500 Nucleic Acids Research, 2006, Vol. 34, No. 2 Animal SECIS elements were previously described as belonging to form 1 and 2 based on whether the unpaired AA sequence is located in the loop or bulge (30). In this classiﬁcation, all four P.falciparum SECIS elements belonged to form 2 SECIS elements. The four SECIS elements were highly conserved across Plasmodia (Figure 1B) and formed structures very similar to those of P.falciparum SECIS elements. Plasmodium selenoproteome Genomic sequences upstream of four SECIS elements were analyzed for the presence of ORFs. We found that PlasmoDB had predicted protein sequences upstream of the SECIS elements, but these were misannotated with respect to exon–intron junctions as well as ORFs. Comparative sequence analyses of four selenoprotein genes in different Plasmodium species helped us to determine their correct gene structures. The organization of predicted P.falciparum selenoprotein genes, which are designated in this study as selenoproteins #1 to #4, is shown in Figure 2. Selenoprotein #1 (Sel1) is organized in three exons, selenoproteins #2 and #3 (Sel2 and Sel3) are encoded by a single exon each and selenoprotein #4 (Sel4) consists of 2 exons. In each Plasmodium selenopro- tein gene, the Sec-encoding UGAs and the SECIS elements were present in the last exon, although the distance between UGA and SECIS was different (Figure 2). Exhaustive BLAST searches against a number of databases produced no homologs outside Plasmodium for all four selenoprotein genes, whereas these sequences were highly homologous within Plasmodia (Figure 3). In addition, Sec residues were conserved across Plasmodium and were ﬂanked by highly conserved regions. In two selenoproteins (Sel1 and Sel4), Sec was present as a C-terminal penultimate residue. This location was reminiscent of Sec in several animal seleno- proteins, such as thioredoxin reductase and selenoproteins K, O and S (17). In addition, prediction of secondary structures revealed similarities between Plasmodium Sel1 and Sel4 on one side, and mammalian SelK and SelS on the other, suggesting a possible common origin of these proteins. Sel2 had Sec in the N-terminal region, and it was present in the form of the CxxU motif (cysteine and Sec separated by two other residues). This motif is also found in several animal selenoproteins, including SelM, SelT, SelV and SelW. Moreover, both animal selenoproteins con- taining the CxxU motif and Sel2 have a predicted a-helix downstream of the motif. These observations suggest that Sel2 might have a redox function, with Sec being a residue that forms a selenenylsulﬁde bond with the Cys. Sel3 has Sec in the N-terminal region, but it was not present within a known motif. The possible location of predicted proteins within Plasmod- ium cells was analyzed by searching for the presence of signal peptides. The ﬁrst 24 amino acids of Sel3 corresponded to a predicted signal peptide (the SignalP probability value of 0.959), whereas other selenoproteins did not have pre- dicted targeting signals. However, using PATS Version 1.2.1, the program that identiﬁes sequences that are targeted to the apicoplast matrix of P.falciparum (31), we found that Sel2 (probability score of 0.982) and Sel3 (probability score of 0.936) were candidate apicoplast proteins. Expression of P.falciparum selenoprotein genes Previous annotations, microarrays with 70 oligomers and Scripps arrays with a higher number of 25 oligomers (all data available in PlasmoDB) suggested that the four predicted selenoprotein genes were transcribed. By carefully analyzing the location of the oligomers used for the arrays, the following conclusions could be drawn: Sel1 was expressed throughout the erythrocytic life cycle with highest levels in schizonts. Sel2 was expressed at constant but relatively low levels throughout the Plasmodium life cycle. Sel3 and Sel4 were also expressed in various stages of the Plasmodium life cycle, with highest levels in trophozoites and rings/schizonts, respectively. These expression proﬁles together with the fact that selenoprotein genes were not clustered within Plasmodium genomes sug- gested that selenoproteins may serve distinct functions. However, these data were not sufﬁcient to determine whether the predicted Sec-encoding UGA codons were indeed present within exons and in the correct reading frame as sug- gested by our in silico analyses, or were parts of introns as annotated in PlasmoDB using Geneﬁnder predictions. The respective PlasmoDB annotation numbers were PF14_0033 for Sel1, PFI1515w for Sel2, MAL8P1.86 for Sel3 and PF14_0251 for Sel4. We designed primer pairs to examine the gene structure of selenoprotein genes and to verify the presence of predicted mRNAs (Figure 2). For Sel2 and Sel3, in which Sec-encoding UGA codons were well within the ORFs (far from the pre- dicted termination signals), the presence and the correct size of the PCR products clearly indicated the correctness of our predictions as indicated in Figure 2, whereas the PCR products were not consistent with the PlasmoDB predictions. In Sel1 and Sel4, Sec was a C-terminal penultimate residue, thus only small deviations from the PlasmoDB predictions occurred. Sel1 primer pairs N-CU, N-C and s-C resulted in bands of 380, 360 and 160 bp, respectively. The 380 bp fragment was cloned and sequenced and found to match our predictions. For Sel2, which we predicted to possess a different C-terminal sequence than that given in PlasmoDB, PCR products could be ampliﬁed with primer pairs N-as and N-C (consistent with our predictions), but not with N-Ca, which would be expected if the PlasmoDB prediction was correct. The N-C fragment of 690 bp was cloned and sequenced and again found to match our prediction. For Sel3, the PCR product of 834 bp was generated with the N-as primer pair and was veriﬁed by sequencing. For Sel4, primer pairs N-as, N-C, N-nC and N-CD gave distinct bands of expected size, which was again consistent with our gene prediction, but with N-gf (Plas- moDB prediction) no product could be detected. These results were further veriﬁed by cloning and sequencing an 444 bp fragment ampliﬁed with the N-nC primer pair. Thus, all four selenoprotein genes were in full agreement with our in silico predictions, including the exonic, in-frame location of the UGA codons. Plasmodium SECIS elements support Sec insertion in mammalian cells Since Plasmodium SECIS elements were similar to known eukaryotic SECIS elements, we tested whether these structures could support Sec insertion in mammalian cells. Mammalian cells provide a convenient, commonly used model system to