Computational search of the human genome for selenoprotein genes containing SECIS elements in the coding region. A total of 23 selenoprotein genes could be detected using this procedure; however, no additional selenoprotein genes containing SECIS elements in the coding region were found.

Analysis of viral genomes for selenoprotein genes. Details of the search are provided in Materials and Methods. The search of 1977 viral sequences revealed three SECIS element structures.

Design of SECIS elements in the coding regions of mammalian selenoprotein genes. (A) Designed GPx1 SECIS element. (B) Designed GPx4 SECIS element. The first amino acids (methionine), Sec, and the SECIS elements are shown in boldface. The amino acids that were mutated to accommodate the designed SECIS elements are highlighted. The newly introduced residues are shown by gray circles, and further changes in the designed GPx1 SECIS are shown by white circles.

An upstream SECIS element allows readthrough at in-frame UGA codons. (A) Constructs were generated in which the fowlpox SECIS element was cloned upstream and in-frame with the ORF of GPx1 separated by a V5 tag. Control constructs contained mutations in either the SBP2-binding region or in the conserved loop region. (B) Schematic illustration of the constructs used in (A). Numbers on the left correspond to lane designations in A.

Expression of wild-type fowlpox virus GPx4 in mammalian cell lines. (A) Western blot analysis of mouse hepatoma cell line Hepa1-6 transfected with wild-type fowlpox virus GPx4 construct (WT FPV GPx) or the corresponding construct in which the SBP2-binding site was disrupted (WT FPV GPx ΔATGA). (B) Transfection of HEK293 with wild-type fowlpox virus GPx4 (FPV GPx), its mutants lacking SBP2-binding site (WT FPV GPx ΔATGA) or the conserved AA in the apical loop (WT FPV GPx ΔAA), and the corresponding Sec-to-Cys mutants.

Amino acid sequence alignment of glutathione peroxidases of the GPx4 family. Accession numbers of the sequences used in the alignment are as follows: NP_566128.1 (Arabidopsis thaliana), AAH22071.1 (Homo sapiens), NP_032188.3 (Mus musculus), AAM18080.2 (Gallus gallus), AAR83433.1 (canarypox virus), CAE52609.1 (fowlpox virus). In-frame SECIS elements in the C-terminal regions of viral proteins are underlined. Location of catalytic Sec and the corresponding Cys is indicated by an asterisk.

SECIS elements in viral glutathione peroxidases. (A) SECIS structures. Left structure, fowlpox virus GPx4 SECIS element. Middle structure, canarypox virus GPx4 SECIS element. Right structure, H.sapiens GPx4 SECIS element. SECIS core and unpaired AA nucleotides in apical loop are shown in boldface. (B) Location of viral GPx4 SECIS elements within mRNAs and comparison with GPx4 genes from other sources. Cys-containing GPx4 homologs from higher plants lack a SECIS element. In the human Sec-containing GPx4, SECIS element is in the 3′-UTR. In the fowlpox virus Sec-containing GPx4 homolog, SECIS element is the in the coding region. In the canarypox virus Cys-containing GPx4, a fossil SECIS element is in the coding region. Location of Cys is shown in white and Sec in black. SECIS elements are indicated by light-gray boxes and ORFs by dark-gray boxes. Left and right black lines represent 5′- and 3′-UTRs, respectively.

Expression of GPx1 containing a designed in-frame SECIS element. (A) Hepa1-6 cells were transfected with WT GPx1 and different in-frame SECIS element containing constructs. The construct GPx1 2.IFS ΔATGA contains an additional modification in the stem-region as shown in Figure 6A. To disrupt the function of the natural SECIS element, mutations were made in either the SBP2-binding region or the apical loop. (B) Transfection of HEK293 cells. No expression was detectable for GPx4 containing a designed in-frame SECIS element. Note the low expression level of the Cys mutant with the designed in-frame SECIS element compared with GPx1. Expressed proteins were detected by western blot analysis using antibodies specific for V5 tag.

Eukaryotic SECIS elements located in coding regions are functional. (A) Western blot analysis of HEK293 cells transfected with GPx1 constructs containing functional in-frame SECIS elements. The indicated constructs were generated by either deleting the stop codon TAA or replacing the GPx1 3′-UTR with the fowlpox virus SECIS element thereby generating a GPx1-FPV fusion protein. (B) The same samples as shown in A were metabolically labeled with ⁷⁵Se and exposed to a PhosphorImager cassette. Location of selenoproteins expressed with the help of coding region SECIS elements is shown by arrows on the right. (C) Western blot analysis of HEK293 cells transfected with GPx4 constructs containing in-frame SECIS elements. GPx4-based constructs were generated by replacing non-homologous sequences with the fowlpox SECIS element thereby generating a GPx4-FPV fusion protein. Lane designations are as follows: Mock, control transfection; WT GPx1, wild-type GPx1 gene; GPx1 ΔATGA, mouse GPx1 gene with disrupted SBP2-binding region; GPx1 ΔTAA, mouse GPx1 gene with its natural stop codon deleted (allowing the ORF to extend beyond the SECIS element which becomes part of the ORF); GPx1 FPV SECIS, the fowlpox SECIS element replacing the 3′-UTR region of the mouse GPx1 gene; GPx1 FPV SECIS ΔATGA, the fowlpox virus SECIS was fused in frame with GPx1 and its SBP2-binding site was disrupted; WT GPx4, cells were transfected with wild-type mouse GPx4 cDNA; GPx4 ΔATGA, the SBP2-binding site in the wild-type GPx4 cDNA was disrupted; GPx4 FPV SECIS, the fowlpox virus SECIS element was fused in frame with GPx4 lacking its own 3′-UTR; GPx4 FPV SECIS ΔATGA, the fowlpox virus SECIS fused to GPx4 was mutated to disrupt the SBP2-binding site.