Human selenoprotein P and S variant mRNAs with different numbers of SECIS elements and inferences from mutant mice of the roles of multiple SECIS elements

Abstract

Dynamic redefinition of the 10 UGAs in human and mouse selenoprotein P (Sepp1) mRNAs to specify selenocysteine instead of termination involves two 3' UTR structural elements (SECIS) and is regulated by selenium availability. In addition to the previously known human Sepp1 mRNA poly(A) addition site just 3' of SECIS 2, two further sites were identified with one resulting in 10-25% of the mRNA lacking SECIS 2. To address function, mutant mice were generated with either SECIS 1 or SECIS 2 deleted or with the first UGA substituted with a serine codon. They were fed on either high or selenium-deficient diets. The mutants had very different effects on the proportions of shorter and longer product Sepp1 protein isoforms isolated from plasma, and on viability. Spatially and functionally distinctive effects of the two SECIS elements on UGA decoding were inferred. We also bioinformatically identify two selenoprotein S mRNAs with different 5' sequences predicted to yield products with different N-termini. These results provide insights into SECIS function and mRNA processing in selenoprotein isoform diversity.

Keywords: codon redefinition; ribosome specialization; selenocysteine; selenoprotein P; selenoprotein S.

Figure 1.

Mouse and human selenoprotein P. (a) Mouse Sepp1 after removal of the signal peptide. The N-terminal region includes the redox site [–44], and heparin binding sites [–86]. The Sec-rich C-terminal region includes the apoER2 binding site (dashed line: exact position unknown) and remaining Sec positions. U (in red) signifies sites of selenocysteine specification. (b) Selenoprotein P products. Ultra-short form terminates before UGA 1 at residue 40 and is undetected by the 9S4 antibody; short form progresses beyond residue 40 and does not include the apoER2 site; and long form extends beyond residue 324 and includes the apoER2 binding site. (c) Human selenoprotein P canonical forms and transcript variants with indication of regions that the designed qPCR primer pairs A1, A2, B1, B2 and C amplify. (d) Sepp1 transcript variants mRNA fold induction. Transcript quantification after normalization with GAPDH and 0 nM Se treatment. Primer pairs A1, A2, C1: long and short isoforms; Primer pairs B1, B2: long isoforms. Values are mean ± s.e.m., n = 3.

Figure 2.

Generation of a knockout mouse line, Sepp1^ΔSECIS1, that has deleted the first SECIS signal of the Sepp1 gene. To generate the Sepp1^ΔSECIS1 allele, a genomic fragment (red line) containing sequence from 5′-CTGAAGCAACAGCTAAAAGA-3′ to 5′-AACACTCCATGCAAACTACA-3′ of the Sepp1 gene was used for constructing the targeting vector. (a) Genomic structure of the WT Sepp1 gene, with its five exons shown in green. The 3′ UTR sequence is shown, with the sequence shown in red being that of SECIS 2. (b) In the targeting vector, a self-excising neo cassette (Ace-Cre-neo, also named ACN) was used to replace the SECIS 1 sequence. (c) During the cross between chimaeric males and WT females, the ACN neo cassette is deleted automatically, resulting in a clean heterozygous Sepp1^ΔSECIS1 allele.

Figure 3.

Generation of a knockout mouse line, Sepp1^ΔSECIS2, that has deleted the second SECIS signal of the Sepp1 gene. To generate the Sepp1^ΔSECIS2 allele, a genomic fragment (red line) containing sequence from 5′-CTGAAGCAACAGCTAAAAGA-3′ to 5′-AACACTCCATGCAAACTACA-3′ of the Sepp1 gene was used for constructing the targeting vector. (a) Genomic structure of the WT Sepp1 gene, with its five exons shown in green. The 3′ UTR sequence is shown, with the SECIS 2 signal highlighted in blue. (b) In the targeting vector, a self-excising neo cassette (Ace-Cre-neo, also named ACN) was used to replace the SECIS 2 sequence. (c) During the cross between chimaeric males and WT females, the ACN neo cassette is deleted automatically, resulting in a clean heterozygous Sepp1^ΔSECIS2 allele.

Figure 4.

Generation of a mouse line with Ser in place of the first selenocysteine (Sec). A genomic fragment (highlighted in red) containing sequence from 5′-CTGAAGCAACAGCTAAAAGA-3′ to 5′-AACACTCCATGCAAACTACA-3′ of the Sepp1 gene was used for constructing the targeting vector. (a) Genomic structure of the WT Sepp1 gene, with its five exons in green. The second exon contains the start codon ATG. Sepp1 has 10 selenocysteines encoded each by a UGA codon. The first of the 10 TGAs that can specify selenocysteine is located within the second exon. The remaining nine TGA sequences are located in exon 5. (b) After homologous recombination in the ES cells, one copy of the endogenous Sepp1 gene is replaced by the modified sequence in the targeting vector, which has the first TGA (Sec) changed into TCA (Ser) and a self-excising neo cassette (Ace-Cre-neo, also named ACN) inserted into the BglI site between first and second exons. (c) During the cross between chimaeric males and WT females, the ACN neo cassette is deleted automatically, resulting in a clean heterozygous allele Sepp1^U59S. U40S referred to below is after the signal peptide is removed.

Figure 5.

Plasma selenium biomarkers of mutant selenoprotein P gene mouse strains. (a) U40S; (b) ΔSECIS 2; (c) ΔSECIS 1. Values are means + 1 s.d., n = 5. Pairs of values with percentages above them are different (p < 0.05) by Student's t-test.

Figure 6.

Analysis of selenoprotein P preparations from plasma of the mutant mice strains by SDS-PAGE. Numbered lanes contain preparations: lane 1, WT; lane 2, U40S; lane 3, ΔSECIS 2; lane 4, ΔSECIS 1. M indicates molecular weight markers. Plasma from each mouse was passed through a column containing a monoclonal antibody (9S4) against the N-terminal portion of Sepp1. Then a Sepp1 fraction was eluted. The amount of Sepp1 fraction that contained 2 µg of protein was loaded onto each lane.

Figure 7.

Effects of deleting SECIS 1 element on tissue selenium concentrations. Mice were fed control diet from weaning and were studied four weeks after weaning. Values are means + 1 s.d., n = 5. Pairs of values with percentages above them are different (p < 0.05) by Student's t-test.

Figure 8.

Sepp1 mRNA and summary of mutant mice findings. Schematics of Sepp1 mRNA including relative positions of UGA 1–10, exon junction complex located 20–26 nucleotides 3′ of UGA 1 and SECIS elements in the 3′ UTR. Dashed line represents 3′ UTR with SECIS elements looping as in the Berry model [33]. The positioning of SECIS 2 and SECIS 1 is intended to illustrate their prime role in facilitating UGA 1, and UGA 2–10, respectively, in specifying selenocysteine.