Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii
2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês
10.1093/emboj/cdf372
ISSN1460-2075
AutoresSergey V. Novoselov, Mahadev Rao, Natalia V. Onoshko, Huijun Zhi, Gregory V. Kryukov, Youbin Xiang, Donald P. Weeks, Dolph L. Hatfield, Vadim N. Gladyshev,
Tópico(s)Organoselenium and organotellurium chemistry
ResumoArticle15 July 2002free access Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii Sergey V. Novoselov Sergey V. Novoselov Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Mahadev Rao Mahadev Rao Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Natalia V. Onoshko Natalia V. Onoshko Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Huijun Zhi Huijun Zhi Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Gregory V. Kryukov Gregory V. Kryukov Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Youbin Xiang Youbin Xiang Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Donald P. Weeks Donald P. Weeks Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Dolph L. Hatfield Dolph L. Hatfield Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Vadim N. Gladyshev Corresponding Author Vadim N. Gladyshev Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Sergey V. Novoselov Sergey V. Novoselov Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Mahadev Rao Mahadev Rao Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Natalia V. Onoshko Natalia V. Onoshko Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Huijun Zhi Huijun Zhi Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Gregory V. Kryukov Gregory V. Kryukov Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Youbin Xiang Youbin Xiang Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Donald P. Weeks Donald P. Weeks Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Dolph L. Hatfield Dolph L. Hatfield Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Vadim N. Gladyshev Corresponding Author Vadim N. Gladyshev Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA Search for more papers by this author Author Information Sergey V. Novoselov1, Mahadev Rao2, Natalia V. Onoshko1, Huijun Zhi2, Gregory V. Kryukov1, Youbin Xiang1, Donald P. Weeks1, Dolph L. Hatfield2 and Vadim N. Gladyshev 1 1Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588 USA 2Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3681-3693https://doi.org/10.1093/emboj/cdf372 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas. Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas. Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals. Introduction Selenocysteine (Sec) is a rare amino acid found in several proteins from various domains of life (Low and Berry, 1996; Stadtman, 1996; Rother et al., 2001a; Hatfield and Gladyshev, 2002). It is inserted into protein co-translationally in response to the codon UGA and the specific Sec insertion machinery. The Sec insertion machinery includes a cis-acting mRNA structure, designated the Sec insertion sequence (SECIS) element, and the trans-acting factors Sec tRNA, selenophosphate synthetase, Sec synthase, Sec-specific elongation factor and a SECIS-binding protein. Sec-containing proteins have been identified in bacteria, archaea and eukaryotes, and the universal use of UGA to designate Sec in these organisms suggests a common origin of the Sec insertion system (Gladyshev and Kryukov, 2001). However, besides selenophosphate synthetase, which is involved in Sec biosynthesis (Stadtman, 1996), there is no overlap between the sets of prokaryotic and eukaryotic selenoproteins. Moreover, selenoproteins in prokaryotes are typically involved in catabolic processes, whereas eukaryotes employ selenoproteins for biosynthetic and antioxidant processes. Details of Sec evolution are scarce and largely unclear (Atkins et al., 1999; Gladyshev and Kryukov, 2001). For example, while all currently known eukaryotic selenoproteins are of animal origin, no selenoproteins have been described in non-animal eukaryotes. Moreover, when genomes of the plant Arabidopsis thaliana and the yeast Saccharomyces cerevisiae were sequenced, their analysis revealed neither selenoprotein genes nor any of the components of the Sec insertion pathway. This lack of Sec-containing proteins contrasts with the essential role of selenoproteins in animals and bacteria (reviewed in Low and Berry, 1996; Stadtman, 1996). For example, disruption of the Sec tRNA gene in mice results in an inability to synthesize selenoproteins and embryonic lethality (Bosl et al., 1997), and mutation in a fruit fly gene for selenophosphate synthetase is also lethal (Serras et al., 2001). Sec-containing proteins are essential for Escherichia coli when grown under anaerobic conditions (Bock et al., 1991). Characterization of Sec-containing proteins and Sec insertion systems in non-animal eukaryotes may elucidate these seemingly contradictory observations in Sec evolution and its essential requirement for some organisms. One report described the presence of a possible Sec-containing glutathione peroxidase 1 (GPx1) in a model plant system, Chlamydomonas reinhardtii (Shigeoka et al., 1991). This protein was isolated directly from green algae, reacted with antibodies raised against mammalian GPx1 and found to contain a stoichiometric amount of selenium. It was not clear whether Sec was present in the protein as no protein or nucleotide sequences were reported for Chlamydomonas GPx1. In this report, we identified and characterized selenoproteins in C.reinhardtii. Remarkably, this organism contains at least 10 natural selenoproteins, including one that is specific to green algae. We also report the identification of Sec tRNA and the finding that Chlamydomonas requires selenium for optimal growth. These data are discussed with respect to evolutionary events that led to the accumulation and loss of the Sec insertion system in eukaryotes. Results Chlamydomonas contains specific selenoproteins Chlamydomonas reinhardtii cells were labeled with 75Se and the cell extracts were analyzed by SDS–PAGE (Figure 1A, left panel) and detection of radioactivity on the gels (Figure 1A, right panel). This revealed four 75Se-labeled proteins that migrated as 52, 22, 18 and 7 kDa bands, with the 7 and 18 kDa proteins being the most abundant soluble selenium-containing proteins (Figure 1A, right panel, lane 5). This 75Se pattern did not match a Coomassie Blue staining profile (Figure 1A, left panel), suggesting the presence of specific Sec-containing proteins rather than labeling of proteins through non-specific selenium incorporation. Furthermore, the radioactive labeling pattern did not match that of animal cells, in which the major 75Se-labeled selenoproteins are a 25 kDa GPx1, a 20 kDa phospholipid hydroperoxide glutathione peroxidase, a 57 kDa thioredoxin reductase 1 and a 15 kDa Sep15 (Gladyshev et al., 1999a). To determine the identity of the Chlamydomonas selenoproteins, a large-scale purification of these proteins was conducted. Figure 1.Detection of Chlamydomonas selenoproteins. Chlamydomonas cells were grown in the presence of 75Se, collected by centrifugation, disrupted by sonication, and the resulting homogenate centrifuged at 18 000 g for 30 min. In (A), fractions were analyzed by SDS–PAGE. Lanes 1–3 (left panel) show homogenate, supernatant and pellet, respectively, stained with Coomassie Blue (protein markers are shown in the left-most lane and their sizes indicated on the left) and lanes 4–6 (right panel) show respective lanes in the same gel exposed to PhosphorImager detection of 75Se (sizes of detected selenoproteins are shown on the right). In (B), the soluble fraction [shown in (A), lane 2] was fractionated on a Q-Sepharose column. Lanes 1 and 2 show the flow-through fractions and contained the 7 and 18 kDa selenoproteins, and lanes 3–9 the fractions that eluted in the salt gradient (see Materials and methods). The 22 kDa selenoprotein shown in lane 5 eluted at ∼150 mM NaCl, and the 52 kDa selenoprotein shown in fractions 7–9 eluted at 400 mM NaCl in buffer B. Proteins were detected by PhosphorImager analysis of an SDS–PAGE gel. Download figure Download PowerPoint Ammonium sulfate fractionation revealed that four proteins were present in a 20–80% ammonium sulfate fraction (not shown), which was subsequently chromatographed on an anion-exchange column (Figure 1B). This procedure separated the 22 and 52 kDa from the 7 and 18 kDa selenoproteins, with the latter two proteins being present in the flow-through fraction. Subsequent purification focused on the 7 and 18 kDa proteins due to their abundance in Chlamydomonas cells. The use of HPLC hydrophobic interaction chromatography as the next purification step resulted in separation of the 7 and 18 kDa proteins (Figure 2A). Finally, the fractions containing these selenoproteins were fractionated separately on a C18 reversed-phase HPLC column (Figure 2B and C). Radioactive fractions corresponding to the 7 kDa selenoprotein eluted at 49% acetonitrile (Figure 2D) and the 18 kDa selenoprotein fraction at 47% acetonitrile (Figure 2E). These fractions were analyzed on SDS–PAGE gels (Figure 2F and G), and the protein bands were cut from the gels and analyzed by Edman degradation. Both selenoprotein sequences were found to be blocked, whereas protein contaminants in the selenoprotein bands were identified by N-terminal sequencing as ubiquitin (7 kDa band) and cyclophilin 1 (18 kDa band). To identify selenoproteins, the two selenoprotein bands cut from SDS–PAGE gels were digested with trypsin and analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (μLC/MS/MS). Twenty-one peptides derived from the 18 kDa selenoprotein band (Table I) and 33 peptides from the 7 kDa band (Table II) were sequenced by MS/MS. Figure 2.Purification of Chlamydomonas 7 and 18 kDa selenoproteins. The 7 and 18 kDa proteins were purified from the radioactive flow-through fractions from Q-Sepharose (see Figure 1B) by concentrating this material with ammonium sulfate, adjusting to a concentration of 0.4 M ammonium sulfate in buffer B, and fractionating on a phenyl–Sepharose HPLC column. Samples were analyzed as shown in (A): lanes 1–7 contain fractions eluted in a 0.4–0 M ammonium sulfate gradient in buffer B and lanes 8–11 show fractions eluted in a buffer B to water gradient as detected by PhosphorImager analysis; or further purified as shown in (B), (D) and (F): fractionation of the 7 kDa selenoprotein on a C18 reversed-phase HPLC column with a 0–70% acetonitrile gradient in 0.05% trifluoroacetic acid, γ-counter detection of 75Se in the eluted fraction, and SDS–PAGE analysis of fraction 33, respectively; and in (C), (E) and (G): fractionation of the 18 kDa selenoprotein on a C18 reversed-phase HPLC column with a 0–70% acetonitrile gradient in 0.05% trifluoroacetic acid, γ-counter detection of 75Se in the eluted fractions, and SDS–PAGE analysis of fraction 31, respectively. Lane 1 in (F) and (G) shows detection of 75Se by PhosphorImager analysis; lanes 2 and 3 show staining with Coomassie Blue of the column fractions and protein standards (masses are shown on the right). Download figure Download PowerPoint Table 1. Identification of Chlamydomonas PHGPx1 by tandem mass spectrometry Protein DDBJ/EMBL/GenBank accession No. Nos of sequenced peptides PHGPx1 (18 kDa) 5 Cyclophilin homolog, amino acid sequence deduced from EST AF052206 9 Cyclophilin 1 AW052206 7 Total: 3 Total: 21 Table 2. Identification of Chlamydomonas SelW1 by tandem mass spectrometry Protein DDBJ/EMBL/GenBank accession No. Nos of sequenced peptides Selenoprotein W1 (7 kDa) 3 Ubiquitin/ribosomal protein UQKM 5 Enolase (2-phospho-D-glycerate hydrolyase) P31683 1 Oxygen-evolving enhancer protein 1 P12853 2 Probable acyl-coenzyme a binding 15368290 4 Probable RSZp22 splicing factor 8286966,14242322 7 Probable RNA-binding protein 14237335 3 Chitinase-like protein 10775248 2 DnaJ (Hsp40)-like protein 15371690 2 Unknown protein 6548300 1 Unknown protein 8287459 1 Chloroplast Cpn21-like protein 6551572 1 FtsH-like protease 6549612 1 Total: 13 Total: 33 Selenoprotein W and glutathione peroxidase are major Chlamydomonas selenoproteins Sequence analyses of MS/MS peptides revealed that the 7 kDa selenoprotein was a homolog of animal selenoprotein W (Whanger, 2000) [three peptides matched an open reading frame (ORF) predicted from Chlamydomonas expressed sequence tag (EST) sequences; Table II]. The 18 kDa protein was identified as a homolog of eukaryotic phospholipid hydroperoxide glutathione peroxidases by matches of five peptides to the ORF predicted from EST sequences (Ursini et al., 1997) (Table I). These two most abundant Chlamydomonas selenoproteins are designated as SelW1 and PHGPx1 in this paper. In addition to the two selenoproteins, MS/MS analy sis identified 12 proteins that were either known Chlamydomonas proteins or Chlamydomonas homologs of proteins characterized in other species. Two additional proteins were also identified that have not been described previously in other organisms, but were represented by EST sequences (Tables I and II). A total of 16 Chlamydomonas proteins were identified by MS/MS analysis. Sec is present in SelW1 and PHGPx1 sequences and is encoded by UGA Based on the three SelW1 and five PHGPx1 peptide sequences, corresponding partial ORFs were constructed from EST sequences. At the time of analysis, SelW1 was not represented by EST sequences to generate a full cDNA sequence. Therefore, SelW1 cDNA was directly cloned from a Chlamydomonas cDNA library as described in Materials and methods. PHGPx1 and SelW1 ORFs contained in-frame UGA codons that corresponded to the Sec codons in their mammalian homologs (Figure 3A and B). Several PHGPx1 and SelW1 peptides that were encoded by nucleotide sequences located downstream of UGA codons were directly sequenced by MS/MS (Tables I and II), confirming that the UGA codon in these genes were read through and did not serve as terminator signals (Figure 3A and B). These observations, along with the fact that the isolated proteins contained selenium, indicated that UGA codons in SelW1 and PHGPx1 genes encoded Sec. Figure 3.Selenoproteins and Sec tRNA in Chlamydomonas. (A) Alignment of human, Arabidopsis, Clostridium, C.elegans, Schistosoma mansoni, yeast and Chlamydomonas PHGPx. The sequences of five tryptic peptides of Chlamydomonas PHGPx1, for which amino acid sequences were determined experimentally, are underlined. The DDBJ/EMBL/GenBank accession Nos are: NP_002076.1, human PHGPx; BF936124.1, Schistosoma PHGPx; AE007667.1, Clostridium PHGPx; AE007667.1, S.cerevisiae PHGPx; NP_497078.1, C.elegans PHGPx; AV623602, Chlamydomonas PHGPx1; BI721156 and AV623602, Chlamydomonas PHGPx2. Sec is present in human, Schistosoma and Chlamydomonas proteins, but is replaced by Cys in other PHGPx homologs. (B) Chlamydomonas cDNA sequence encoding selenoprotein W1. The amino acid U represents selenocysteine-14, which is encoded by TGA (underlined). The sequences of three tryptic peptides, for which amino acid sequences were determined experimentally, are also underlined. In the 3′-UTR, the position of the SECIS element is shown. (C) Chromatography of Chlamydomonas [75Se]selenocysteyl-tRNA. Chlamydomonas cells (2.5 g) were labeled with 75Se, and the labeled tRNA extracted and chromatographed on an RPC-5 column as described in Materials and methods. The large peak and trailing shoulder of 75Se-containing tRNA were pooled, as shown by the hatched area in the figure, prepared for ribosomal binding studies and the ribosomal binding studies carried out as described in Materials and methods. Binding to ribosomes in the absence of codon (designated None) or in the presence of codon is shown in the figure. Total CPM added to each reaction were 3200. Download figure Download PowerPoint Chlamydomonas Sec tRNA decodes UGA The finding of two specific Sec-containing proteins was surprising in that neither plants nor yeast are known to contain such proteins. Since Sec is inserted into all known natural selenoproteins by Sec tRNA, we isolated and characterized the Chlamydomonas Sec tRNA that recognizes UGA. Chlamydomonas cells were labeled with 75Se and the resulting labeled tRNA chromatographed on an RPC5 column (Figure 3C). The major peak and trailing shoulder were pooled as shown in the figure. The coding properties of this labeled tRNA were determined and it was found to recognize UGA, but not UGU, UGC (Cys codons) or UGG (Trp codon). These data strongly suggest that this 75Se-labeled tRNA recognizes specifically UGA. The amino acid attached to this tRNA was deacylated and identified as Sec as described in Materials and methods (data not shown). Chlamydomonas therefore contains Sec tRNA that decodes UGA. Identification of seven additional selenoprotein genes Chlamydomonas EST and genomic databases were analyzed for the presence of homologs of all known selenoproteins. Partial sequences derived from Chlamydomonas EST clones were identified that corresponded to several animal selenoproteins. Subsequently, whenever possible, EST clones were obtained and sequenced, and the remaining sequences were directly cloned from four Chlamydomonas cDNA libraries. These procedures resulted in the identification of seven additional Chlamydomonas selenoproteins. The ORFs of each of these selenoproteins were found to contain UGA at positions corresponding to Sec residues in animal proteins. The seven selenoprotein sequences were identified as: (i) a homolog of a recently identified mammalian selenoprotein M (further designated as SelM1) (Korotkov et al., 2002) (Supplementary figure A available at The EMBO Journal Online); (ii) a second selenoprotein M homolog (SelM2) (Supplementary figure A); (iii) a selenoprotein T homolog (SelT1) (Kryukov et al., 1999) (Supplementary figure B); (iv) a second selenoprotein W homolog (Supplementary figure C) (SelW2); (v) a second glutathione peroxidase homolog (PHGPx2) (Figure 3A); (vi) a homolog of a recently identified selenoprotein K (SelK1) (G.V.Kryukov and V.N.Gladyshev, unpublished data) (Supplementary figure D); and (vii) a homolog of animal thioredoxin reductase (TR1) (Sun et al., 1999) (Supplementary figure E). Identification of Chlamydomonas SECIS elements In animals, Sec is inserted in response to UGA when selenoprotein genes contain a stem–loop structure in 3′-UTRs, designated as the SECIS element (Low and Berry, 1996). Archaea also contain SECIS elements in their 3′-UTRs, but the secondary structure is different from that of animals (Rother et al., 2001b). In contrast, bacteria have a third type of SECIS element and it is located in the coding region of selenoprotein genes immediately downstream of Sec-encoding UGA codons (Bock, 2001). To determine which form of SECIS element is present in Chlamydomonas, we analyzed coding and 3′-UTR regions in the alga selenoprotein genes for which full cDNA sequences were available (SelK1, TR1, SelM1, SelM2 and SelW1) and identified SECIS elements in these genes (Figure 4A, five structures from the left) as well as a consensus RNA structural element (Figure 4A, right structure and B). The putative SECIS elements were found to reside in the 3′-UTR of selenoprotein genes. Overall structures of Chlamydomonas SECIS elements were found to be similar to those of animals. Both were composed of a conserved UGAN…NGAN sequence that formed a core (quartet) of non-Watson–Crick interacting nucleotides at the base of a strong helix (Figure 4B) (Low and Berry, 1996; Walczak et al., 1996). On top of the helix, unpaired nucleotides were present in either an apical loop or a bulge. Animal SECIS elements have been classified as Form 1 and Form 2 structures based on whether unpaired nucleotides are located in the loop or bulge (Grundner-Culemann et al., 1999), and both structures were interconvertible by mutations that remove or create a mini-helix in the apical loop. Interestingly, Chlamydomonas SECIS elements also occur as Form 1 (SelK1 and TR1 genes) and Form 2 (SelM1, SelM2 and SelW1 genes) structures (Figure 4A). These findings clearly indicate a common origin of Chlamydomonas and animal SECIS elements, and also distinguish the algal SECIS elements from those of bacterial and archaeal origin. Figure 4.SECIS elements in Chlamydomonas selenoprotein genes. (A) SECIS structures. SECIS element structures are shown for selenoproteins indicated below the structures. Proposed Chlamydomonas SECIS element consensus is shown on the right. (B) Alignment of the SECIS elements of Chlamydomonas selenoproteins genes. TR1 and SelK1 structures are Form 1 structures and the others are Form 2 structures. Human SelR SECIS element is aligned for comparison. Conserved nucleotides are shown in bold. Locations of helix 1, helix 2, quartet and apical loop are indicated. (C) Expression of Chlamydomonas SelK1 in mammalian cells. NIH 3T3 cells were transfected with a plasmid encoding GFP-chlamySelK (lane 1) or with GFP-C3 Vector (Clontech) as control (lane 2). Transfected cells were grown in the presence of [75Se]selenite and the resulting 75Se-labeled proteins were resolved by SDS–PAGE and visualized with a PhosphorImager. The location of GFP–SelK1 product is shown on the left. Locations and molecular weights of major selenoproteins, thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1), are also indicated. (D) Multiple alignment of Chlamydomonas MsrA1. The accession numbers are: NP_495540.1, C.elegans MsrA; P54149, bovine MsrA; NP_036463.1, human MsrA; NP_290851.1, E.coli MsrA; AAK83645, A.thaliana MsrA; 11342533, Fragaria ananassa MsrA; 7446683, Schizosaccharomyces pombe MsrA; AF494053, C.reinhardtii MsrA1. Download figure Download PowerPoint In spite of an apparent common origin of animal and Chlamydomonas SECIS elements, the length of a helix that separated the core (quartet) and the unpaired nucleotides in the apical loop or bulge was 12–13 nucleotides in algal structures instead of 11–12 nucleotides as in the animal structures (Low and Berry, 1996; Walczak et al., 1996). Moreover, the identities of the nucleotide located immediately upstream of the quartet (A or G in animals) and the unpaired nucleotides in the loop/bulge (AA or CC in animals) were often different in that Chlamydomonas SECIS elements could tolerate additional nucleotides present in these positions (Figure 4A and B). Expression of Chlamydomonas SelK1 in mammalian cells To test whether the Chlamydomonas Sec insertion system was indeed similar to that of animals, we expressed Chlamydomonas SelK1 in mouse NIH 3T3 cells. This protein was selected because its SECIS element, which occurs as the Form 1 structure, was most closely related to animal SECIS elements (Figure 4A, left structure). We developed a construct encoding SelK1 fused in-frame downstream of GFP with the purpose of obtaining a selenoprotein form whose mass is different from those of natural mouse selenoproteins and could therefore be easily detected in transfected cells. When NIH 3T3 cells transfected with the GFP–SelK1 construct were labeled with 75Se, a band corresponding to a fusion protein was detected (Figure 4C). The data suggest that Sec was inserted into Chlamydomonas SelK1 by the mammalian Sec insertion system in response to a Chlamydomonas SECIS element. Since SECIS elements in other Chlamydomonas selenoprotein genes were more distantly related to animal SECIS elements, it is possible that Sec insertion into these algal selenoproteins in mammalian cells could be less efficient. Nevertheless, our data clearly demonstrate that the Chlamydomonas Sec insertion system functionally overlaps with that of animals. Identification of a Chlamydomonas-specific selenoprotein The large number of selenoprotein genes detected in Chlamydomonas cells was surprising as most organisms have only a few such genes. For example, the genome of Caenorhabditis elegans contains only a single selenoprotein gene (thioredoxin reductase) (Buettner et al., 1999; Gladyshev et al., 1999b), whereas the Drosophila genome encodes three selenoprotein genes (selenophosphate synthetase, G-rich and BthD) (Castellano et al., 2001; Martin-Romero et al., 2001). Eighteen selenoproteins were previously identified in mammals (Hatfield and Gladyshev, 2002), including homologs of all nine Chlamydomonas selenoproteins described above. Interestingly, in terms of a set of identified selenoproteins, Chlamydomonas appeared to be more similar to mammals than to any other organisms (for example, using BLAST analysis, the closest homolog of Chlamydomonas TR1 was human TR1). We previously developed a program, called SECISearch, which allowed the identification of selenoprotein genes by searching for sequence, structural and thermodynamic characteristics of SECIS elements in animal nucleotide sequence databases (Kryukov et al., 1999; Martin-Romero et al., 2001). Since animal and Chlamydomonas SECIS elements were closely related, it was attractive to employ this program with the Chlamydomonas databases to search for additional algal selenoprotein genes. However, SECISearch could not be directly applied for identifying Chlamydomonas selenoprotein genes because of the differences in the length of helix 2, in the nucleotides in the apical loop and in the nucleotide preceding the core (quartet) between Chlamydomonas and animal SECIS elements. Therefore, we modified the program as described in Materials and methods to recognize SECIS elements in Chlamydomonas selenoprotein genes and applied the new program, Chlamy SECISearch, to the Chlamydomonas EST database. A computational screen of 113 064 available sequences revealed only 84 unique sequences that satisfied primary sequence, secondary structure and energetic criteria of Chlamydomonas SECIS elements, including four sequences that corresponded to the already identified selenoprotein SelM1, SelK1, SelW1 and TR1 genes (Table III). The SECIS element in the SelM2 gene was not detected as it did not satisfy the algorithm employed. Table 3. Analysis of Chlamydomonas EST database with Chlamy SECISearch Total No. of sequences 113 064 (∼80 000 000 nucleotides) Fit primary sequence and secondary structure consensuses 1502 Satisfy energy and fine structural criteria 243 Redundancy removal 84 Correspond to known selenoproteins 4 New selenoproteins 1 (MsrA1) The remaining sequences were analyzed manually using Mammalian Selenoprotein Signature criteria (e.g. conservation of Sec and the presence of homologs containing cysteine in place of Sec) (Kryukov and Gladyshev, 2002) revealing at least one new SECIS element (Table III; Figure 4A, second structure from the right). The corresponding EST clone was obtained and its sequence determined, revealing a gene encoding methionine-S-sulfoxide reductase (MsrA1) (Figure 4D). The MsrA1 gene contained an in-frame UGA codon at the position corresponding to the active site cysteine present in all known MsrAs (Weissbach et
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