the ctYB-1 cDNA once (lane 2). In the latter experiment, most of the cDNA probe corresponded to the lower DNA band explaining the relatively high intensity of this band. We conclude that Southern blot analysis suggested that the ctYB-1 gene is a single copy gene.

The significance of the many YB variants is still unclear. Many different roles are ascribed to YB pro- teins and the YB proteins are present throughout development and in essentially all cell types in the organism. To better understand the role of each of individual variant it is essential to study when and where they appear in the organism and in what context they are located within the cell.

banding

and

the

results

To localize the ctYB-1 gene on C. tentans chromo- somes, in situ hybridization was performed on polytene chromosomes using squash preparations of salivary gland cells. The ctYB-1 cDNA was labelled with dig- oxigenin and used as a probe and visualized with a fluorescently labelled digoxigenin antibody. As shown in Fig. 1B, only a single band was observed, and it was mapped close to the end of chromosome III (region 1C)2A). The location of the probe was estab- lished using cytological analysis of the whole chromo- some set in 10 specimens (the presence and position of a nucleolus, distribution of constrictions along the pattern). Taken chromosome together, from Southern blot analysis and in situ hybridization show that there is only one ctYB-1

In this study, we investigated two size variants of the Y-box protein YB-1 in the dipteran Chirono- mus tentans, and in particular we analysed the beha- viour of the variants in salivary glands [24]. The nuclei of salivary gland cells contain polytene chromosomes with transcriptionally active regions blown up as puffs. A few giant puffs, called Balbiani rings (BRs), generate a transcript of exceptional size (35–40 kb), which is packed with proteins to an mRNP particle, 50 nm in diameter. The flow of this large mRNP can be fol- lowed from gene to polysomes using an electron microscope [24]. Nuclear BR mRNP particles and cytoplasmic BR polysomes can be isolated and ana- lysed for mRNA-associated proteins. The C. tentans YB-1, designated ctYB-1, has two size variants, p40

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A

B

C

Fig. 1. Evidence for a single ctYB-1 gene. (A) Southern blot analysis of C. tentans genomic DNA using a 32P-labelled ctYB-1 cDNA probe. Genomic DNA was digested with the indicated restriction enzymes and hybridized with the ctYB-1 probe. The positions of the molecular size markers (in kb) are indicated on the left. (B) In situ hybridization of a digoxigenin-labelled ctYB-1 cDNA probe to C. tentans polytene chromo- somes. The probe was detected with a rhodamine-labelled anti-DIG Ig. A single red band can be seen close to the end of one chromosome (III). DNA was stained with DAPI (blue). (C) RT-PCR on salivary gland total RNA using primers specific to p40 (lane 1) and p50 (lane 2) coding sequences. The positions of the molecular size markers (in bp) are indicated on the left.

gene, suggesting that the two ctYB-1 variants are enco- ded in the same gene.

and stomach (lane 3) p50 is the predominant variant, whereas in intestine (lane 4), colon (lane 5) and ima- ginal discs (lanes 6–8) both variants are present in approximately equal amounts. As expected, tissue cul- ture cells were devoid of the p50 variant (lane 9). The higher mobility of the two variants in the intestine sample may be due to divergent processing but is per- haps more likely caused by site-specific degradation during preparation of the sample.

To explore whether there are separate transcripts corresponding to the two size variants of YB-1, we performed RT-PCR experiments with salivary gland RNA and proper primers (see Experimental proce- dures). As shown in Fig. 1C, we found that the PCR products corresponded in size to the coding sequence of p40 mRNA and that of p50 mRNA (lane 1 and 2, respectively). We conclude that the two YB-1 size vari- ants are likely to be splicing isoforms.

When salivary gland extract was treated with alka- line phosphatase, we noted a slight but distinct shift in mobility for both p40 and p50, indicating that both variants are to some extent phosphorylated (Fig. 2B).

Both ctYB-1 variants are expressed in many larval tissues

ctYB-1 splicing variants are present in nucleus as well as in cytoplasm and are associated with BR mRNA in both compartments

We have previously shown that p40 alone is present in tissue culture cells, whereas both p50 and p40 are expressed in salivary gland cells [6]. To reveal how the two YB-1 splicing isoforms are expressed in other lar- val tissues, we prepared protein extracts from various tissues of C. tentans fourth-instar larvae and carried out SDS ⁄ PAGE and western blot analysis using an antibody recognizing both the p40 and p50 variants [6]. As shown in Fig. 2A, p40 and p50 were present in all samples, but in different proportions (lanes 1–8). In salivary glands (lane 1), Malpighian tubules (lane 2)

It has previously been shown using immunocytology and immunoelectron microscopy that ctYB-1 is present in the nucleus and is abundant in the cytoplasm [6]. To also elucidate whether the two splicing variants of ctYB-1 appear in both compartments, nuclear and cytoplasmic extracts from salivary gland cells were studied using SDS ⁄ PAGE and western blot analysis (Fig. 3). Both proteins were present in the nucleus, but

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B

Fig. 2. Tissue distribution of ctYB-1 isoforms. (A) Western blot analysis of ctYB-1 splicing variants in protein extracts from C. tentans tissues. Extracts were subjected to SDS ⁄ PAGE followed by western blotting using a p40 ⁄ 50 antibody. The following larval tissues were examined: salivary glands (lane 1), Malpighian tubules (lane 2), cardia chamber of stomach (lane 3), intestine (lane 4), colon (lane 5), imaginal disc from thorax (lane 6), male genital imaginal disk (lane 7) and female genital imaginal disk (lane 8). An extract from C. tentans tissue culture cells was studied in parallel (lane 9). Approximately the same amount of protein was loaded in each lane as shown with a Coomassie brilliant blue-stained control gel (data not shown). (B) Alkaline phosphatase treatment of a protein extract from salivary glands. Lane 1, nontreated extract; lane 2, treated extract.

there was essentially no cross-contamination between the nuclear and cytoplasmic samples.

To study the YB-1 variants in relation to a specific mRNA, nuclear BR mRNP particles were extracted from salivary gland cells and sedimented in a sucrose gradient according to Wurtz et al. [27]. The BR RNPs appeared as a 300S peak (Fig. 4A) and were collected and bound to oligo(dT) cellulose. The proteins were released, separated by SDS ⁄ PAGE, and analysed by western blotting using the p40 ⁄ 50 antibody. Both p40 and p50 could be detected in BR RNP (Fig. 4B). Thus, both ctYB-1 isoforms copurify with the nuclear BR RNPs and are likely to be bound to nuclear BR mRNA.

Fig. 3. The two ctYB-1 splicing variants are present in both nucleus and cytoplasm of salivary glands cells from C. tentans. Nuclear (N) and cytoplasmic (C) samples were extracted from salivary glands as described in Experimental procedures. The nuclear sample and 1 ⁄ 50 of the cytoplasmic sample were subjected to SDS ⁄ PAGE and subsequent western blot analysis. Antibodies against p40 ⁄ 50 (lanes 1–2), hrp45 (lanes 3–4) and tubulin (lanes 5–6) were used. The hrp45 protein served as marker for nucleus and tubulin for cytoplasm. The molecular size (kDa) markers are indicated on the left.

In the cytoplasm, BR mRNA is associated with giant BR polysomes ((cid:2) 100 ribosomes per polysome) [28]. Polysomes from salivary gland cells were released and sedimented in a sucose gradient (Fig. 5A). To ver- ify the accuracy of the fractionation we examined the polysomes in one heavy fraction (fraction 8) and one light fraction (fraction 11) in the electron microscope. As expected, in fraction 8 we observed giant polysomes and in fraction 11 smaller ones (Fig. 5A, inserts). Pro- teins in each tube were examined by SDS ⁄ PAGE and western blot analysis (Fig. 5B). Both p40 and p50 were present in fractions containing the giant BR polysomes (Fig. 5B, lanes 7–8) as well as in fractions containing smaller polysomes (Fig. 5B, lanes 10–11). Both vari- ants were indeed associated with polysomes, because disruption of polysomes with EDTA (Fig. 5A) also shifted p40 and p50 to the top of the gradient (Fig. 5C). We conclude that the two ctYB-1 splicing

were much more abundant in the cytoplasm (lane 1 versus 2; cf. added amounts). As expected, the control protein hrp45 was confined to the nucleus (lanes 3–4) and tubulin to the cytoplasm (lanes 5–6), showing that

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A

A

B

B

C

Fig. 4. Both p40 and p50 are recorded in BR mRNP particles. (A) Sucrose gradient sedimentation of BR RNP particles. Isolated saliv- ary glands were incubated in the presence of [a-32P]ATP, and the labelled BR RNP particles were released by homogenization and centrifuged in a sucrose gradient. Fractions were collected from the bottom of the gradient, and the radioactivity was determined in each fraction by Cerenkov counting. The BR particles appeared as a 300S peak in the gradient. (B) Western blot analysis of YB-1 vari- ants in BR RNPs. The 300S peak fractions (4–9) were incubated with oligo(dT) cellulose, and the proteins were eluted with sample buffer, resolved by SDS ⁄ PAGE and analysed be western blotting using a p40 ⁄ 50 antibody.

variants in giant polysomes are likely to be associated with the BR mRNA in polysomes.

Fig. 5. Both p40 and p50 are present in polysomes. (A) Sucrose gra- dient analysis of polysomes from salivary glands. Polysomes were extracted from salivary glands and sedimented in a sucrose gradient as described in Experimental procedures. Fraction numbers are from bottom to top of the gradient. The absorbance in each fraction was measured at 254 nm. Electron micrographs of polysomes from different parts of the gradient are shown as inserts. Untreated extract (––––); extract treated with 20 mM EDTA (– – –). (B,C) West- ern blot analysis of YB-1 variants along the sucrose gradient. Pro- teins in each sucrose gradient fraction were resolved by SDS ⁄ PAGE and analysed by western blot analysis using a p40 ⁄ 50 antibody. Untreated extract (B), EDTA-treated extract (C).

The two ctYB-1 splicing variants appear on the same BR mRNA molecule

heavy chain of the antibody (lane 3). We concluded that p40 and p50 appear on the same BR mRNA molecule (heavy polysomes). The ability of the p50 antibody to coimmunoprecipitate p40 not only from BR polysomes but also from lighter polysomes sug- gests that the two ctYB-1 variants are also present on other types of mRNA.

Discussion

To study whether p40 and p50 appear together on the same BR mRNA molecule, we raised an antibody against the last 59 amino acids of the C-terminus of p50. Although the p40 ⁄ 50 antibody recognized both proteins in a polysomal extract (Fig. 6A, lane 2), the p50 antibody stained only p50 (Fig. 6A, lane 1). We then collected heavy and light polysomes and immuno- precipitated the polysomes with the p50 antibody bound to Sepharose beads. The immunoprecipitated proteins were eluted from the beads, separated by SDS ⁄ PAGE, and analysed by western blotting using the p40 ⁄ 50 antibody (Fig. 6B). Both the p40 and p50 variants were precipitated with the p50 antibody from the heavy (lane 1) as well as light polysomes (lane 2). The third band in the western blot corresponds to the

In this study we analysed expression of the p40 and p50 isoforms of the C. tentans Y-box protein ctYB-1. We have shown using in situ hybridization and South- ern blot analysis that there is probably only one ctYB-1 gene in C. tentans, suggesting that the two isoforms

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A

B

MSY3S mRNA is expressed at high levels in skeletal muscle. MSY3S mRNA is much more abundant than MSY3L mRNA is somatic tissues. Regulation of the expression of the protein variants can take place on many levels, but in the case of splicing variants control is usually exerted on the alternative splicing level [31].

Both ctYB-1 variants are associated with BR mRNA from gene to polysomes

We have previously shown using immunoelectron microscopy and UV cross-linking experiments that YB-1 binds to BR mRNA cotranscriptionally, remains associated with the mRNA during its transfer from the nucleus to cytoplasm, and most likely also enters poly- somes coupled to the mRNA [6]. Our demonstration in this study that the two isoforms p40 and p50 are recorded in both nuclear BR mRNPs and in BR poly- somes suggests that they are both coupled to mRNA in the nucleus as well as in the cytoplasm. Further- more, the two variants remain bound to the mRNA during translation.

Fig. 6. The two YB-1 variants appear in the same polysome. (A) SDS ⁄ PAGE and western blot analysis of a polysomal extract from salivary glands using a p50 antibody (lane 1) and a p40 ⁄ 50 antibody (lane 2). (B) SDS ⁄ PAGE and western blot analysis of heavy and light polysomes immunoprecipitated from the corresponding sucrose gradient fractions with a p50-antibody. The heavy poly- somes comprised fractions 7 and 8 and light polysomes frac- tions 10 and 11 (cf. Fig. 5A). An IgG preparation served as control. The p50 antibody was coupled to Sepharose beads, and after incu- bation with the polysome samples the bound proteins were eluted with SDS, precipitated and analysed by SDS ⁄ PAGE and western blotting using a p40 ⁄ 50 antibody. Heavy polysomes (lane 1), light polysomes (lane 2), and the IgG control (lane 3).

are splicing variants. This conclusion was further cor- roborated by the identification of products of predicted in RT-PCR experiments. Furthermore, we sizes revealed that the p40 and p50 isoforms are ubiqui- tously expressed in several larval tissues, although in different proportions. They are both localized in the nucleus as well as in the cytoplasm. It could be esta- blished that the two ctYB-1 isoforms are associated with BR mRNA both in nuclear mRNP and in cyto- plasmic polysomes. It was finally shown that the two variants appear on the same BR mRNA molecule during translation.

Differential expression of the ctYB-1 variants

Biochemical analysis of nuclear BR mRNPs and BR polysomes suggests that the relative amounts of the two variants are not changed during the transfer of the major BR mRNA from nucleus to cytoplasm; exchange of proteins in the BR mRNP particle during translocation although the nuclear pore [24] does not seem to involve the YB-1 variants. It is noteworthy that the ratio of the variants is the same in BR poly- somes (heavy polysomes), non-BR polysomes (light polysomes), and in the total cell extract. As YB-1 appears cotranscriptionally on a large number of tran- scripts, maybe all, it seems likely that the two variants are present on essentially all mRNA transcripts and presumably in roughly the same proportions. This is in agreement with the observation that YB proteins bind single-stranded RNA rather unspecifically. However, some sequence preference has been reported [21,32– 35], and there might well be subtle differences between individual mRNAs as to their content and organiza- tion of the YB-1 isoforms.

tissues

investigated,

although the

The two ctYB-1 variants appear on the same BR transcript

The two splicing variants of ctYB-1 are present in all larval relative amounts vary to some extent. The p50 variant is the predominant isoform in salivary glands, Malpighian tubules and stomach, whereas p40 and p50 are present in about equal amounts in intestine, colon and imagin- al disks. Such differential expression has also been seen for other YB proteins. The somatic variants of the YB proteins MSY1, MSY3S ⁄ MY1a and MSY3L ⁄ MY1 represent a striking example [23,29,30]. MSY1 mRNA is expressed at high levels in kidney and heart, whereas

The main result of this study is that the two variants of ctYB-1 are associated with the same BR mRNA molecule. This does not necessarily imply that the iso- forms are functionally equivalent. The Drosophila RNA-binding protein How, which controls tendon cell differentiation, provides an interesting example of dif- ferent roles for isoforms during mRNA biogenesis. A

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electrophoresis, the DNA fragments were transferred to a positively charged nylon membrane (Amersham Bioscienc- es, Little Chalfont, UK) and UV cross-linked. The part of ctYB-1 cDNA that is identical in p40 and p50 (nucleotides 1–774) was labelled with 32P-dCTP with the NEBlot kit (New England BioLabs, Ipswich, MA) and used as the probe.

RT-PCR

Reverse-strand cDNA was generated from total RNA isola- ted from salivary glands, using a first-strand cDNA synthe- sis kit (Roche, Basel, Switzerland). PCR products corresponding to the cDNA coding sequences of p40 and p50 were amplified by PCR in the presence of 1.5 mm MgCl2, 200 lm dNTPs and 10 pmoles of the same forward primer (5¢-ATGACAGACACCGAAGCACC), but differ- ent reverse primers (5¢-ATTGCCGCCACGTGG for p40 and 5¢-TTGGACTGGTTGACCACC for p50). PCR pro- ducts were separated by electrophoresis on 0.8% agarose gels and visualized with ethidium bromide.

In situ hybridization of polytene chromosomes

nuclear-specific form, How(L), with 36 extra amino acids in the C-terminus, binds to target mRNA, pre- vents its efficient export to the cytoplasm and induces its rapid degradation. Later on in development a sec- ond isoform, How(S), competes with How(L) for bind- ing to target mRNA, releases the inhibited nuclear export and stabilizes the mRNA; the differentiation arrest induced by How(L) is released [36,37]. Interest- ingly, How(S) and How(L) are coprecipitated with tar- get mRNA [37], suggesting that these two variants are together on the same transcript. In the case of ctYB-1, it remains to be established whether the two isoforms are functionally redundant or whether they play differ- ent roles in BR mRNA biogenesis and ⁄ or translation. The fact that p40 is sufficient to maintain epithelial cells in culture argues in favour of redundancy, but the proper function of YB-1 in the salivary gland of the intact organism could require that both isoforms are present on the same BR mRNA transcript. YB-1 is likely to control the level of translation by changing the conformation of the mRNP template [38], and therefore it is essential how the YB-1 protein is organ- ized in relation to the other proteins associated with mRNA. As the C-terminal domain of YB-1 is respon- sible for interactions with other proteins [2,22], it is possible that the two ctYB-1 isoforms with their diver- gent C-termini could modify the mRNP template differently and that both are required for proper trans- lation.

Experimental procedures

Culturing conditions

Salivary glands were fixed in 45% acetic acid for 5 min and squashed on a slide. The slides were frozen in liquid nitro- gen, the coverslips flipped off, and the preparations were incubated twice in 95% ethanol for 10 min. The slides were kept at 70 (cid:2)C for 30 min in 2· NaCl ⁄ Cit, washed in 2· NaCl ⁄ Cit, treated with 0.07 m NaOH for 90 s, dehydrated and air-dried. Hybridization of the chromosome prepara- tions with a digoxigenin-labelled ctYB-1 probe was carried out as described previously [25]. The probe was detected with a rhodamine-labelled anti-DIG Ig (Roche). DNA was stained with DAPI. Images were taken in an LSM 5120 microscope (Zeiss, Jena, Germany).

Preparation of tissue extracts

Chironomus tentans larvae were raised under laboratory conditions. Fourth-instar larvae were used for the experi- ments. A C. tentans epithelial cell line of embryonic origin was grown in suspension at 24 (cid:2)C as described previously [39].

Antibodies

Salivary glands and other tissues were dissected from fourth-instar larvae, washed with NaCl ⁄ Pi containing 1 mm phenylmethylsulfonyl fluoride and sonicated. Extracted pro- teins were precipitated with acetone, resuspended in sample (10% glycerol, 2% SDS, 40 mm dithiothreitol, buffer 0.02% bromophenol blue in 50 mm Tris ⁄ HCl, pH 6.8), fractionated by SDS ⁄ PAGE (10% polyacrylamide) and analysed by western blotting as described previously [26].

Rabbit p50 antibody was raised against the most C-ter- minal 59 amino acids of p50, affinity purified and used at a dilution of 1 : 1000. Rabbit p40 ⁄ 50 antibody [6] and hrp45 antibody (raised against amino acids 309–322) were affinity purified and diluted 1 : 5000 and 1 : 1000, respectively. Tubulin mAb (Sigma, St Louis, MO) was diluted 1 : 1000.

Preparation of nuclear and cytoplasmic extracts

Southern blot analysis

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Isolated salivary glands were transferred to TKM buffer (100 mm KCl, 1 mm MgCl2 in 10 mm triethanolamine-HCl, pH 7.0), washed once with fresh TKM and incubated in TKM-Triton (TKM containing 1% Triton) twice for 30 s. Restriction enzyme-digested genomic DNA (10 lg) was size in 0.5· TAE. After fractionated in a 0.8% agarose gel

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bers, each with a grid, were put in a tabletop centrifuge and centrifuged for 20 min at 18 000 g with a Microfuge R22 with F241.5P rotor. The grids were removed from the chambers and rinsed briefly in TKM buffer. Samples were stained sequentially with 1% phosphotungstic acid in 95% ethanol for 30 s and 1% uranyl acetate in 95% ethanol for 1 min, washed in a flow of 95% ethanol and air-dried. Spec- imens were examined in a 120 kV Technai microscope at 80 kV. The images were recorded with a 2048 · 2048 TVIPS TemCam-F224HD CCD camera (TVIPS, Gauting, Germany; pixel size 24 lm). The glands were placed in fresh TKM-Triton and disrupted by gentle pipetting to release the nuclei and cytoplasmic components. The released material was collected and centrifuged for 10 min at 2000 g at 4 (cid:2)C with a Microfuge R22 with F241.5P rotor (Beckman Coulter). The resulting supernatant constituted the cytoplasmic extract. The nuclei- containing pellet was washed three times with TKM-Triton, resuspended in TKM-Triton containing 420 mm NaCl, incubated for 30 min on ice and centrifuged for 5 min at 18 000 g at 4 (cid:2)C with a Microfuge R22 with F241.5P rotor. The supernatant was retained as the nuclear extract.

Isolation of BR RNPs and analysis of YB-1

Immunoprecipitation of polysomes

The polyclonal p50 antibody, covalently coupled to NHS Sepharose (Amersham Biosciences), was added to sucrose gradient fractions containing heavy or light polysomes and incubated for 2 h at 4 (cid:2)C with gentle agitation. The beads were washed with NaCl ⁄ Pi containing 0.2% NP-40, and the proteins were eluted with 0.5% SDS, precipitated with acet- one, and analysed by western blotting.

Acknowledgements

BR RNP particles were 32P-labelled in vivo, released from salivary gland cells by homogenization and centrifuged in a sucrose gradient as described previously [27]. The BR RNPs, present as a 300S peak in the gradient, were collec- ted and incubated with oligo(dT) cellulose (Sigma) for 20 min at room temperature, washed with TKE buffer (100 mm KCl, 10 mm EDTA in 10 mm triethanolamine- HCl, pH 7.0), and eluted with sample buffer. BR RNP pro- teins were resolved by SDS ⁄ PAGE and analysed by western blotting.

Isolation of polysomes and analysis of YB-1

The study was supported by the Swedish Research Council, Human Frontier Science Program Organiza- tion, and Knut and Alice Wallenberg Foundation. DN obtained a fellowship from the Swedish Institute.

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Extraction and analysis of polysomes from salivary glands cells were carried out as described previously [28] with some modifications. Isolated salivary glands were incuba- ted in TKM buffer (100 mm KCl, 3 mm MgCl2 in 20 mm triethanolamine-HCl, pH 7.0), containing 0.5% Tween 20, 0.5% sodium deoxycholate, 0.1% b-mercaptoethanol, and 50 lgÆmL)1 cycloheximide for 10 min at 2 (cid:2)C. After centrifu- gation at 2000 g for 5 min at 2 (cid:2)C with a Microfuge R22 with F241.5P rotor, the supernatant polysome extract was loaded onto a gradient of 15–60% (w ⁄ v) sucrose in TKM containing 50 lgÆmL)1 cycloheximide and centrifuged at 37 300 r.p.m. for 30 min in an AH 650 rotor at 2 (cid:2)C (centrifuge model Dis- covery 90 from Sorvall). The gradient was collected in 15 fractions with concomitant measurement of the absorbance at 254 nm. The proteins were precipitated with trichloroace- tic acid and submitted to SDS ⁄ PAGE and western blotting.

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