In silico identification of novel selenoproteins in the Drosophila melanogaster genome

Abstract

In selenoproteins, incorporation of the amino acid selenocysteine is specified by the UGA codon, usually a stop signal. The alternative decoding of UGA is conferred by an mRNA structure, the SECIS element, located in the 3'-untranslated region of the selenoprotein mRNA. Because of the non-standard use of the UGA codon, current computational gene prediction methods are unable to identify selenoproteins in the sequence of the eukaryotic genomes. Here we describe a method to predict selenoproteins in genomic sequences, which relies on the prediction of SECIS elements in coordination with the prediction of genes in which the strong codon bias characteristic of protein coding regions extends beyond a TGA codon interrupting the open reading frame. We applied the method to the Drosophila melanogaster genome, and predicted four potential selenoprotein genes. One of them belongs to a known family of selenoproteins, and we have tested experimentally two other predictions with positive results. Finally, we have characterized the expression pattern of these two novel selenoprotein genes.

Fig. 1. dSelG. (A) Gene structure for dselG and in-tandem CG1840 paralog plotted using gff2ps (Abril and Guigó, 2000). Coordinates correspond to the AE002593 (X) scaffold. The extra coding region is shown in red as predicted by geneid and the annotated coding exons are in blue. (B) dselG form 2 SECIS. (C) Alignment of dSelG and CG1840 paralogs using CLUSTAL_W (Thompson et al., 1994).

Fig. 2. dSelM. (A) Gene structure for dselM and single exon CG15147, CG13186 paralogs. Coordinates correspond to the AE002593 (X), AE002690 (2L) and AE002787 (2R) scaffolds, respectively. (B) dselM form 2 SECIS. (C) Alignment of dSelM and CG15147, CG13186 paralogs.

Fig. 3. ⁷⁵Se-labeling of the D. melanogaster selenoproteins expressed in mammalian cells. (A) Lane 1: ⁷⁵Se-labeling of cells transfected with empty vector. Lane 2: ⁷⁵Se-labeling of cells transfected with dselG. Lane 3: ⁷⁵Se-labeling of cells transfected with dselM. (B) High magnification of the region corresponding to the dselG labeling.

Fig. 4. In situ hybridization in embryos, imaginal discs and brain. (A, C and E) dselM expression pattern in sincytial blastoderm, cellular blastoderm and gastrulation embryonic stages, respectively; (B, D and F) the corresponding sense controls (scale bar is 50 mm); (G) dselM expression in brain and neuroblast staining in the inset; (H) the brain sense control (scale bar 100 mm, inset scale bar 2.5 mm); (I) dselM expression in wing disc; (J) wing disc sense control (scale bar 50 mm); (K, M and O) dselG expression pattern in sincytial blastoderm, cellular blastoderm and gastrulation embryonic stages, respectively; (L, N and P) are the corresponding sense controls (scale bar is 50 mm).

Fig. 5. SECIS and gene prediction. (A) General form 1 SECIS divided into structural units. Form 2 has an extra short stem–loop in the apical loop. (B) PatScan SECIS pattern to search for both form 1 and form 2 SECIS. The extra stem–loop in form 2 is not taken into account when searching. (C) The two possible ways of geneid prediction for an ideal two exons gene: as a normal gene or as a selenoprotein gene with a TGA in-frame and a SECIS. Exon defining signals are shown. (D) False positive selenoprotein genes with either a TGA in-frame or a SECIS. These partial predictions are not permitted in the gene prediction.

Fig. 6. General schema for selenoprotein identification.