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. 2014;11(11):1402-13.
doi: 10.1080/15476286.2014.996472.

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

Jodi L Bubenik  1 , Angela C Miniard, Donna M Driscoll
Affiliations

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

Jodi L Bubenik et al. RNA Biol. 2014.

Abstract

Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Sec-containing proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3' untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.

Keywords: DTT, dithiothreitol; Dio1, deiodinase 1; Dio2, deiodinase 2; GPx1, glutathione peroxidase 1; PHGPx, phospholipid hydroperoxide glutathione peroxidase; REMSA, RNA electrophoretic mobility shift assay; RNA-protein interactions; RRL, rabbit reticulocyte lysate; SBP2, SECIS binding protein 2; SECIS, Selenocysteine Insertion Sequence; SECIS-binding protein 2; Sec, selenocysteine; selenium; selenocysteine; selenoprotein; translation.

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Figures

Figure 1.
Figure 1.
In vitro translated SBP2-CT is more active than recombinant protein. (A) Schematic diagram of the luc/UGA/PHGPx reporter construct. (B) The reporter mRNA was in vitro translated in the presence of increasing amounts of purified recombinant SBP2-CT as indicated. Translation products were analyzed for luciferase activity. The error bars represent one standard deviation. (C) The luc/UGA/PHGPx reporter RNA was in vitro translated in the presence of either purified recombinant SBP2-CT or in vitro translated SBP2-CT, as indicated. Translation products were analyzed for luciferase activity. The error bars represent one standard deviation. (D) The luc/UGA/PHGPx reporter was in vitro translated in the presence of either purified recombinant SBP2-CT or in vitro translated SBP2-CT, in the presence of 35S-methionine. Two microliters of the translation products were analyzed by SDS-PAGE. TR-Luc, truncated luciferase; FL-luc, full-length luciferase; *, radio-labeled in vitro translated SBP2-CT.
Figure 2.
Figure 2.
SBP2-CT is selective for different SECIS elements. The UGA recoding assays were performed in RRL using luc/UGA/Dio1 reporter in the presence of in vitro translated SBP2-CT, with PHGPx, Dio2 and GPx1 SECIS elements added as competitor RNAs, as indicated. Translation products were analyzed for luciferase activity. The error bars represent one standard deviation.
Figure 3.
Figure 3.
RBD is selective and binds the SECIS element with high affinity. (A) REMSA analysis was performed using [32P]-labeled PHGPx SECIS RNA and increasing amounts of purified, recombinant SBP2-RBD protein. The RNA and protein were allowed to bind to equilibrium and then complex formation was analyzed by non-denaturing gel electrophoresis. (B) Quantification of binding data from REMSA in panel A. (C) Competition REMSAs using [32P]-labeled PHGPx SECIS RNA as the probe and increasing amounts of PHGPx, Dio2 and GPx1 SECIS RNAs as competitors as indicated. P, unbound probe; S, shift due to functional SECIS:SBP2 interaction.
Figure 4.
Figure 4.
Vertebrate SBP2 RBD contains 5 conserved cysteines. A multiple sequence alignment of the second part of the RBD (612-756, rat numbering) including primate, rodent, ungulate, marsupial, reptile, avian, and fish and additional mammalian sequences. The red and blue highlighting indicates completely and partially conserved cysteine residues respectively. The gray boxes indicate regions of identity across the domain. The locations of the 5 conserved vertebrate cysteines are indicated and numbered with respect to the rat sequence.
Figure 5.
Figure 5.
RNA-binding activity is not affected by the loss of an individual cysteine (A) SDS-PAGE analysis of in vitro translated SBP2-RBD and individual cysteine mutant proteins labeled with 35S-methionine. (B) REMSAs were performed with equimolar amounts of the in vitro translated proteins incubated with PHGPx SECIS RNA as described in Figure 3. P, unbound probe; S, functional SECIS/SBP2 interaction; *, shift caused by interaction with an endogenous protein in the RRL.
Figure 6.
Figure 6.
Multiple cysteine mutations impair the RNA-binding activity of SBP2. (A) Schematic representation of the multiple cysteine (C) to serine (S) mutations within the RBD from amino acid 612 to 777. All numbering refers to the rat sequence. (B) SDS-PAGE analysis of in vitro translated wildtype and mutant SBP2-RBD proteins labeled with 35S-methionine. (C) REMSAs of wildtype and mutant translated proteins using PHGPx SECIS RNA as the probe as described in Figure 3. P, unbound probe; S, functional SECIS/SBP2 interaction; *, shift caused by interaction with an endogenous protein in the RRL.
Figure 7.
Figure 7.
The subcellular localization of SBP2-CT is not affected by single cysteine mutations. (A) McArdle 7777 cells were examined for the localization of transiently transfected SBP2-CT, wildtype and single cysteine mutants, as described in Materials and Methods. Images were taken at 40x magnification. The SBP2-CT proteins were detected with an antibody directed against the V5 epitope tag, and the nuclei were detected with 4',6-diamidino-2-phenylindole (DAPI) stain. (B) Forty-8 hours post transfection, cells were treated with 2 ng/mL amount of Leptomycin B for 2 hours to prevent nuclear export, and the subcellular localization was determined as in A. (C) Forty-8 hours post transfection cells were treated with 1 mM hydrogen peroxide for 2 hours and the subcellular localization was detected as in A.
Figure 8.
Figure 8.
Nucleo-cytoplasmic shuttling is not perturbed when RNA-binding is impaired. McArdle 7777 cells were examined for the localization of transiently transfected SBP2-CT 4X(Cys to Ser) mutant. Images were taken at 40x magnification. The SBP2-CT 4x(Cys to Ser) mutant protein was detected with an antibody directed against the V5 epitope tag, and the nuclei were detected with 4',6-diamidino-2-phenylindole (DAPI) stain. Leptomycin B and hydrogen peroxide treatments were performed as described in Figure 7.
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