Structural analysis of new local features in SECIS RNA hairpins

Abstract

Decoding of the UGA selenocysteine codon for selenoprotein translation requires the SECIS element, a stem-loop motif in the 3'-UTR of the mRNA carrying short or large apical loops. In previous structural studies, we derived a secondary structure model for SECIS RNAs with short apical loops. Work from others proposed that intra-apical loop base pairing can occur in those SECIS that possess large apical loops, yielding form 2 SECIS versus the form 1 with short loops. In this work, SECIS elements arising from eight different selenoprotein mRNAs were assayed by enzymatic and/or chemical probing showing that seven can adopt form 2. Further, database searches led to the discovery in drosophila and zebrafish of SECIS elements in the selenophosphate synthetase 2, type 1 deiodinase and SelW mRNAs. Alignment of SECIS sequences not only highlighted the predominance of form 2 but also made it possible to classify the SECIS elements according to the type of selenoprotein mRNA they belong to. Interestingly, the alignment revealed that an unpaired adenine, previously thought to be invariant, is replaced by a guanine in four SECIS elements. Tested in vivo, neither the A to G nor the A to U changes at this position greatly affected the activity while the most detrimental effect was provided by a C. The putative contribution of the various SECIS motifs to function and ligand binding is discussed.

Figure 1

Experimentally supported secondary structure models for the SECIS RNAs of the human SelN, SelD, SelX, SelY, SelZ, mouse Sel15, rat PHGPx and XlDI3. The structures were manually folded and tested by enzymatic and/or chemical probing. Potential base pairings in the apical loops are drawn. Sequences were taken from the references indicated in the text. Enzymatic and Pb(II) cleavages are indicated by arrowheads: red, RNase T1; green, RNase T2; blue, RNase V1; black, Pb(II). The sizes of the arrowheads are roughly proportional to the intensity of cleavage. Circles indicate bases that are reactive towards DMS or CMCT under native (orange) or semi-denaturing (purple) conditions. Moderate reactivities are highlighted in yellow (native) or light purple (semi-denaturing). The form 2 SECIS models arising from the probing experiments are displayed on the right for each SECIS hairpin. For hSelD and hSelX, the equilibrium arrows schematize the occurrence of the transient form 1 and form 2 hairpins. The non-Watson–Crick quartet is boxed in red. Owing to the variable lengths of helix 1 in the various SECIS, the numbering starts at the fifth base pair below internal loop 1 in each SECIS element.

Figure 1

Experimentally supported secondary structure models for the SECIS RNAs of the human SelN, SelD, SelX, SelY, SelZ, mouse Sel15, rat PHGPx and XlDI3. The structures were manually folded and tested by enzymatic and/or chemical probing. Potential base pairings in the apical loops are drawn. Sequences were taken from the references indicated in the text. Enzymatic and Pb(II) cleavages are indicated by arrowheads: red, RNase T1; green, RNase T2; blue, RNase V1; black, Pb(II). The sizes of the arrowheads are roughly proportional to the intensity of cleavage. Circles indicate bases that are reactive towards DMS or CMCT under native (orange) or semi-denaturing (purple) conditions. Moderate reactivities are highlighted in yellow (native) or light purple (semi-denaturing). The form 2 SECIS models arising from the probing experiments are displayed on the right for each SECIS hairpin. For hSelD and hSelX, the equilibrium arrows schematize the occurrence of the transient form 1 and form 2 hairpins. The non-Watson–Crick quartet is boxed in red. Owing to the variable lengths of helix 1 in the various SECIS, the numbering starts at the fifth base pair below internal loop 1 in each SECIS element.

Figure 2

Enzymatic and Pb(II) probing of the SECIS RNAs shown in Figure 1. (A) hSelD; (B) hSelN; (C) hSelX; (D) hSelY; (E) hSelZ; (F) mSel15; (G) rPHGPx; (H) XlDI3. The RNAs were digested with RNases T1, T2 and V1 for 5 and 10 min. (I) hSelX SECIS was cleaved at 20°C with 4, 7.5 and 10 mM Pb(II) acetate for 2 and 5 min (lanes 2–7, respectively). (J) rPHGPx SECIS was cleaved at 37°C for 2 min with 1.25 (lane 2), 2.5 (lane 3) and 5 mM (lane 4) Pb(II) acetate. Lanes marked C are control reactions; L, alkaline ladder; T1, RNase T1 ladder. The numbering corresponds to that in Figure 1.

Figure 2

Enzymatic and Pb(II) probing of the SECIS RNAs shown in Figure 1. (A) hSelD; (B) hSelN; (C) hSelX; (D) hSelY; (E) hSelZ; (F) mSel15; (G) rPHGPx; (H) XlDI3. The RNAs were digested with RNases T1, T2 and V1 for 5 and 10 min. (I) hSelX SECIS was cleaved at 20°C with 4, 7.5 and 10 mM Pb(II) acetate for 2 and 5 min (lanes 2–7, respectively). (J) rPHGPx SECIS was cleaved at 37°C for 2 min with 1.25 (lane 2), 2.5 (lane 3) and 5 mM (lane 4) Pb(II) acetate. Lanes marked C are control reactions; L, alkaline ladder; T1, RNase T1 ladder. The numbering corresponds to that in Figure 1.

Figure 2

Enzymatic and Pb(II) probing of the SECIS RNAs shown in Figure 1. (A) hSelD; (B) hSelN; (C) hSelX; (D) hSelY; (E) hSelZ; (F) mSel15; (G) rPHGPx; (H) XlDI3. The RNAs were digested with RNases T1, T2 and V1 for 5 and 10 min. (I) hSelX SECIS was cleaved at 20°C with 4, 7.5 and 10 mM Pb(II) acetate for 2 and 5 min (lanes 2–7, respectively). (J) rPHGPx SECIS was cleaved at 37°C for 2 min with 1.25 (lane 2), 2.5 (lane 3) and 5 mM (lane 4) Pb(II) acetate. Lanes marked C are control reactions; L, alkaline ladder; T1, RNase T1 ladder. The numbering corresponds to that in Figure 1.

Figure 3

Chemical probing with DMS and CMCT. (A) hSelD; (B) hSelX; (C) hSelY; (D) rPHGPx. A short migration is also displayed. (E) XlDI3. RNAs were treated for 5 min at 20°C with 0.5 µl DMS under native (lanes 2) or semi-denaturing conditions (lanes 4), or with 10 mg/ml CMCT for 10 min at 25°C, under native (lanes 6) or semi-denaturing (lanes 8) conditions. Lanes 1, 3, 5 and 7 are controls. A, G, C, U are sequencing lanes. Numbering is from Figure 1.

Figure 3

Chemical probing with DMS and CMCT. (A) hSelD; (B) hSelX; (C) hSelY; (D) rPHGPx. A short migration is also displayed. (E) XlDI3. RNAs were treated for 5 min at 20°C with 0.5 µl DMS under native (lanes 2) or semi-denaturing conditions (lanes 4), or with 10 mg/ml CMCT for 10 min at 25°C, under native (lanes 6) or semi-denaturing (lanes 8) conditions. Lanes 1, 3, 5 and 7 are controls. A, G, C, U are sequencing lanes. Numbering is from Figure 1.

Figure 4

Activities of the A1017G, A1017C and A1017U GPx SECIS mutants in transfected COS-7 cells, determined with the glutathione peroxidase activity assay. The average values above the bars, subtracted from the endogenous GPx level, are given with respect to the wt GPx activity taken as 100%.