Volume 35, Issue 4, Pages 479-489 (August 2009) Eukaryotic Initiation Factor 4a3 Is a Selenium-Regulated RNA-Binding Protein that Selectively Inhibits Selenocysteine Incorporation  Michael E. Budiman, Jodi L. Bubenik, Angela C. Miniard, Lisa M. Middleton, Carri A. Gerber, Ayla Cash, Donna M. Driscoll  Molecular Cell  Volume 35, Issue 4, Pages 479-489 (August 2009) DOI: 10.1016/j.molcel.2009.06.026 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Identification of a 48 kDa SECIS-Binding Protein (A) Schematic representation of the GPx1 and PHGPx SECIS elements. The SECIS core, AAR motif, and internal loop region are indicated. The location of the AAR motif determines whether the element is a type 1 SECIS (Apical Loop) or type 2 SECIS (Bulge). (B) Radiolabeled GPx1 or PHGPx SECIS probes (1 nM) were used with McArdle 7777 nuclear extract (50 μg) in UV crosslinking assays. The 48 kDa protein that binds only to the GPx1 probe is indicated by the arrow. (C) Biotinylated GPx1 or PHGPx SECIS RNAs were incubated with McArdle 7777 nuclear extract. The bound proteins were eluted and analyzed by SDS-PAGE and Coomassie blue staining as described in the Experimental Procedures. The arrow indicates the 48 kDa protein identified by LCMS. Molecular Cell 2009 35, 479-489DOI: (10.1016/j.molcel.2009.06.026) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 eIF4a3 Selectively Interacts with the GPx1 SECIS (A) Radiolabeled GPx1 or PHGPx SECIS RNAs were UV crosslinked in the presence of varying amounts (0–400 ng) of recombinant rat eIF4a3. The arrow indicates the position of the crosslinked product. (B) Crosslinking of eIF4a3 to the radiolabeled GPx1 probe was competed with increasing concentrations (1–200 nM) of unlabeled GPx1 or PHGPx SECIS RNAs. (C) Quantification of the competition experiments. The IC50s were calculated as the concentration of RNA required to decrease the crosslinking signal by 50%. The data from four independent observations are represented as the mean ±SD. Molecular Cell 2009 35, 479-489DOI: (10.1016/j.molcel.2009.06.026) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Selective Inhibition of UGA Recoding Activity by eIF4a3 (A) Varying amounts of eIF4a3 (25–200 ng) were added to in vitro translation assays containing 100 ng of luc/UGA/GPx1, luc/UGA/PHGPx, or luc/UGU/GPx1 reporter mRNA. The luciferase results were expressed relative to reactions that were performed in the absence of eIF4a3. The data are represented as mean ±SEM. (B) REMSA assay using the 32P-labeled GPx1 SECIS element, which was incubated with increasing amounts of eIF4a3 protein as indicated. The samples were then analyzed by native gel electrophoresis and autoradiography. Representative REMSA assays for the SelR, TR1, and PHGPx SECIS elements are shown in Figure S2. (C) Graph illustrating the apparent Kds of eIF4a3 for the GPx1, SelR, TR1, and PHGPx SECIS elements, which were calculated as the concentration of protein required to achieve 50% binding of the RNA. Kd values from three independent experiments are represented as the mean ±SD. (D) Recoding assays were performed as described in (A) using reporter constructs containing the SelR and TR1 SECIS elements. The data are represented as mean ±SEM. Molecular Cell 2009 35, 479-489DOI: (10.1016/j.molcel.2009.06.026) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 GPx1 SECIS-Binding Activities of eIF4a3 and SBP2 In Vitro (A) The radiolabeled GPx1 SECIS was incubated with SBP2 (0 or 200 nM) and increasing concentrations of eIF4a3 (0–200 nM), as indicated, and analyzed by UV crosslinking. Two different crosslinked products are indicated: eIF4a3-GPx1 SECIS, open triangle; SBP2-GPx1 SECIS, filled triangle. (B) Preformed complexes between radiolabeled GPx1 SECIS (1 nM) and varying amounts of eIF4a3 (0–200 nM) were challenged with SBP2 (200 nM). Samples were analyzed by UV crosslinking. (C) Recoding assays with the luc/UGA/GPx1 and luc/UGA/PHGPx reporter RNAs were performed in the absence or presence of 100 ng purified eIF4a3. The RNAs and eIF4a3 were not preincubated or were preincubated for 20 min prior to in vitro translation, as indicated. The data are represented as mean ±SEM. Molecular Cell 2009 35, 479-489DOI: (10.1016/j.molcel.2009.06.026) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Characterization of the eIF4a3-GPx1 SECIS Interaction (A) Schematic representation of the GPx1 SECIS, with the SECIS core mutations indicated. (B) The mutant RNAs in (A) were tested as competitors (0–200 nM) in UV crosslinking assays containing eIF4a3 and the radiolabeled wild-type GPx1 SECIS probe. The IC50s of the mutant RNAs, which were calculated as described in the legend to Figure 2, are shown. (C) Schematic representation of the mutations in the internal loop of the GPx1 SECIS. (D) Graphic representation of the results of competition experiments in which cold wild-type and mutant SECIS RNAs were tested for their ability to compete for binding of eIF4a3 to the 32P-labeled GPx1 SECIS element. The IC50s were calculated as described in (B), and the values are represented as the mean ±SD. Molecular Cell 2009 35, 479-489DOI: (10.1016/j.molcel.2009.06.026) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Effect of Selenium on mRNA and Protein Expression (A) Total RNA was extracted from McArdle 7777 cells grown with (+Se) or without (−Se) 30 nM sodium selenite supplementation after 3 days. GPx1, PHGPx, GAPDH, and eIF4a3 mRNA levels were determined by qRT-PCR. The level of mRNA was expressed relative to the amount of mRNA from +Se cells. The data are represented as the mean ±SEM. (B) Cytoplasmic or nuclear extracts were analyzed by western blotting using different antibodies as indicated. (C) The graph represents quantification of western blot data from three independent experiments. The results are normalized to GAPDH expression and expressed as mean ±SD. (D) qRT-PCR crossing point values of GAPDH, GPx1, and PHGPx mRNAs coimmunoprecipitated with anti-eIF4a3 or isotype control antibody, and the values are expressed as the mean ±SEM. (E) Relative enrichment of GAPDH, GPx1, and PHGPx mRNAs in the eIF4a3 immunoprecipitation compared to the isotype control represented as mean ±SEM. Molecular Cell 2009 35, 479-489DOI: (10.1016/j.molcel.2009.06.026) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Manipulation of eIF4a3 Regulates GPx1 Expression in Cells (A) McArdle 7777 cells were treated with different siRNAs for 72 hr as described in the Experimental Procedures. Cell lysates were analyzed by western blotting using the indicated antibodies. (B) Quantification of the western blot data was from three independent experiments. The results are normalized to GAPDH and represented as mean ±SD. (C and D) McArdle 7777 cells were transiently transfected with empty vector DNA (mock) or a plasmid encoding the human eIF4a3 cDNA. The western blots (C) and quantification (D) were performed as described in (A) and (B). Molecular Cell 2009 35, 479-489DOI: (10.1016/j.molcel.2009.06.026) Copyright © 2009 Elsevier Inc. Terms and Conditions