the Xenopus multifinger protein Xfin - Europe PMC

4218-4225 Nucleic Acids Research, 1993, Vol. 21, No. 18

RNA binding properties and evolutionary conservation of the Xenopus multifinger protein Xfin Massimiliano Andreazzoli, Stefania De Lucchini*, Mario Costa1 and Giuseppina Barsacchi Laboratori di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia e Biochimica, Universita' di Pisa, Via Carducci 13, 56010 Ghezzano, Pisa and 1lstituto di Farmacologia Medica, Universita' di Pisa, Via Roma 55, 56100 Pisa, Italy Received June 14, 1993: Revised and Accepted August 10, 1993

ABSTRACT Xfin is a Xenopus zinc finger protein which is expressed in the cytoplasm of the oocyte and throughout embryogenesis, as well as in the cytoplasm of some specific and highly differentiated cell types (De Lucchini et al., Mech. Dev. 36, 31 -40, 1991). In this paper we present a characterization of some structural features of the protein and of its nucleic acid binding properties. We found that Xfin is a phosphoprotein, is present in the soluble fraction of the cytoplasm, and is actively phosphorylated in cytosolic extracts. Several putative phosphorylation sites are present in the cDNA-derived protein sequence, mostly located at specific positions within the Zn-fingers. In an in vitro assay a fusion protein containing part of the finger region of Xfin exhibits specific binding to a poly (G) RNA homopolymer, while it does not bind DNA. The RNA binding activity of the protein is significantly enhanced by phosphorylation. A putative Xfin homolog, which appears to be evolutionarily conserved with regard to size, cytoplasmic expression and antigenic specificity, is present in representatives of five Vertebrate classes. Taken together, these results may suggest that, by virtue of its RNA binding activity modulated through phosphorylation, Xfin could serve some evolutionarily conserved function in post-transcriptional regulation processes.

INTRODUCTION The C2H2 zinc finger domain is a highly conserv,ed nucleic acid binding motif, originally discovered in the Xeuopus transcription factor HIIA (TFIIIA) (1), which regulates transcription of somatic and oocyte 5S RNA genes (2, 3). This structural motif was subsequently found in several Drosophila developmental regulator genes acting as sequence-specific transcription factors, as exemplified by Krulppel (4). Proteins carrying this conserved domiiain (zinc finlger proteins: ZFPs) are widely distributed in eukaryotic organisms: for example, in the human genome it has been estimated that there are several hundred zinc finger encoding genes (5). and a large *

To whom coi-respondence

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pool of maternal transcripts coding for ZFPs has been detected the Xeuioplus oocyte (6). Evolutionarily conserved modules of unknown function, such as the FAX (7) and the KRAB domains (8. 9), are located at the amino-terminus of ZFPs: these domains define subfanmilies of ZFPs. It has been speculated that the modular design of ZFPs would allow the emergence of new regulatory functions during evolution (10). Even though some ZFPs have been proven to play a key role in the control of developmient and/or cell proliferation by acting as transcription factors (e.g. Krhippel, (4): Krox-20, (11, 12); for a review on ZFPs see (13) and references therein), the function of the vast majority of ZFPs remains unknown. For example, it was not possible to demonstrate an involvement of the maternal storage of ZFP-encoding imiRNAs in the complex events of Xeniopus early embryogenesis ( 14), neither to establish other cellular functions for their protein products. The case of TFIIIA shows that zinc finger proteins can play additional roles besides transcription regulation: TFIIIA possesses in fact the remarkable feature of being able to bind both 5S DNA and its 5S RNA product, thus controlling 5S RNA transcription, as well as 5S RNA storage and transport during Xenopus oogenesis (1 5. 16). The other Xenopus ZFP involved in 5S RNA storage and transport, p43, is able to interact with 5S RNA only, showing no affinity for 5S RNA genes (17): this finding testifies the existence of ZFPs specialized in RNA binding functions. Recently, another Xeuioplus RNA binding ZFP, termed XFG 5-1, has been identified on the basis of its ability to bind RNA homopolymets iti v'itro (18). In addition to the examples cited, a role in pre-mRNA synthesis or processing has been proposed for the zinc finger-related protein A33, which is found associated with the nascent transcripts on the lampbrush chromosome loops of the salamander Pleurodeles (19). It has been suggested for TFIIIA that RNA binding may have been the sole function of an hypothetical ancestral protein, which would have subsequently evolved into a multifunctional one (20): perhaps RNA binding was the primitive property of ancestral in

ZFPs.

RNA-protein interactions are important determinants of gene expression through their actions in post-transcriptional regulation during both development and differentiation. For example,

Nucleic Acids Research, 1993, Vol. 21, No. 18 4219 maternal mRNAs, important in several animal species to direct the program of early development, are kept translationally inactive in the oocyte mainly by interactions with specific proteins (21 -24). The poly (A) tail of mRNA plays an important role in regulation of translation; recently it has been shown that the regulated poly (A) addition to maternal mRNAs requires a sequence-specific RNA binding activity (25). In principle, RNA binding ZFPs could be part of the cellular machinery which controls events in development and differentiation through posttranscriptional regulation. We are focusing our study on a KRAB-ZFP from Xenopus called Xfin, which contains as many as 37 zinc fingers clustered in six 'hands' (26). The Xfin protein is present in the cytoplasm of Xenopus oocytes but also in the cytoplasm of some specific cell types (27, 28). We therefore speculated that Xfin could have a specific cytoplasmic function, possibly involving RNA binding properties (27). In the present work, we determined several characteristics of the Xfin protein to help define its cellular function. We report here that (1) Xfin is phosphorylated in cytosolic extracts, (2) the protein shows a RNA binding activity which is enhanced by phosphorylation, (3) a putative Xfin homolog is present in representatives of five Vertebrate classes. Together with the cytoplasmic localization of the protein, these results suggest that Xfin may serve an evolutionarily conserved function in post-transcriptional regulation processes: this function could be accomplished through its RNA binding activity, modulated by phosphorylation.

MATERIALS AND METHODS Protein extracts, subcellular fractionation and sucrose gradients Xenopus ovary S100 extracts (100,000 g, I h supernatant) were prepared essentially as described in ref. 29, starting from total ovary.

Total protein extracts, SDS-PAGE and Western blots were performed as previously described (27). Sucrose gradients of S100 extracts and subcellular fractionation of DU145 or HOS cells were performed by standard methods (see ref. 30). For rat testis, the postnuclear supernatant was used as a crude cytoplasmic preparation without any further purification.

Alkaline phosphatase treatment and in vitro phosphorylation S100 extracts in dephosphorylation buffer (50 mM Tris -HCI, pH 8; 0.1 mM EDTA, pH 8.5) were incubated at 37°C for 0-5 h with 20 U of calf intestinal alkaline phosphatase (Boehringer). In control reactions the mixture was adjusted to 0.1 M Na2HPO4, an inhibitor of phosphatase activity, prior to the start of incubation. As a further control S100 extracts were incubated with dephosphorylation buffer at 37°C for 0-5 h in the absence of the enzyme. In all cases, the reactions were stopped by addition of SDS loading buffer and the samples analyzed by SDS-PAGE and immunoblots. For phosphorylation experiments, a ,Bgal fusion protein, containing the 13 N-terminal fingers of Xfin, was purified from polyacrylamide gels and renatured by dialyzing the protein against 20 mM Tris pH 7.7, 50 mM KCI, 10 mM MgCl2, 1 mM EDTA, 10 ,M ZnSO4, 20% glycerol, ImM DTT, 0.2 mM PMSF,

1

mM sodium

metabisulphite. The renatured fusion

protein was incubated with S100 extracts containing 4 mM MgCl2, in the presence of 15 ,uCi of (-y32P)-ATP at 37 °C for

1 h and subsequently immunoprecipitated with anti-fgal polyclonal antibodies (Sigma). As a control, 3-galactosidase (Sigma) was treated exactly in the same way. The immunoprecipitates were separated electrophoretically and the dryed polyacrylamide gel exposed to X-ray film.

Nucleic acid binding assay The assay was performed basically as described in ref. 18. The construction of fusion proteins used in the assay is described elsewhere (27). The fusion proteins were immunoprecipitated from E. coli extracts with either anti-3gal or anti-MS2 antibodies. When indicated competitors RNAs (poly A, poly C, poly G, poly U and tRNA), or DNAs (total fragmented DNA and poly d[IC]) were added prior to the start of incubation. Control experiments were done using 3-galactosidase instead of the fusion protein in the binding assay. In one set of experiments the fusion protein 3gal-finger was incubated in phosphorylation conditions and used in the nucleic acid binding assay, as follows. Preclearified soluble E.coli extracts expressing the fusion protein were incubated at 37°C for 1 h with 2.5 volumes of Xenopus S100 extract and addition of 10 mM ATP and 4 mM MgCl2. At the end of incubation the fusion protein was purified by immunoprecipitation and used directly for the assay. Controls were performed using in the assay either ,B-galactosidase or an uninduced E. coli extract previously incubated in the same phosphorylation conditions. Cell lines The following cell lines were used: SW 480, SW 620, HCT 8 (human colon tumor lines); PC 3M, DU 145, LNCaP (huma., prostate tumor lines); HOS (human osteosarcoma cell line); U87MG, SNB 19 (human glioblastoma lines); A549 (human lung tumor line); V79 (chinese hamster fibroblast line) (American Type Colture Collection, ATCC, Rockville, MD); A2780, CP70 (human ovary tumor lines) (Dr Reed, N.C.I., Bethesda, USA); H411 (rat hepatoma line) (31); B3.2 (Xenopus kidney line) (32). LNCaP-t and DU145-t in Fig. 8 indicate cell lines transformed with plasmid Homer 6, containing a ras oncogene (33). Cell culture and transformation was performed by standard procedures.

Proteinase K protection assay Mitochondria were resuspended to a protein concentration of lmg/ml and incubated for 20 min. with proteinase K (5 jtg/ml) at 4°C. Where indicated 0.5% Triton X-100 was added prior to the start of incubation. Proteinase K digestion was stopped by addition of 1mM PMSF and the samples prepared for SDS-PAGE.

Animal species Animal species used were the following: planarian, Dugesia benazii; musca, Musca domestica larvae; fish, Carassius carassius; newt, Triturus vulgaris meridionalis; lizard, Podarcis sicula campestris; pigeon, Columba livia; Long Evans rats (Nossan, Italy); Xenopus laevis (NASCO, USA).

RESULTS Characterization of the native state of Xfln in Xenopus ovary Cytoplasm from Xenopus ovary was fractionated into a mitochondrial, a microsomal membrane and a cytosolic (5100) fraction. The different subcellular fractions were treated with the previously described anti-Xfin antibodies (27): these antibodies

4220 Nucleic Acids Research, 1993, Vol. 21, No. 18

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Figure 1. Left panel: Western blot showing a time course of alkaline phosphatase treatment of Xenopus SI 00 extract. Samples were taken after 0, 1. 3 and 5 hours (hr) of treatment, either in the presence (+) or absencc (-) of alkaline phosphatase (A.P.) as indicated at the bottom of panel. Right panel: Control experiments performed with S1OO extracts treated for 3 hours with alkaline phosphatase in the presence (+) or absence(-) of 0.1 M Na,HPO4. The lane to the right (A.P.-. Na2HPO4-) was loaded with the sample bef'ore treatment. The arrows points to the position of the different isoforms.

detect the protein only in the soluble fraction of the cytoplasm (data not shown). To characterize the native state of Xfin in X'noeopus oocyte, S 100 extracts were separated on sucrose gradients run in nondenaturing conditions. The Xfin sedimentation profile results in a single peak at about 4.3 S (not shown). Two forms of the protein were discriminated by the sucrose gradient (data not shown). We decided to further investigate the relationship between these two forms with the idea that post-translational modifications might be involved.

Xfln is a phosphoprotein Since phosphoproteins usually appear as a doublet in SDSPAGE, we decided to determine whether Xfin is indeed phosphorylated. To test this hypothesis, SlOO extracts were treated with calf intestine alkaline phosphatase for different lengths of time. Control samples were incubated at 37°C for 3 and 5 hours in the same incubation buffer, but in absence of alkaline phosphatase (Fig. 1). As shown, after one hour of treatment the upper band disappears, while the lower band is unaffected. We conclude that the slow migrating form is probably due to phosphorylation. After 5 hours there is a slow reappearance of the slow migrating form. We interprete this observation as an active rephosphorylation of Xfin by endogenous kinases present in the SlOO extracts and therefore competing with the activity of the alkaline phosphatase. This explanation is also confirmed by the increase in intensity of the upper band in mock experiments compared to the sample treatment (Fig. 1). The incubation buffer in fact contains magnesium ions necessary for kinase activity. The fact that the disappearance of the slow migrating isoform is due to alkaline phosphatase is also confirmed by a control experiment where 0. 1 M sodium phosphate-an alkaline phosphatast inhibitor-was added in the incubation buffer, as shown in Fig. 1 (right panel): as a result of the sodium phosphate treatment, the slow migrating form is unaffected. A computer analysis of the Xfin sequence has revealed the presence of several putative recognition sites for different kinases (Fig. 2A). Most of these sites, representing presumptive targets for protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaM kinase ll), map at specific positions within the fingers and the H/C links. A schematic representation of the position of phosphorylation sites within the fingers and H/C links and their relative frequencies is given in Fig. 2B. Further evidences that Xfin is phosphorylated are given by an independent experiment where a fusion /gal protein was labelled with -y 32p in oocyte S1OO extracts. The fusion protein contains

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Figure 2. (a) Map of potential sites for protein kinases in Xfinl. The KRAB domain, the single Zn-fingers as well as the six hands' (I-VI) of the protein are represented. The positions of' consensus recognition sites for protein kinase C (PKC), calnmodulin-depcndent kinase II (CaMK), protein kinase A (PKA), p34 d,2 (CDC2), and casein kinase II (CK II) are indicated above the protein. Only those sites that bcst fit with the consensus are shown. For ease of representation the putativc partially overlapping recognition sites for PKC and CaMKII in the H'C link are indicated by a dot. (b) Schematic diagram of a consensus Xfioi Zn-finger. showing the positions of potential phosphoacceptor amino acids (circled) within the finger and the H/C link. Sites occurring only once in the Zn-finger- structure are not indicated. Encircled threonines fall within a putative phosphorylation site for PKC with a frequency of 25 out of 37 fingers (larger circle), and of 4 out of' 37 (smiialler circle).

the 13 N-terminal fingers of Xfin with several potential kinase recognition sites. The labelling was done by incubating the purified fusion protein, isolated from a polyacrylamide gel and renatured in the presence of Zn ions (see Materials and Methods) with SlOO extracts with f 32P-ATP. The protein was then immunoprecipitated by use of commercial anti-/gal antibodies, and loaded on a polyacrylamide gel. A control was performed by using /-galactosidase instead of the fusion protein. The purified fusion protein used for the experiment was visualized by Coomassie stainin, (Fig. 3, lane 1). Two bands are present, the higher corresponding to the complete protein, and the lower representing a degradation product. After incubation in phosphorylation conditions in S100 extracts, the same two bands appear to be labelled (Fig. 3, lane 3); it seems however that a further degradation of the intact protein has occured during the incubation, since the lower band is more heavily labelled. A degradation band, measuring about 55 kD, is also present. Taken together, these data provide evidence that the two Xfin isoforms characterized by different electrophoretic mobility are due to phosphorylation; moreover, they suggest that the kinase(s) involved in Xfini phosphorylation is (are) present in cytosolic SlOO extracts.

RNA binding activity of Xfln In order to understand the function of Xfitn, we analyzed the nucleic acid binding properties of this protein. For this analysis ani izi vitro nucleic acid binding assay, described for the Xenzopus poly (A) binding protein (34), the Zn-finger protein XFG 5-1 (18), and the Zn-finger protein Bvr 3 from S. pombe (35), was

performed. Two fusion proteins, encompassing different regions of Xfin, were employed in our assay. The first is a 3gal fusion protein containing the 13 N-terminal fingers of Xfin (referred to as fgal-

Nucleic Acids Research, 1993, Vol. 21, No. 18 4221

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COrET1T0(R G9) Figure 3. Phosphorylation of the /3gal-finger fusion protein. Lane 1: Coomassie stained 10% polyacrylamide gel showing the purified fusion protein /3gal/finger; the higher band corresponds to the complete protein while the lower is a degradation product. Lanes 2 and 3: autoradiograph of a 10% polyacrylamide gel loaded with either purified /gal/finger (lane 3) or /-galactosidase (lane2) that had been previously incubated in phosphorylation conditions with S100 extracts in the presence of (-y32P)-ATP and subsequently immunoprecipitated with anti /-gal antibodies. Arrows indicate the position of the fusion protein and of the degradation product. The numbers to the right refer to the position of a molecular weight marker (in kD).

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Table I. Nucleic acid binding activity of Xfin fusion proteins and relative controls PROTEIN

DNA p(A)

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extract

1.5

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0.5 3

0.7 2

9

(not phosphorylated) phosphorylated

0.8

1.5

6

4

46

MS2/spacer

10

/-gal/finger The indicated values represent the percentage of nucleic acid bound by each protein in the in vitro binding assay described in the text, calculated on the average of four independent experiments. The asterisk indicates that the protein (or extract) was incubated with Xenopus ovary S100 extracts in phosphorylation conditions before being used in the assay; n.d., not determined.

finger); the second contains one of the short spacer regions that divide consecutive sets of fingers, and is expressed as a MS2 fusion protein (referred to as MS2-spacer in the text). The production of the two constructs is decribed elsewhere (27). After immunoprecipitation from the soluble fraction of an E. coli extract with either anti-3gal or anti-MS2 antibodies, the fusion proteins were incubated with different 5' labelled RNA homopolymers or fragmented total DNA. The percentage of input nucleic acid bound to the proteins was quantified by scintillation counting after extensive washing (see Table I), and the binding specificity evaluated by competition with increasing amount of various unlabelled RNAs and DNAs. Controls were performed by incubating f-galactosidase in the same conditions (see Table I). As expected, no affinity for any of the tested nucleic acids was observed for the /-galactosidase as well as for the MS2-spacer fusion protein: in our assay, both these proteins bind radioactivity at a background level (Table I). As shown in Table I, the /galfinger fusion protein does not bind DNA, poly(A), poly(C) and

Figure 4. Graphs showing competition experiments to test the poly(G) binding specificity of the fusion protein /3gal/finger. Four independent experiments were performed for each point. The standard error bars are indicated. (a) The immunoprecipitated fusion protein was incubated with 5' end labelled poly(G) in the presence of increasing amount of the indicated competitor RNAs or DNAs. (b) Competition experiments carried out as in (a) with the difference that the fusion protein /gal/finger was previously phosphorylated by incubation in S100 extracts.

poly(U) homopolymers significantly above background, but it binds poly(G). Poly(C) and poly(U) binding can be competed by addition of any other homopolymer (data not shown), thus confirming the non-specificity of binding. Poly(G) binding specificity was evaluated by competition experiments; as shown in Fig. 4a, the binding is not competed by poly(A), double stranded total DNA or poly d(IC), while competition occurs after addition of poly(G) and, to some degree, of tRNA. Poly(U) and poly(C) homopolymers were not used for competition, since they did not allow us to distinguish the competition effect from that of duplex formation. Since phosphorylation has been shown to regulate proteinnucleic acid interactions (see, for the Xenopus RNA binding proteins, ref. 23) and because the majority of phosphorylation sites in Xfin fall within fingers or H/C links, we decided to perform the same in vitro assay using the jgal-finger fusion protein, previously phosphorylated in Xenopus oocyte S100 extracts (see Materials and Methods). Under these conditions a

4222 Nucleic Acids Research, 1993, Vol. 21, No. 18

Figure 5. (A) Zoo' blot probed with anti-Xfin antibodies. The species used for the blot are listed in Materials and Methods. The arrow indicates the position of Xfin (120 kD). (B) Western blot analysis ot adult organs froom the same Vertebrate species shown in (A). except that thc smiiooth newt, Tritlurul.s vulgtiris, was used as amphibian representative. m: skeletal muscle h. hebart: li. liver: k, kidney: g, gut: a.b.. air bladdel-; i..- immature (onad: lu, lung: te. testis, br. brain: Xl.. Xeoiopus klevis ovary 5100 extract used for positive control and reference molecular weigth. The blots were stained with anti-Xfin antibodies; the integrity of total proteins from each extract was checked by Coomassic staining (not shown). (C) Rat testis subccllular fractionation. 180 ug of total proteins were loaded on each lane. TOT: total extract; CYT: cytoplasmic fraction; NU: crude nuclei fraction. X.l.: Xenolps ovary S 100 extract: DU 145: hblman cell line. The molecular weigth in kD of antigens stained bv the Xfini antihody, as determllined from reference standard proteins. arc indicated on the lcft.

significant increase of at least fivefold is observed for the poly(G) binding activity of the protein (Table I). The specificity of the binding was evaluated by competition with different unlabelled nucleic acids (Fig. 4b). As shown, the poly(G) binding specificity of the phosphorylated protein appears to be higher with respect to that of the unphosphorylated one, since in this case no competition by tRNA is observed, even if this is present at high concentrations. As controls, 3-galactosidase and an unininduced E. coli extract, not expressing the fusion protein, were incubated in phosphorylation conditions and used in the in ivitro binding assay (Table I). These controls demonstrate that the increase in poly(G) binding is due to the finger portion of the fusion protein, and show only a very slight increase in background for these controls due to the incubation in S 100 extracts.

Evolutionary conservation Since no other Zn-finger protein of Xfin feature and size has been described to date, we wondered whether Xfin is a peculiar Xenopus protein, or if it is a more widespread eukaryotic protein. For this purpose we undertook an analysis of the phylogenetic distribution of Xfin-related proteins in eukaryotes. Protein extracts from various vertebrates and invertebrates were examined on immunoblots using anti-Xfin antibodies directed against a portion of the Zn-finger domiiaini of Xfini (27). These polyclonal antibodies are very specific for Xfini in Xenopus, showing no cross-reaction with other proteins, despite the presence of about 100 Zn-finger genes in this species (6). Fig. 5A shows that the same antibodies specifically recognize one band (sometimes appearing as a doublet) in every representative of five vertebrate classes, while

Figure 6. (A) Western blot of total protein extracts of various human cell lines (see Materials and Methods) probed with anti-Xfio2 antibodies. X.l.: Xensopus S100 ovary extract used for positive control and reference molecular weight. The molecular weights in kD ot the two antigens recognized by the antibody are indicated on the left. (B) Subccllular fractionation of DU 145 cells analyzed by

Western blot with anti-Xfin arntibodies: on the right are indicated in kD the molecular weights of the two proteins (the 120 kD one appearing sometimes as a doublet). MIT: nitochondria rich fraction: CYT: cytoplasmic fraction; NU: purified nuclei: TOT: total extract. Each lane was loaded with 500,000 cells CqLIvldiu[.

no specific protein is detected in the invertebrates tested. The presence of double bands might suggest the occurence of phosphorylated isoforms. as described for Xenopus (this paper). As a control the same extracts were reacted with preimmune or

Nucleic Acids Research, 1993, Vol. 21, No. 18 4223

Figure 8. Western blot analysis of Xfin-related proteins in cell lines from different species. V79: chinese hamster fibroblast cell line; H4: rat hepatoma H411 cell line; B3.2: Xenopus kidney cell line; S100: Xenopus ovary S1OO extract. The molecular weights, in kD, of the detected antigens are indicated on the left.

Figure 7. (A) Proteinase K digestion of mitochondria. The mitochondria rich fraction (Mit.) from DU 145 cells was incubated at 4°C with proteinase K (Prot. K) in the absence (-) or presence (+) of Triton X-100. The samples were then separated by SDS-PAGE, transferred to a nitrocellulose filter and analyzed by immunoblotting with anti-Xfin antibodies. The arrowhead indicates the 160 kD antigen; the lower band represents contaminating 120 kD protein present in the postmitochondrial supernatant. (B) Coomassie staining showing the total proteins of the samples described above.

anti-3gal sera (not shown); in none of these cases was a specific signal detected. Remarkably, the antibody recognizes a protein of about the same size of Xfin in all vertebrates except in fishes, where a larger protein is detected. No specific reaction was observed when antibodies directed against a region of Xfin not containing fingers were used on a similar blot (not shown), while these antibodies (anti-Xfin2) were shown to be effective in Xenopus (27). This result suggests that the Zn-finger domains of the protein have been conserved during evolution. We subsequently analyzed the expression of the Xfin related proteins in various organs of five representative Vertebrates (Fig. SB). As in the case of Xenopus (27), the putative homologous proteins are expressed in several organs, embryologically unrelated. We then asked if during evolution Xfin-related proteins maintained the same subcellular localization. To answer this question we looked at the subcellular distribution of the mammalian counterpart of Xfin in the rat testis, which is evolutionarily the most distant from Amphibia among the species analysed (Fig. SC). Each lane was loaded with equal amount of proteins from either rat testis total extract, cytoplasmic fraction or nuclear fraction. The cytoplasmic fraction is enriched in the antigen while the nuclear fraction shows only a very faint band that could represent some cytoplasmic contaminat on. Taken together these data suggest that Xfin, and in particular its Zn-finger domain, has been conserved during Vertebrate evolution and that a strong selective pressure must be acting to keep the protein of approximately the same size and in the same cellular compartment.

Expression in cell lines To further characterize some features of Xfin-related protein expression, we extended our analysis to cell lines. Employing the anti-Xfin antibodies already described, we tested a variety of different established human cell lines (Fig. 6A). In all cases a 120 kD antigen, which precisely comigrates with Xfin, is detected by the antibody. Besides this major band, another protein at 160 kD is recognized by the antibody, although with different intensities from one cell line to another. Upon subcellular fractionation of the cell line DU 145, the 120 kD protein is found in the cytoplasm. Surprisingly, the 160 kD antigen is instead found to cofractionate with the mitochondria rich fraction (Fig. 6B). To determine whether this polypeptide is localized inside or outside of the mitochondria we performed a proteinase K digestion of the mitochondria rich fraction. The presence of the antigen was then visualized by Western blotting (Fig. 7A). An equal amount of either untreated mitochondria, mitochondria treated with proteinase K or proteinase K and the detergent Triton X-100 was loaded in adjacent lanes. As shown (Fig. 7A), the 160 kD protein does not appear to be protected by the mitochondria envelope from proteinase K digestion. Controls were performed by running part of the samples in the same gel and visualizing the proteins by Coomassie staining (Fig. 7B). As can be seen, several proteins are protected by proteinase K digestion. These data allow us to conclude that the 160 kD protein related to Xfin present in human cell lines is located on the outside of the mitochondria. The same data about the subcellular distribution of the 120 and 160 kD proteins were obtained using the cell line HOS (data not shown). Finally, we asked whether the occurrence of the 160 kD antigen is typical of human cell lines. To this purpose we analyzed by Western blot rat, hamster and Xenopus cell lines. Anti-Xfin antibodies detect the 120 kD antigen in all the tested lines, while the 160 kD protein is present, although in low amount, in rat and hamster, but it is absent in the Xenopus cell line (Fig. 8).

DISCUSSION The Xenopus Zn-finger protein Xfin shows a complex pattern of expression; it is in fact expressed throughout oogenesis and embryogenesis (27), in Xenopus cultured cells (this paper), as well as in specific, highly differentiated cell types, such as the

4224 Nucleic Acids Research, 1993, Vol. 21, No. 18 cones in the neural retina (28). The fact that the Xfini protein is cytoplasmic in all cell types where it was detected, led us to hypothesize that Xfin may represent an RNA binding protein functioning through RNA/protein interactions. In an attempt to gain insights into the Xfin function, we decided to first characterize some structural features of the protein, such as its native state. We found that Xfini is present in the soluble fraction of the oocyte cytoplasm (S100 extracts), and that it sediments as a single peak of about 4.3S in sucrose gradients. Two stable forms of the protein were observed, differing slightly in molecular weight and sedimentation coefficient. By means of two independent experiments we have demonstrated here that the two forms of the protein are due to different phosphorylation states, and that both the native protein and a bacterially expressed fusion protein are actively phosphorylated in Xenopus oocyte S100 extracts, thus suggesting the involvement of endogenous kinase(s). The fact that we do not detect intermediate phosphorylation forms might suggest the occurrence of a processive phenomenon, i.e. the phosphoprotein carrying the first phosphate molecule being a preferred substrate for further modifications. Examples of phosphorylated Zn-finger proteins include the ubiquitous transcription factor SP 1, which is phosphorylated by a dsDNA-dependent kinase (36), SW15, a yeast transcription factor exhibiting a phosphorylation-dependent nuclear transport (37), and the product of the Drosophila segmentation gene Kruppel (38). In the reported examples of SPl and SWJ5, phosphorylation is shown to regulate protein activities such as transactivation or nuclear translocation. Here we demonstrate that phosphorylation can directly influence the binding properties of a ZFP: this is indeed the case for Xfitn. To address directly the question of the nucleic acid binding activity of Xfini, we performed an in vitro assay using a bacterially expressed fusion protein containing the 13 N-terminal fingers. We reasoned that a truncated fusion protein could effectively mimic the activity of the complete Xfi,n molecule, since corresponding fingers in different hands exhibit a better homology with respect to consecutive finger motifs (26). Remarkably, we found that this fusion protein is not able to bind DNA in the in vitro assay, but it specifically binds a poly(G) RNA homopolymer. It is also worth noting that the binding activity is entirely due to the finger domain of the protein: one of the 'spacer' regions separating consecutive sets of fingers does not show any binding property. Poly(G) may not be the natural target of Xfin. No information to date is available about the physiological targets of RNA binding ZFPs, besides 5S RNA, which is bound by TFIIIA and p43 (17, 20, 39). By inference from the available data it seems that specific RNA recognition depends primarily on the secondary/tertiary structure of the target RNA, rather than on its sequence (39); in the case of Xfin, it is conceivable that G residues in the target RNA(s) may be important either as contact points for the protein or to give the RNA(s) a characteristic structure recognized by Xfin. The RNA binding activity of Xfin is enhanced by phosphorylation: in fact, in our in vitro assay we observed a fivefold increase, at least, in the RNA binding activity of the phosphorylated, truncated form of Xfin. The affinity of the phosphorylated, full-length protein in vivo could be even greater. We show that the kinase(s) involved in Xfin phosphorylation is (are) present in the oocyte S100 extracts: this would argue in favour of a physiological role of phosphorylation.

Numerous putative phosphorylation sites are present in the cDNA-deduced XJfin aminoacidic sequence (Fig. 2a; see references 40 and 41 for the consensus sequences for protein kinases). Most of these sites would be targets for protein kinase C (PKC) and calmodulin-dependent kinase II (CaM kinase II). Remark-ably, several putative phosphorylation sites occupy specific positions whithin the fingers and the H/C links (see

Fig. 2b), thus retaining the ability of directly influencing the binding properties of the protein. In particular, a threonine in the tip region of the fingers is found to fall within a presumptive phosphorylation site for PKC. This position may be important in ZFP/DNA interactions, because the aminoacid at that position could make contact with the phosphate backbone, thus stabilizing the

protein-DNA complex (42). It is worth noting that specific ZFP-RNA recognition could involve mainly phosphate backbone interactions (39). Another putative phosphorylation target, a conserved threonine in the H/C link, is found to fall several times within a putative recognition sequence for PKC and CaM kinase II. Given the conservation of the H/C link, this threonine might represent a phosphoacceptor aminoacid common to other ZFPs. The fact that a fusion protein containing part of the finger region of Xfin, but not other domains, is actively phosphorylated in oocyte S 100 extracts would argue in favour of the possibility that at least some of the sites located on fingers and/or H/C link may be used by the endogenous kinase(s). It is known that PKC and phosphoinositol phosphate play an essential role in both maturation and fertilization events (see, for Xenopus, refs. 43 and 44), as well as in early embryogenesis (45): in our opinion, an attractive possibility is that Xfi,n activity might be regulated through the PKC/inositol phosphate pathway. As a model, we propose that phosphorylation might represent a molecular switch for Xfini activity, the phosphorylated form being presumably capable of tightly binding RNA molecules; dephosphorylation would release the ZFP from the RNA. This mechanism would be particularly suitable in the case of an involvement of Xfini in RNA transport or masking activity. We may speculate that certain features in RNA metabolism could be shared between different cell types, thus explaining the expression of the protein in different cell types. A possibly important cellular function of Xfini is further suggested by the occurrence, shown in this paper, of a possible Xfitn homolog in representatives of five Vertebrate classes, as judged on the basis of a specific reaction with the anti-Xfini

antibody, conserved molecular weight, and similar subcellular

distribution. We are aware that sequence comparison and a functional analysis will be necessary to determine the full extent of Xfin conservation. Interestingly, the finger domain of the protein seems to be the one conserved among different Vertebrate species, since antibodies raised against a non-finger region do not show any specific reaction in the species tested, while they recognize the Xeniopus protein. Since the finger region of Xfinl is also the one responsible for the RNA binding properties of the protein, its conservation, as shown immunologically, would argue in favor of a possible maintenance of the binding characteristics of the protein during evolution. A protein related to Xfin, as judged on the basis of antibody reaction, but differing ftom XJini for both molecular weight (about 160 kD) and subcellular distribution is found in mammalian cell lines. At present, we (do not have a reasonable explanation for the occurrence of this protein in nmammalian cell lines, while the same antigen is absent trom a Xenopus cell line. Interestingly, subcellular fractionation and proteinase K protection assay

Nucleic Acids Research, 1993, Vol. 21, No. 18 4225 could be envelope. Given the antibody crossreaction, the 160 kD antigen could share at least some epitopes with the finger domain of Xfin: it is tempting to speculate that the 160 kD antigen is a ZFP that might

suggests that in human cells the 160 kD protein associated to the cytoplasmic side of the mitochondrial

mediate transport of certain RNAs to the mitochondria. In summary, our data provide evidence that Xfin possesses a phosphorylation-modulated RNA binding activity and thus it may function through protein-RNA interactions. In a simple model, Xfin might accomplish binding and releasing RNA by switching from a highly phosphorylated to an unphosphorylated form. Data indicating the evolutionary conservation of the finger domain of the protein, in contrast to the non-finger regions, lead us to speculate that the binding characteristics of the protein might be conserved as well, and that selective constraints may have maintained its target element(s). The emerging picture points towards the possibility that Xfin and Xfin-related protein(s) may play a conserved function in vertebrates.

ACKNOWLEDGEMENTS We wish to thank Profs Mario Del Tacca and Romano Danesi for having generously provided us with human and hamster cell lines, as well as for advice in cell culture techniques; Dr Fabrizio Loreni for his kind gift of the Xenopus cell line; Prof. Gennaro Ciliberto for the rat hepatoma line and Prof. Susan A.Gerbi for critical reading of the manuscript. This work was supported by grants from MURST and CNR (Progetto Finalizzato Ingegneria Genetica), Roma, Italy.

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