Nonsense-mediated decay of mRNA for the selenoprotein phospholipid hydroperoxide glutathione peroxidase is detectable in cultured cells but masked or inhibited in rat tissues

Abstract

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Figure 1

Hepatocytes and testes from rats fed a Se-deficient diet for 12 wk have the same level of PHGPx mRNA as the corresponding tissues from rats fed a Se-supplemented diet. Rat pairs and hepatocyte RNAs were identical to those described in Moriarty et al. (1998). (A) Total RNA (25 μg) was electorphoresed in agarose, transferred to a nylon membrane, and hybridized to ³²P-labeled cDNAs for rat PHGPx (a gift from Donna Driscoll; Pushpa-Rekha et al., 1995), β-actin (Moriarty et al., 1998), and GPx1 (Moriarty et al., 1998). The levels of PHGPx and GPx1 mRNAs were normalized to the level of β-actin mRNA and presented as a percentage of the normalized level in Se-fed animals, which was defined as 100%. N.D., not detectable. Results are shown for one pair of animals. (B) RT-PCR analysis of PHGPx and β-actin mRNAs was as described (Moriarty et al., 1997, 1998), except that PHGPx mRNA was analyzing using primers described in MATERIALS AND METHODS. The left-most four lanes assay a serial dilution of testis RNA and demonstrate that the analysis is quantitative.

Figure 2

rPHGPx nascent peptide generated by the coupled transcription-translation of rPHGPx (TGA) cDNA by using wheat germ extracts does not accumulate at the codon preceding the Sec codon when the Sec codon is recognized as nonsense. The structure of the SP6-rat(r)PHGPx cDNA and positions of important codons are shown. The diagonally stripped box specifies the bacteriophage SP6 promoter, open boxes represent PHGPx exons, and the darkened region of the last exon indicates the SECIS. ATG(27) specifies the translation initiation codon used primarily in somatic tissues (Pushpa-Rekha et al., 1995), TGA(72) specifies the sole Sec codon [which in derivative constructs was changed to either TGT(72) or TAA(72)], and TAA(197) specifies the normal termination codon. Test pSP-rPHGPx plasmids (9 μg) harboring either the usual Sec (TGA) codon, a Cys (TGT) codon, or a nonsense (TAA) codon and reference luciferase DNA (2 μg; Promega) were incubated in the presence of [³⁵S]methionine in 25 μl of the TNT Wheat Germ Lysate System (Promega). By so doing, cDNAs were transcribed by SP6 RNA polymerase, and product RNAs were then translated. A fraction (12 μl) was precipitated by reducing the pH to 5.0 by using acetic acid and subsequently electrophoresed in acrylamide. The level of PHGPx from each plasmid was normalized to the level of luciferase, and normalized values were then calculated relative to the amount of normalized protein from the TGA-containing construct (after accounting for the number of radioactive amino acids per molecule), which was defined as 1. The asterisk denotes an unidentified protein that was produced in extracts without exogenous DNA (−).

Figure 3

RNA blot hybridization indicates that pmCMV-PHGPx harboring either the TGA Sec codon or one of several nonsense codons generates mRNA that is reduced in abundance in NIH3T3 and H35 cells. Structures of the mCMV-PHGPx gene and positions mutated in the various derivative alleles are shown. The diagonally stripped box specifies the mCMV promoter, open boxes represent PHGPx exons, intervening lines represent introns, and the right-most bold line represents PHGPx 3′-flanking DNA. Codons for translation initiation, Sec, and translation termination are specified as determined for rPHGPx sequences (see legend to Figure 2). NIH3T3 or H35 cells that had been propagated in MEM plus 10% FBS were either not transfected (−) or transiently transfected with the designated pmCMV-PHGPx test plasmid and the pmCMV-TPI reference plasmid, total RNA was isolated, and PHGPx and TPI transcripts were quantitated by blot hybridization. The level of each PHGPx mRNA was normalized to the level of TPI mRNA and subsequently calculated as a percentage of the normalized level of PHGPx UGU (72) mRNA, which was defined as 100. Values did nor vary by >7–8% in three independently performed experiments.

Figure 4

RNA blot hybridization indicates that pmCMV-PHGPx harboring either the Sec codon or a TAA codon in its place generates mRNA that is subject to NMD. Cos cells were transfected with the specified pmCMV-PHGPx test plasmid, the pmCMV-Gl reference plasmid, and either pCI-Neo-hUPF1 Wt or pCI-Neo-hUPF1 R844C, and PHGPx and Gl transcripts were quantitated as described in the legend to Figure 3. The level of each PHGPx mRNA was normalized to the level of Gl mRNA and subsequently calculated as a percentage of the normalized level of PHGPx UGU (72) mRNA in the presence of Wt hUpf1 protein (p), which was defined as 100. Values did nor vary by >7–8% in two independently performed experiments.

Figure 5

pPHGPx harboring either the TGA Sec codon or one of several nonsense codons generates mRNA that is subject to NMD in NIH3T3 and H35 cells. NIH3T3 and H35 cells were treated and analyzed as described in the legend to Figure 3 except that pPHGPx plasmids were used in the place of pmCMV-PHGPx plasmids. Values did nor vary by >5–7% in two independently performed experiments.

Figure 6

Se deficiency augments the NMD of PHGPx mRNA harboring the Sec codon in NIH3T3 and H35 cells. NIH3T3 and H35 cells were treated and analyzed as described in the legend to Figure 4 except that the cells were cultured 12 h after transfection in either Se-deficient (−Se) or Se-supplemented (+Se) medium. Values did nor vary by >5–7% in two independently performed experiments.

Figure 7

Se deficiency also augments the NMD of endogenous PHGPx mRNA in untransfected H35 cells. Untransfected H35 cells were cultured as described in the legend to Figure 6 except that insulin and transferrin were replaced by 10% FBS where specified and the concentration of sodium selenite was as specified. For each lane, the level of endogenous PHGPx mRNA was normalized to the level of endogenous TPI mRNA, and normalized levels were expressed relative to the level in the absence of sodium selenite (SeO₃), which was defined as one for cells cultured in insulin + transferrin as well as cells cultured in the presence serum.