A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells Sergey V. Novoselov*†, Alexey V. Lobanov*, Deame Hua*, Marina V. Kasaikina*, Dolph L. Hatfield‡, and Vadim N. Gladyshev*§ *Department of Biochemistry, University of Nebraska, Lincoln, NE 68588; and ‡Section on the Molecular Biology of Selenium, Laboratory of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Edited by W. Ford Doolittle, Dalhousie University, Halifax, NS, Canada, and approved February 21, 2007 (received for review December 12, 2006) Selenoproteins are an elite group of proteins containing a rare fully applied in genomic searches to identify these stem–loop amino acid, selenocysteine (Sec), encoded by the codon, UGA. In structures, and thereby selenoprotein genes, in nucleotide sequence eukaryotes, incorporation of Sec requires a Sec insertion sequence databases (5, 7, 17–19). SECIS) element, a stem–loop structure located in the 3؅-untrans- It has been established that the quartet is involved in the) lated regions of selenoprotein mRNAs. Here we report identifica- interaction with SECIS-binding protein 2 (SBP2) (20, 21), which, in tion of a noncanonical form of SECIS element in Toxoplasma gondii turn, is essential for the formation of a complex with the Sec-specific and Neospora canine, single-celled apicomplexan parasites of hu- elongation factor, known as EFsec, and tRNA[Ser]Sec (22, 23). This mans and domestic animals. This SECIS has a GGGA sequence in the protein–RNA complex functions by inserting Sec in response to SBP2-binding site in place of AUGA previously considered invari- UGA codons (24). Recently, additional components of the complex ant. Using a combination of computational and molecular tech- were reported, including the L30 protein that interacts with the niques, we show that Toxoplasma and Neospora possess both SECIS element in vitro and in vivo and competes with SBP2 for canonical and noncanonical SECIS elements. The GGGA-type SECIS SECIS binding (25). In addition, SECp43 and SLA have roles in element supported Sec insertion in mammalian HEK 293 and NIH selenoprotein biosynthesis through interaction with tRNA[Ser]Sec 3T3 cells and did so more efficiently than the natural mammalian in a supramolecular complex (26, 27). SECIS elements tested. In addition, mammalian type I and type II It has been found that the four consecutive non-Watson–Crick SECIS elements mutated into the GGGA forms were functional but base pairs are part of a recurrent motif, the kink-turn (K-turn), and manifested decreased Sec insertion efficiency. We carried out constitute the functional motif of the SECIS structure, which is computational searches for both AUGA and GGGA forms of SECIS recognized by SBP2. It has been proposed that the invariant U in elements in Toxoplasma and detected five selenoprotein genes, the conserved AUGA sequence forms a non-Watson–Crick base including one coding for a previously undescribed selenoprotein, pair with another U in 5ЈDI SECIS RNA (12, 28). Similarly, it is designated SelQ, and two containing the GGGA form of the SECIS thought this U may form a base pair with the corresponding BIOCHEMISTRY element. In contrast, the GGGA-type SECIS elements were not nucleotide independent of its identity. The analysis of crystal detected in mammals and nematodes. As a practical outcome of the structures of RNA–protein complexes suggested the residue at this study, we developed pSelExpress1, a vector for convenient expres- position binds in a pocket of RNA-binding proteins (29–31). It was sion of selenoproteins in mammalian cells. It contains an SBP2 gene also proposed that the SECIS core is a K-turn-like motif, which and the most efficient tested SECIS element: an AUGA mutant of provides greater flexibility and allows switching between open and the GGGA-type Toxoplasma SelT structure. closed kinked conformations. In turn, this feature triggers a major conformational change in the SBP2-bound complex (25, 28). genome ͉ RNA structure ͉ selenocysteine ͉ selenoprotein In our study, we found that the U in the quartet, previously considered an invariant residue, is not conserved in some apicom- elenocysteine (Sec)-containing proteins (selenoproteins) are plexan selenoprotein genes. Instead, a previously undescribed form Srare but widely distributed in all domains of life (1), including of SECIS element was found that has a GGGA sequence in place bacteria (2, 3), archaea (4), and eukaryotes (5–7). The human of the conserved AUGA sequence. We characterized this structure genome possesses 25 genes encoding such proteins (7). The class in detail using a combination of computational and molecular of selenoproteins is defined by the occurrence of Sec, the 21st approaches. amino acid encoded by the UGA codon. Selenoproteins use the high reactivity of Sec, which is located in catalytic centers and Results and Discussion serves redox function analogous to the functions of redox-active Identification of a Noncanonical Form of Eukaryotic SECIS Element. A Cys residues (8). In addition to the UGA codon, a cis-acting search for Toxoplasma selenoprotein genes was carried out by element is present within selenoprotein genes, which is also essential for recognition of UGA as the Sec codon. This element is a stem–loop structure, known as the Sec insertion sequence Author contributions: S.V.N. and V.N.G. designed research; S.V.N., A.V.L., D.H., and M.V.K. (SECIS) located in coding regions of bacterial and in 3Ј-UTRs performed research; S.V.N., A.V.L., D.L.H., and V.N.G. analyzed data; and S.V.N., D.L.H., and of archaeal and eukaryotic selenoprotein genes (9, 10). V.N.G. wrote the paper. The principal features of the eukaryotic SECIS element include The authors declare no conflict of interest. a segment containing four non-Watson–Crick base pairs This article is a PNAS Direct Submission. UGAN....NGAN (designated the quartet or core), an unpaired Abbreviations: Sec, selenocysteine; SECIS, Sec insertion sequence. A preceding the quartet, and an unpaired AA or CC motif in the †Present address: Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, apical loop or bulge that is separated from the quartet by 11–12 Moscow Region 142292, Russia. nucleotides (11–14). Although having low sequence conservation, §To whom correspondence should be addressed. E-mail: [email protected]. the secondary structure of eukaryotic SECIS elements is strictly This article contains supporting information online at www.pnas.org/cgi/content/full/ conserved and thermodynamically stable (15, 16). Based on these 0610683104/DC1. observations, several algorithms have been developed and success- © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610683104 PNAS ͉ May 8, 2007 ͉ vol. 104 ͉ no. 19 ͉ 7857–7862 Downloaded by guest on September 26, 2021 Fig. 1. SECIS elements identified in Toxoplasma and Neospora.(A) Canonical (shown in white background) and the GGGA-type (shown in gray background) SECIS elements identified in T. gondii and N. caninum are shown. The SECIS core and the unpaired AA nucleotides in the apical loop are shown in bold. (B) Selenoprotein Q (SelQ). EST sequences (GenBank accession nos. CN615432.1 and CF268978.1) were used for sequence reconstruction. Locations of the initiator AUG codon, Sec-encoding UGA codon, stop signal, and the SECIS element are indicated. homology analyses involving all known selenoproteins as queries. Sec insertion (i.e., the 40-kDa selenoprotein band appeared to be This procedure identified homologs of four mammalian selenopro- more enriched). Thus, the new type of SECIS element is not only teins: SelW, SelK, SelS, and SelT [supporting information (SI) Figs. functional, but its function could be stimulated by mammalian 6–9]. Their genes had predicted Sec residues encoded by UGA SBP2. Moreover, when certain constructs were used, the GGGA codons. Analysis of the 3Ј-UTRs in these selenoprotein genes form of SECIS element appeared to be more efficient than the revealed the presence of canonical SECIS elements in SelK and natural mouse SelH element (e.g., compare lanes 1–4 and 9 and 10, SelW genes (Fig. 1A). However, no suitable structure was found in Fig. 2 B and C). the SelT 3Ј-UTR. The use of relaxed settings and the loose pattern of SECISearch did not yield candidate SECIS structures in the The New Type of SECIS Element Is Highly Efficient in Sec Insertion. Toxoplasma SelT gene. Another way to monitor efficiency of Sec insertion is to probe The lack of a standard SECIS element in the SelT gene suggested lysates of transfected cells in Western blot assays with anti-GFP the presence of a noncanonical structure. Manual analysis of the antibodies to determine the ratio between full-length and truncated SelT 3Ј-UTR using MFOLD revealed a SECIS-like structure that forms of the fusion protein (Fig. 2 B Lower and C Lower). The satisfied all SECIS element requirements with one notable excep- truncated form is generated by termination of protein synthesis at tion: the SBP2-binding site (e.g., SECIS core or quartet) had a the UGA codon because of competition of Sec insertion and GGGA sequence instead of AUGA (Fig. 1A). The U in the AUGA translation termination, whereas the full-length protein is made sequence is considered invariant and is present in all known when UGA is read as the Sec codon, and translation continues until eukaryotic SECIS elements. To examine whether the GGGA the true stop signal. The ratio of full-length and truncated forms of sequence in the SECIS core represented a sequencing error, we fusion proteins that resulted from transfections with various GFP- analyzed additional protozoan sequences. EST sequences of Neos- SelH chimeras differed in the cell lines used in the study.