Immunological identity of proteins that bind stored 5S RNA in Xenopus oocytes

Experimental

Immunological

Cell Research 153 (1984) 299-307

Identity of Proteins that Bind Stored 5s RNA in Xenopus Oocytes

PERRY BARRETT, ROSALYN M. JOHNSON and JOHN SOMMERVILLE Department

of Zoology. University of St. Andrews, F(fe KY16 9TS. Scotland

St. Andrew.s,

In small oocytes of Xenopus laevis, the three most abundant proteins are isolated as basic polypeptides with molecular weights of 48 kD (P48), 43 kD (P43) and 40 kD (P40, also known as transcription factor IIIA). All three proteins share common properties in being able to bind specifically ribosomal 5s RNA molecules and influence, in different ways, their rates of production and utilization. It has been shown by biochemical analysis and immunological characterization that the three proteins are structurally distinct and are most probably the products of different genes. Immunostaining and radio-immunoassays indicate that both P48 and P43 have diverged considerably in structure between the amphibian genera Xenopus and Triturus. Antibodies raised against the transcription factor for Xenopus laevis 5s RNA genes (P40/TFIIIA) do not cross-react with the transcription factor isolated from oocytes of the closely related species Xenopus borealis. A protein equivalent of TFIIIA is not found in 5s RNA-containing RNP storage particles of Wturus oocytes. The functions of the three Xenopus oocyte proteins in transporting 5.5 RNA between different cellular compartments are considered in the light of these variations.

The three most abundant proteins of previtellogenic oocytes of Xenopus laeuis have molecular weights of 48 kD (P48), 43 kD (P43) and 40 kD (P40). Each of these proteins can bind 5s RNA [l-3], and P48 and P43 can bind tRNA [3]. P48 and P43 occur in a ribonucleoprotein particle that sediments at 42s and contains stored 5s RNA and tRNA [4-6], P40 occurs in a 7s particle that contains stored 5s RNA [l]. Apart from their role in complexing and stabilizing RNA during early oocyte development, these proteins (or their analogues in other amphibia) have been implicated in additional, yet related processes: P40 acts as a factor that positively regulates transcription of 5s RNA genes [7, 81; P48 has a nuclear function in early stages and may transport 5s RNA from its site of synthesis to the cytoplasm [9, IO]; modified forms of P48 and P43 influence the efficiency of transfer of 5s RNA to the nucleolus for ribosome assembly [l I]. Here we consider the chemical and immunological relationships between these three functionally related proteins and demonstrate that they must be products of different genes. Furthermore, we show that P48 and P43 from Xenopus oocyte 42s particles are antigenically distinct from the analogous proteins of newt (Triturus) species and that P40 from 7s particles is antigenically distinct between X. laevis and X. borealis. Copyright @ 1984 by Academic Press, Inc. All rights of reproduction in any form reserved OO14-482784 $03.00

300 Barrett,

Johnson and Sommerville MATERIALS

AND

METHODS

Animals Xenopus lueuis was bred in our own laboratory, Xenopus borealis was kindly supplied by Dr P. J. Ford, Department of Molecular Biology, University of Edinburgh; Triturus cristatus carnifex was obtained from Bioserv Ltd., Worthing, Sussex; and Triturus uulguris was caught locally.

Isolation

of RNP Particles

Ovary containing previtellogenic oocytes was homogenized and 42s and 7S RNP particles were isolated by sucrose gradient centrifugation as described previously [3, IO].

Chemical Analysis For amino acid analysis electrophoretically purified polypeptides were prepared from RNP particles as described previously [lo], dialysed extensively against distilled water and hydrolysed in 6 N HCI, 0.1 M thioglycolic acid at 110°Cfor 24 h under an atmosphere of NZ. The residues were analysed using an automated single-column analyser. For cyanogen bromide cleavage patterns the electrophoretically purified polypeptides were dissolved in 70% (v/v) formic acid and incubated with a 100 molar excess of CNBr at 20°C for 48 h. The cleavage products were precipitated with 3 vol ethanol at -20°C overnight and the dried pellets were raised in electrophoresis sample buffer [ 121. Electrophoresis was run through 20% polyacrylamideSDS gels at 150 V for 16 h. Polypeptide fragments were located by staining the gel with silver salts ]131.

Antibodies Antibodies were raised against electrophoretically purified P48 and P43 by reconstructing RNP complexes with equimolar amounts of protein and tRNA as described previously [lo] and by injecting 100-200 ug amounts as multiple emulsions into rabbits [9]. Antibodies were raised against P40 by isolating 7S RNP particles from early previtellogenic ovary. The 7S RNP peak was purified by recentrifugation through a second sucrose gradient and this naturally formed RNP complex was used as antigen. P40 was the only polypeptide seen in stained protein gels using this preparation. In no instance were antibodies detected that reacted with the RNA component.

Radio-immunostaining

and Radio-immunoassays

Polypeptides derived from 42s and 7S RNP particles were separated on 12% polyacrylamide-SDS gels [12] and electrophoretically transferred to nitrocellulose paper (Schleicher & Schuell, BA83 0.2 urn pore size) in 192 mM glycine, 50 mM Tris, 20% methanol adjusted to pH 8.3 with HCl [14]. Transfers were made at 30 V, 0.1 A for 16 h. Tracks stained with amido black showed that the polypeptides were transferred in proportion to the amounts loaded. The transferred polypeptides were immunostained as described previously [ 151 with antiserum dilutions of 1 : 20 in 0.9% (w/v) NaCl, 10 mM Tris-HCl, pH 7.4, 5% (w/v) bovine serum albumin. After incubation with antiserum at 20°C for 2 h, the transfers were washed and incubated at 20°C for 45 min in the presence of 0.2 &i/ml [“‘Ilprotein A (>30 mCi/mg, Amersham International, Amersham, Bucks, UK). Autoradiographs were made using Kodak X-omat RP film exposed in contact with dried transfers and intensifier screens at -70°C for 2-6 days. For the radio-immunoassays, 42s and 7S particles were adjusted to 10 ugiml in 0.1 M NaCI, 10 mM sodium phosphate, pH 7.0 (PBS). Aliquots of 100 ul of this antigen solution were added to the wells of microtitre plates (Linbro, Flow Laboratories Inc.) and incubated at 32°C for 1 h then at 4°C overnight. After washing the plates thoroughly in five changes of PBS, free reactive sites were blocked by incubating with diluent (3 % w/v bovine serum albumin in PBS) for 2 h at 32°C. Antisera were diluted in the wells to give a series of 5-fold dilutions, final volume 100 ~1, and incubated at 32°C for 4 h. The plates were washed with PBS as before and incubated overnight at 18°C with 100 ul per well of ?labelled protein A (2.5~ lo5 cpm/ml). After repeated washing with PBS, individual wells were cut out and counted to determine the radioactivity bound. Exp Cd Res 153 (1984)

Oocyte 5S RNA-binding proteins

301

Assay of Transcription Inhibition by Antibodies Immunoglobulin was prepared and microinjected into germinal vesicles of oocytes as described previously [16]. The recombinant plasmid pXlo8 that contains X. laevis genes encoding oocyte-type 5s RNA [17] was kindly supplied by Dr Adrian Bird, Mammalian Genome Unit, University of Edinburgh. The injection mixture contained 160 &ml pXlo8, 5.5 mgiml IgG, 4 mCi/ml ~I[~‘P]GTP (750 Ci/mmole, New England Nuclear) in 44 mM NaCI, 0.5 mM KCI, 7.5 mM Tris-HCI, pH 7.6. Batches of 50-stage 2/3 oocytes of X. laevis were each injected with 20 nl injection mixture and incubated for 18 h at 18°C in Barths solution [17]. RNA extraction and electrophoresis were as described previously [3]. Autoradiographs were set up as described in the previous section and exposed for 3 days.

RESULTS Ribonucleoprotein (RNP) fractions sedimenting at 42s and at 7S-10s were isolated from homogenates of previtellogenic ovary by sucrose gradient centrifugation. Analytical electrophoresis on SDS-polyacrylamide gels resolves two major polypeptide bands from 42s RNP with M, values of 48 kD (P48) and 43 kD (P43) and one major polypeptide band from 7S RNP with an M, value of 40 kD (P40). This latter protein previously has been assigned values of 45.3 kD [l], 42 kD [6] and 37 kD [18] and has been given the functional description of transcription factor (TFIIIA, 7, 8, 18). The polypeptides were eluted in the presence of 1% sodium dodecyl sulfate (SDS) from macerated slices of preparative gels and the purity was checked by re-electrophoresis. All three proteins have poor solubilities, especially after denaturation in SDS. Consequently, they were held in low concentration (0.1%) of SDS and preferably also in 8 M urea, to prevent precipitation prior to further analysis. On renaturing the polypeptides in the presence of 5S RNA (or tRNA where appropriate) specific RNP complexes are formed and solubility is regained thereafter in the absence of SDS [3, IO]. It has been demonstrated previously [6] that P48, P43 and P40 are all basic with P43 and P40 being difficult to resolve completely by 2-D electrophoresis. To demonstrate that structurally different proteins had been isolated for use as antigens, each protein preparation was analysed for amino acid composition and for pattern of cyanogen bromide cleavage. Amino acid analysis of three separate preparations of each of P48, P43 and P40 (not shown) confirmed that they were completely distinct proteins which could not be produced, one from the other, by simple modification such as proteolytic cleavage. This lack of relatedness is demonstrated more simply by comparing cyanogen bromide cleavage patterns (fig. 1). In this gel the cleavage products do not appear in stoichiometric amounts due to the differential effects of silver staining. Also the slight difference in banding pattern of fragments from P40 with previously published patterns for TFIIIA [ 18, 191presumably are due to this differential staining. Nevertheless, it is obvious that there is no similarity between the cleavage patterns of the three proteins P48, P43 and P40. Antibodies were raised against P48, P43 and P40 by injecting purified proteins as RNP complexes into rabbits [lo]. The antisera were reacted with electrophoExp Cell Res 153 (1984)

302 Barrett, Johnson and Sommerville

Fig. 1. Cyanogen bromide cleavage patterns of P48, P43 and P40. Staining intensity with silver salts is

not necessarily proportional to the amounts of material in the bands. M, marker polypeptides with M, values X lo-‘. Fig. 2. Radio-immunostaining of 42s and 7s RNP particle polypeptides using antibodies directed against P48, P43 and P40 with ‘*‘I-labelled protein A. a, 42s particle proteins stained with Coomassie Blue; b, stained 7S particle protein: c, d, transfors of proteins shown in a, b, immunostained with antiP48; e, f, transfers immunostained with anti-P43; g. h, transfers immunostained with anti-P40.

retie transfers of polypeptides from polyacrylamide gels on to nitrocellulose paper [14, 151. Bound antibody was detected by further binding of ‘251-labelled protein A to the antigen-antibody complexes followed by autoradiography. As can be seen from fig. 2, anti-P48 reacts strongly with P48 from 42s RNP particles but not with P43 from 42s particles (fig. 2, c) nor with P40 from 7s particles (fig. 2, d). Similarly, anti-P43 is specific for P43 (fig. 2, e) and anti-P40 is specific for P40 (fig. 2, h). This lack of cross-reaction indicates that the three proteins are antigenically dissimilar. Furthermore, the antibodies used are polyclonal and react with a range of determinants in each protein. For instance, all three antibody preparations bind between two and four of the CNBr fragments (see fig. 1) derived from the protein used as antigen and both anti-P48 and anti-P43 each bind two naturally-produced cleavage products of P48 and P43, respectively [ 111. Therefore none of the antigenic determinants detected so far is shared between any of the three proteins. It might be expected that proteins which appear to have critical functions in RNA transcription and metabolism, particularly in recognizing and binding distinct DNA sequences [7] and RNA conformation [IO] should be structurally conserved. It has been demonstrated previously that analogous proteins are found in 42s and 7s particles isolated from a range of amphibian and teleost species (for review, see [20]). Cross-reactivity of antibodies raised against X. laevis proteins was tested in reactions with both RNP particles and separated Exp Cell Res 153 (1984)

Fig. 3. Radio-immunoassays showing the capacities of RNP species to bind antibodies directed against P48, P43 and P40. P43 from X. laeuis; (c) P48 from T. cristafrrs; (4 P40 from X. and particles from 0. X. laeuis; 0, X. borealis: 0, T. cristutm A. X. luevis;

particles from Xenopus and Triturus Antisera to ((I) P48 from X. lueuk; (h) laevis were assayed for binding to 42s n , T. uulguris; and to 7s particles from

V. X. borealis.

proteins isolated from the other amphibian species, X. borealis, Triturus cristatus and T. vulgaris. Radio-immunoassays were performed to determine the binding of antibodies to antigens in their native (RNP) configuration. The results show that, whereas binding of anti-P48 to 42s particles from the two Xenopus species is similar, binding to 42s particles from the Triturus species is much reduced, the level of cross-reaction being equivalent to an antibody-binding capacity less than li20th of the homologous reaction (fig. 3 a). Similar results are obtained using anti-P43 (fig. 3 6). The simplest interpretation is that both 42s particle proteins have diverged in structure between the Xenopus and Triturus lineages. This view is confirmed by using antibodies directed against T. cristatus particle proteins, which reveal antigenic homology with particles from T. vulgaris but lack of homology with particles from the Xenopus species (fig. 3 c). Exactly comparable results were obtained using the radio-immunostaining procedure, demonstrating that proteins that have been denatured, and at best only partially renatured in the absence of RNA, are recognized to the same relative extent as are native structures. A series of immunostainings is shown in fig. 4. Using antibodies raised against the protein constituents of 42s RNP particles, in general, cross-reaction occurs only within the Xenopus and Triturus genera. The one exception is an occasional and slight cross-reaction of antibodies to X. laevis P48 with the analogous protein from Triturus 42s particles (not shown). Thus the same patterns of protein identity are recognized in both the assay and staining techniques. Surprisingly, however, results with the 7S particle protein are quite different. Exp Cd

Res 153 11984)

304 Barrett, Johnson and Sommerville

Fig. 4. Radio-immunostaining

of RNP particle proteins from Xenopus and Tvirurus species using antibodies directed against P48, P43 and P40. a, 42s particle proteins from X. laeuis, X. borealis, T. cristarus and T. vulgaris immunostained using antiserum to P48 from X. laeuis. b, 42s particle proteins as in a, immunostained using antiserum to P43 from X. lueuis. c, 42s particle proteins as in a, immunostained using antiserum to P48 from T. cristutus; d, 7S particle proteins from X. luevis and X. borealis and 42s particle proteins from T. cristafus and T. vulgaris immunostained using antiserum to P40 from X. lueuis. M,, molecular weight X IO-’ of marker polypeptides. Fig. 5. Assay of 5S RNA synthesis after microinjection of antibodies into germinal vesicles of X. laevis. In addition to 5S RNA genes (pXlo8) and a[32P]GTP, each batch of oocytes was injected with either a, pre-immune IgG; b, IgG from anti-P40; c, IgG from anti-TFIIIA of X. borealis. After incubation, oocytes were homogenized and samples containing the same amount of radioactivity ( IO5 cpm) were taken for RNA extraction and electrophoresis. Comparison of the three treatments is made by autoradiography.

Using 7s particles in radio-immunoassays (fig. 36) and the separated protein in radio-immunostaining (fig. 4 6), no cross-reaction was detected between X. laevis anti-P40 and the X. borealis protein. It has been noted also that antibodies raised against a gel-purified 7S particle protein from X. borealis do not cross-react with the X. laevis protein in radio-immunostaining experiments (pers. comm., K. Rose & P. J. Ford). Thus the protein that binds 5S RNA to form a 7s RNP particle has diverged in structure to such an extent that it is no longer recognized by antibodies directed against the equivalent protein from the other Xenopus species. That our preparation of anti-P40 is indeed directed against TFIIIA is indicated by its inhibitory action on transcription of 5s RNA genes (fig. 5). The assay system involves simultaneous injection of pXlo8 DNA, IgG and a[32P]GTP into germinal vesicles of living oocytes of X. laevis. RNA extracted from oocytes injected with pre-immune IgG shows a strong signal of 32P-labelled 5s RNA (fig. 5, a). Whereas antibodies directed against TFIIIA of X. borealis have no inhibitory effect (fig. 5, c), there is a 4- to 5-fold reduction in synthesis of 5s RNA in oocytes injected with anti-P40 (fig. 5, b). That inhibition is not general is shown by similar levels of labelling in all three tracks of tRNA due to transcription of endogenous genes. Thus the effects of anti-P40 appear to be directed primarily Exp Cell Res 153 (1984)

Oocyte 5S RNA-binding proteins

305

against transcription of 5s RNA genes and show species specificity in the binding of protein factors. The quantification of transcription inhibition and the effects of other antibodies will be described in detail elsewhere. The true significance of the 7S storage particle is questioned by the further finding that no substantial amounts of 5s RNA accumulate in this RNP form in Triturus oocytes (unpubl. data). Consequently no protein analogous to P40 (TFIIIA) can be detected in the newt species, even when whole homogenates of Triturus oocytes are analysed, and no further comparison could be made. Neither of the 42s particle proteins of T. cristatus or T. vulgaris binds anti-P40 (figs 3d, 46). DISCUSSION By all criteria examined, the three proteins that are associated with stored 5s RNA in Xenopus oocytes, P48, P43 and P40, are structurally distinct. This agrees with the findings that chymotrypsin digestion patterns for P43 and TFIIIA (P40) are different [8] and that antibodies directed against TFIIIA do not cross-react with 42s RNP particles [18]. More recently it has been demonstrated that a preparation of anti-P48 does not cross-react with the other particle proteins [21]. The results presented here establish that there can be no simple precursorproduct relationship between any pair of proteins. The characterization of this set of proteins is critical in establishing the roles that they play in the transcription and metabolism of 5s RNA and tRNA. During amphibian oogenesis, 5s RNA and tRNA molecules move between various cellular compartments. For instance, 5s RNA is transported from transcription sites to nucleoplasm, then to cytoplasmic storage particles and tinally to ribosome assembly sites (nucleoli). Each of these steps probably is governed by the form of protein bound to the RNA. In principle, transfer of RNA between compartments can occur through either modification of the binding protein or exchange of binding proteins. Both processes are known to occur: in previtellogenie oocytes 5s RNA is bound to P48 in 42s RNP particles [3] and to P40 in 7s RNP particles [l]. During vitellogenesis about 80% of the 5s RNA is transferred to the nucleoli and incorporated into ribosomes in association with protein that is neither P48 nor P40 (i.e., exchange has occurred) whereas the remainder of the 5s RNA persists as a 7s storage particle 1221in association with an M,. 33 kD cleavage product of P48 [3] (i.e., modification by cleavage has occurred). Although cleavage products are produced from P48 and from P43 [ 1I], we can detect in immunoblotting experiments no higher molecular weight precursors of these proteins themselves. Even analysis of whole homogenates of ovary containing very early oocyte stages reveals no immunostained bands of higher molecular weight. These observations also suggest that there is no stable polyprotein precursor that contains more than one of P48, P43 and P40. It is reasonable to suppose that P48, P43 and P40 are each encoded by separate Exp Cell Res 153 (1984)

306 Barrett,

Johnson and Sommerville

genes which, because of their related functions, may have arisen by duplication but have now diverged to an extent whereby they express proteins of distinct antigenic structure. Divergence in structure is seen also when the functionally analogous proteins from different amphibia are compared. There appears to be little antigenic relatedness between the proteins isolated from toads (Xenopus) and the corresponding proteins isolated from newts (Triturus). However, within the genus Xenopus and within the genus Triturus, P48 and P43 appear to have diverged little in structure, both proteins being indistinguishable between species in the immunological assays performed in this report. This contrasts with the finding that P40 shows no antigenic relatedness between the two closely-related Xenopus species and has no detectable analogue in the genus Triturus. The immunological distinction between P40 of X. laevis and X. borealis in antibody binding and transcription inhibition is particularly strange because of their identical function. It is expected that at least the RNA-binding sites are conserved structural features of the proteins. It is possible that recognition of these determinants is precluded by the use of RNP structures as antigens. The role of two separate RNP storage forms of 5s RNA in Xenopus oocytes, with P40 in the 7S particle and with P48 (and P43 and tRNA) in the 42s particle, is not apparent. The one type of (42s) storage particle in Triturus represents a simpler situation but does not exclude the presence of small amounts of a P40 analogue acting as a transcription factor of 5s RNA genes in Triturus oocytes. The observed difference might simply be due to the presence of a large pool of P40 in Xenopus oocytes and of a small pool of an equivalent protein in Triturus oocytes. Nevertheless, until the overall significance of the different RNA-protein interactions are fully understood, generalizations covering various different organisms should be avoided. Furthermore, caution should be exercised in the interpretation of in vitro transcription assays [18, 231 that utilize transcription factors isolated from one organism (X. laevis) with genes isolated from another, albeit closely related, organism (X. borealis). We thank Dr Peter Ford for helpful discussion and for supplying us with antibodies to TFIIIA of borealis. This work is supported by a grant from the Science and Engineering Research Council of Great Britain.

Xenopus

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