The cDNA for a protein that is related to translation elongation factor 1α (EF-1α) has been cloned from the sea urchin Anthocidaris crassispina. The sea urchin EF-1α-related protein (AcEFP) seems somewhat unique in structure compared to EF-1α of other organisms so that it is uncertain if AcEFP is a genuine EF-1α. Still, it possesses many features of typical EF-1α reported so far, suggesting that AcEFP is involved in protein synthesis or its regulation. Genomic Southern analysis indicated that the sea urchin genome contains one or at most two copies of the AcEFP gene. A single transcript of 2.2 kb is expressed ubiquitously in adult tissues examined and, during embryogenesis, zygotically after the blastula stage. Whole mount in situ hybridization showed that the AcEFP gene is also widely expressed in embryos, with relatively high expression in the gut and oral ectoderm, both of which are proliferating tissues in embryos. The expression of AcEFP was not affected in embryos by agents that destabilize the extracellular matrix (ECM), suggesting that expression of AcEFP is relatively independent of the integrity of ECM in sea urchin embryos.
Protein synthesis involves a variety of different factors, among which elongation factor 1α (EF-1α) promotes the GTP-dependent binding of aminoacyl-tRNA to the acceptor site of the ribosome (Hershey, 1991). Besides its essential role in protein synthesis, there are several reports showing the involvement of EF-1α in a number of other functions: complex formation with mitotic apparatus (Kuriyama et al., 1990; Ohta et al., 1990), interaction with cytoskeletal elements (Yang et al., 1990; Shiina et al., 1994), with the endoplasmic reticulum (Hayashi et al., 1989), and with ribosomal subunits (Berchtold et al., 1993). Furthermore, a possible implication has been inferred in cellular senescence and aging (Cavallius et al., 1986; Shepherd et al., 1989). All these facts suggest the coupling of protein synthesis and other cellular functions.
Meanwhile, it seems rather common that there are multiple EF-1α genes or EF-1α-related genes in a single organism. In Xenopus, four EF-1α or related proteins have been identified so far (Deschamps et al., 1991), which have different developmental expression patterns and/or different roles in development. This is also the case for the multiple EF-1α genes in Artemia (Maassen et al., 1985), Drosophila (Hovemann et al., 1988), and mammals (Ann et al., 1992). Thus, different EF-1α genes are regulated differentially and strictly in embryogenesis, serving a variety of aspects in embryonic cells. Since embryogenesis is a complicated process that requires strict regulation of protein synthesis, clarification of the expression and function of EF-1α and its related proteins in embryos will contribute to our understanding of development.
The sea urchin is an excellent model animal for understanding early embryogenesis of animals, though information on EF-1α is still limited despite a number of findings showing the important roles of translational regulation in sea urchin embryos (Brandis and Raff, 1978; Hille and Albers, 1979). There have been a few reports on sea urchin EF-1α-related proteins. In one report, a partial cDNA was obtained, but the sequence was not shown (Peeler et al., 1990). In another report, the cDNA sequence is also partial, and the gene product seems rather unique in structure compared to typical EF-1α of other animals as described below (Kuriyama et al., 1990). Ohta et al. purified a mitotic apparatus-associated protein that has EF-1α-like activities and antigenicity, though no structural data were presented (Ohta et al., 1990). Here, we show the cDNA structure for EF-1α-related protein of the sea urchin Anthocidaris crassispina and its expression in embryos and adults.
MATERIALS AND METHODS
Collection of gametes of A. crassispina and culture of embryos were performed as described before (Yamasu et al., 1995).
Total RNA was isolated from adult tissues or embryos at different developmental stages by the acid guanidinium phenolchloroform method (Chomczynski and Sacchi, 1987). Southern and northern analyses were done according to the standard protocol (Sambrook et al., 1989) using as probe the 5′-terminal cDNA fragment (+1 to +270/BamHI site) labeled with [α-32P]dCTP. Final wash was done with 0.2×SSC/0.1% SDS for 15 min at 65°C.
Whole mount in situ hybridization was performed as described previously (Onodera et al., 1999) using as probe the entire AcEFP cDNA.
Nucleotide sequences of cDNAs and deduced amino acid sequences were analyzed by Genetyx-Mac (Ver. 9.0; Software Development Co., Ltd.). The following amino acid sequences of EF-1α registered in GenBank were used for comparative analyses: Homo sapiens (EF-1α-1, X03558; EF-1α-2, X70940), Xenopus laevis (EF-1α-S, M25697; EF-1α-O, M75873; EF-1α-O1, Z19545; 42Sp50, X56699), Danio rerio (X77689), Drosophila melanogaster (EF-1α F1, X06869; EF-1α F2, X06870), Onchocerca volvulus (M64333), Arabidopsis thaliana (X16430), Tetrahymena pyriformis (D11083), Dictyostelium discoideum (X55972), Neurospora crassa (D45837), Saccharomyces cerevisiae (M10992). In addition, the sequence for a EF-1α-related protein (SU5 antigen) of another sea urchin Strongylocentrotus purpuratus deposited in the PIR data base (A37159) was also employed as well for comparison.
Screening of cDNA clones for the sea urchin EF-1α-related protein
A PCR fragment with high similarity to EF-1α cDNAs of other organisms was fortuitously obtained in our previous study searching for protein tyrosine kinase genes (Sakuma et al., 1997). A λZAPII cDNA library from sea urchin prism embryos (Yamasu et al., 1995) was screened using this fragment as probe. The longest cDNA clone obtained (pEF29) contains a fragment of 1754 bp and includes an ORF of 462 amino acids (aa) with an estimated size of 50.8 kDa (Fig. 1). Ten bp upstream to the initiation codon is seen an in-frame termination codon (data not shown), showing that the ORF codes for a full structure of the protein.
Structural analysis of the sea urchin EF-1α cDNA
The deduced protein (AcEFP for EF-1α-related protein of A. crassispina) shows significant homology to EF-1α that has been reported so far (Table 1). Curiously, however, the similarities of the AcEFP protein to EF-1α of other animals (78–81%) are not clearly distinguishable from those to EF-1α of fungi (76–79%) in contrast to the higher similarities among typical EF-1α of other animals such as vertebrates, arthropods, and nematodes (Table 1; additional data not shown; see also Fig. 2). Actually, though vertebrates and echinoderms including sea urchins constitute one phylogenetic group (deuterostome), EF-1α of another group (protostome) show higher identities of more than 83% to vertebrate EF-1α. This raises the possibility that AcEFP might be a specialized protein related to, but different from, the authentic EF-1α.
Comparison of eukaryotic EF-1α proteinsa
In its entirety, however, the AcEFP protein shows all the features of typical animal EF-1α (Fig. 1): 1) The 12-aa segment found in AcEFP from +214 to +225 with respect to the start codon is unique to animal and fungal EF-1α sequences (Nordnes et al., 1994). 2) The full complement of the three PKC phosphorylation sites, which is present only in those of animals and fungi (Nordnes et al., 1994), is also seen in the sea urchin protein (+242, +316, +383). 3) The 2-aa insertion in the sea urchin protein (+158 to +159) has been seen in animal EF-1α but not in fungal EF-1α. 4) The actin binding sequence in human EF-1α can also be seen in the sea urchin protein (Yang et al., 1990). 5) Basic amino acids considered necessary for binding to tRNA can also be found in the sea urchin protein (Berchtold et al., 1993). 6) The amino acid required for binding to the endoplasmic reticulum (Hayashi et al., 1989) also occurs in the sea urchin protein (D at +306).
Kuriyama et al. (1990) obtained monoclonal antibody (SU5) that recognizes a centrosphere protein of 50 kDa and cloned a partial cDNA for this antigen. The SU5 antigen also show significant similarity to EF-1α, though the antigen has several distinct features such as a one amino-acid insertion at a site that corresponds to between +103 and +104 of AcEFP and lack of the first PKC target site among the three (Fig. 1). Despite these structural uniqueness, the SU5 antigen is most close to AcEFP (Figs. 1, 2, Table 1), suggesting that the SU5 antigen and AcEFP diverged during the evolution of the sea urchin after the duplication of a common ancestor gene.
To test if other genes related to EF-1α are expressed in sea urchin embryos, we performed RT-PCR on cDNA from gastrulae employing degenerate primers designed for the two regions (GEFEAG, QDVYKI; Fig. 1) which are highly conserved among EF-1α of diverse arrays of organisms, and the PCR fragment of the expected size (ca.430 bp) was cloned into plasmid. Ten randomly selected clones showed essentially the same sequences as the AcEFP cDNA clone, demonstrating that AcEFP represents the main EF-1α family gene at least in gastrulae of A. crassispina.
In the genomic Southern analysis (Fig. 3A), only one or a few intense bands were seen for different restriction enzymes, showing that there are at most two genes for AcEFP in the genome.
Expression of the AcEFP gene in embryos and adults
Zygotic expression of the AcEFP gene was first detected as a 2.2 kb band as early as in hatching blastulae and increased in amount until the early gastrula stage, when the expression leveled off and remained constant through the pluteus stage (Fig. 3B). Longer exposure revealed faint bands of the same size from the unfertilized egg through the cleavage stage (data not shown). The spatial regulation of the gene in the embryo was addressed by the whole-mount in situ hybridization (Fig. 4). From mesenchyme blastulae (data not shown) to early gastrulae (Fig. 4B), the expression was observed rather ubiquitously. In later stages such as the prism stage, the transcript was still widely observed, though relatively intense expression was detected in the gut and oral ectoderm. In keeping with this, we observed a slight increase in the amount of the transcript after treatment with lithium ions, which are known to respecify the prospective ectoderm to endoderm in sea urchin embryos (data not shown). In adult sea urchins, the transcript of AcEFP exist in all the tissues examined (Fig. 3C), with relatively high expression in immature testes and low expression in coelomocytes. Generally speaking, the expression levels in adult tissues are lower than that in prism embryos.
ECM-independent expression of AcEFP gene during embryogenesis
To test if the AcEFP gene is regulated by external signals, embryos were treated with β-aminopropionitrile (BAPN) or cis-hydroxyproline, which destabilizes the collagen polymer, leading to disruption of the embryonic ECM (Wessel and McClay, 1987). Both treatments significantly reduced the expression of a gut-specific tyrosine kinase gene, AcSrc1 (Onodera et al., 1999), in a dose-dependent manner (Fig. 3D). In contrast, repression of AcEFP expression by hydroxyproline and BAPN was only limited. Thus, the expression of AcEFP does not depend strongly on the integrity of ECM in the embryo.
EF-1α-related protein of the sea urchin
In the present study we have cloned a cDNA for a sea urchin protein that is highly related to EF-1α. Comparison of this protein, AcEFP, with EF-1α of other organisms renders it doubtful that AcEFP is the sea urchin EF-1α. On the other hand, since the rate of base substitution is known to be rather high in the sea urchin genome (Hall et al., 1980), the possibility cannot be excluded that AcEFP is the sea urchin EF-1α. Actually AcEFP possesses all the features of EF-1α known till now, and RT-PCR identified only the mRNA for AcEFP in gastrulae, favoring the view that AcEFP is the genuine EF-1α. Though this issue awaits further study, it is highly likely that AcEFP is in some ways involved in protein synthesis.
Comparison with other EF-1α-related proteins from sea urchins
From eggs of different sea urchin species, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, GTP-binding proteins of 51 kDa with EF-1α-like activities and antigenicity were isolated as mitotic apparatus-associated proteins (Ohta et al., 1990). This is not surprizing since EF-1α is now known to associate with the cytoskeleton (Shiina et al., 1994; Yang et al., 1990). It is possible that the 51-kDa protein corresponds to AcEFP of A. crassispina, though it should be addressed in the future. Meanwhile, Kuriyama et al. obtained monoclonal antibody (SU5) which recognizes a centrosphere protein of 50 kDa and cloned a partial cDNA for this antigen (Kuriyama et al., 1990). Despite its unique structural features, the SU5 antigen is most close to AcEFP (Figs. 1, 2, Table 1), suggesting that the duplication of the ancestor gene of AcEFP and SU5 antigen took place after the branching of the sea urchin or echinoderm from other phylogenetic groups in the deuterostome. Since SU5 antigen seems to have accumulated more amino acid substitutions than AcEFP, SU5 antigen might have acquired novel functions after gene duplication such as for the regulation of mitotic spindle formation in sea urchin cells. We failed to obtain cDNAs for any SU5-type protein from A. crassispina by the RT-PCR probably because it is not expressed abundantly at the gastrula stage or the primers used here are not suitable for amplification of cDNA for such highly deviated proteins. Though cloning of another partial cDNA clone for sea urchin EF-1α-related gene was reported previously, no sequence data was provided, making it impossible to be compared with AcEFP (Peeler et al., 1990).
In fact, it is well-known that a given single organism contains multiple EF-1α genes and related genes. In Xenopus, one of the four EF-1α-related proteins (42Sp50) is considered involved in long-term storage of 5S RNA and aminoacyltRNA (Deschamps et al., 1991; Viel et al., 1991). It is possible, therefore, that the sea urchin also possesses multiple EF-1α family proteins with different roles. Possiblity of the presence of multiple genes closely related to AcEFP was, however, excluded by the result of the genomic Southern analysis (Fig. 3A). In adult sea urchins, the transcript of AcEFP exists in all the tissues examined (Fig. 3C), with relatively high expression in immature testes and low expression in coelomocytes. In embryos, AcEFP is expressed also ubiquitously, with some bias to the gut and oral ectoderm. Its universal expression is in line with a view that AcEFP is involved in various aspects of protein synthesis per se or its regulation. The bias of the expression to the gut and oral ectoderm is known for SpS24, a small ribosomal subunit protein in the sea urchin S. purpuratus, which is also involved in protein synthesis (Angerer et al., 1992). Both tissues are highly proliferative in sea urchin embryos (Angerer et al., 1992 and references therein), and may require active translation for their growth. Among sea urchin genes expressed during embryogenesis, some are known to depend on ECM for its full expression (Spec1, LpS1) while others (SM50, LyUSF) do not (Benson et al., 1991; George et al., 1996). Our present result suggested that the expression of AcEFP is partially independent of the external signal. In fact, we observed that AcEFP expression still persisted, though at a reduced level, when embryos were dissoicated into cells and cultured succeedingly (unpublished data).
Translational control in sea urchin embryos
Translational control plays important roles during sea urchin embryogenesis, especially in the enhancement of protein synthesis after fertilization. Total rate of protein synthesis increases more than 100-fold (Regier and Kafatos, 1977), and enhancement of the rate of translational elongation was estimated to be 2.5-fold (Hille and Albers, 1979). Since AcEFP belongs to the EF-1α family, it will be involved in the protein synthesis itself or its regulation. Thus, cloning of the AcEFP cDNA will certainly contribute to the elucidation of this longstanding issue of the sea urchin development.
We thank Dr. Takashi Suyemitsu for his support of our research and reviewing the manuscript, and Dr. Hirokazu Inoue for his critical reading of the manuscript. We also thank Mr. Takuya Ogawa for his technical assistance. This research was supported in part by Grants-in-Aid to K.Y. (Nos. 08780699, 09780677) from the Ministry of Education, Science and Culture, Japan.