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. 2019 Sep 27;294(39):14185-14200.
doi: 10.1074/jbc.RA119.009369. Epub 2019 Jul 26.

Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations

Wenchao Zhao  1 , Simon Bohleber  1 , Henrik Schmidt  1 , Sandra Seeher  1 , Michael T Howard  2 , Doreen Braun  1 , Simone Arndt  1 , Uschi Reuter  1 , Hagen Wende  3 , Carmen Birchmeier  3 , Noelia Fradejas-Villar  1 , Ulrich Schweizer  4
Affiliations

Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations

Wenchao Zhao et al. J Biol Chem. .

Abstract

Recoding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3'-UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNA-binding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA recoding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2.

Keywords: SECIS-binding protein 2 (SECISBP2); Sbp2; brain; deiodinase; liver; neurobiology; neuroinflammation; selenium; selenocysteine; selenocysteine insertion sequence (SECIS); selenoprotein; thermal instability; thyroid hormone; translation.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Generation of Secisbp2 alleles in mice carrying pathogenic mutations found in patients. A, strategy to build the targeting vector containing a R543Q mutation in exon 12 and a FRT-flanked neo cassette in intron 12 for double homologous recombination in embryonic stem cells. After germline transmission, the neo cassette is removed by germline FLPe expression. Sanger sequencing confirmed the heterozygous base substitutions in the Arg-543 codon and in the Leu-540 codon, creating a diagnostic DraI restriction site. DTA refers to a diphtheria toxin A chain used for negative selection against off-site integration. B, strategy to build the targeting vector containing a C696R mutation in exon 14 and a FRT-flanked neo cassette in intron 14 for double homologous recombination in embryonic stem cells. After germline transmission, the neo cassette is removed by germline FLPe expression. Sanger sequencing confirmed the heterozygous base substitutions in the Cys-696 codon and in the Leu-694 codon creating a diagnostic AflII restriction site. C and D, verification of correct gene targeting by Southern blotting using hybridization probes located outside of the targeting vector sequence. C, Secisbp2 R543Q allele. The SpeI digestion was combined with the 3′ probe and yielded the expected fragments of 12,148 bp (WT) and 14,048 bp (RQ). The EcoRI digestion was combined with the 5′ probe and yielded fragments of 9129 bp (WT) and 4209 bp (RQ). D, Secisbp2 C696R allele. The BglII digestion was combined with the 5′ probe and yielded the expected fragments of 8328 bp (WT) and 10,228 bp (CR). The HindIII digestion was combined with the 3′ probe and yielded the expected fragments of 10,177 bp (WT) and 7340 bp (CR). DNA marker sizes in bp are indicated.
Figure 2.
Figure 2.
Selenoprotein expression in liver-specific Secisbp2 mutant mice. Control mice (WT) were compared with Alb-Cre; Secisbp2fl/fl (KO), Alb-Cre; Secisbp2C696R/fl (CR), and Alb-Cre; Secisbp2R543Q/fl (RQ) mice. A, semiquantitative RT-PCR analysis for selected selenoprotein mRNAs. ΔΔCt values are calculated in relation to 18S rRNA. Means are given ± S.D. (error bars), n = 2. B, Western blot analysis for selected selenoproteins. Two individual liver extracts are analyzed for each genotype. C, Western blot analysis for Secisbp2. The nonspecific bands demonstrate equal loading. Images were copied directly from the camera output files and displayed without modifications. The complete image files are available as Fig. S2.
Figure 3.
Figure 3.
In vitro functional analysis of recombinantly expressed mouse Secisbp2 R543Q and C696R. Reporter constructs containing a Sec-dependent luciferase cDNA and SECIS elements cloned into the 3′ UTR as given are in vitro translated, and the amount of luciferase produced was measured by an activity assay. A, Secisbp2 R543Q is functional, whereas C696R is not functional at all. Recombinant Secisbp2 at 160 nm was incubated with constructs containing murine Gpx1, Gpx4, and Txnrd1 SECIS elements, and luciferase activity was measured. A rat Gpx4 SECIS element lacking the AUGA sequence of the essential kink-turn served as negative control. Further negative controls were no mRNA added, no Secisbp2 protein added, and a luciferase carrying a UAA codon at the position of the UGA/Sec codon. A log2 scale is used to better appreciate the small increment of luciferase activity in the controls lacking Secisbp2 or the SECIS quartet over lack of mRNA or a definite stop codon indicating a small SECIS/Secisbp2-independent UGA read-through. B, kinetics of Secisbp2R543Q:SECIS binding compared with Secisbp2WT for several murine SECIS elements. The concentration of recombinant Secisbp2 was 80 nm, and 0.1–20 nm mRNA was used. The apparent KD of Secisbp2R543Q was significantly reduced for Dio1 and Gpx1 SECIS only, and the maximal amount of luciferase was decreased when using the mutant as compared with Secisbp2WT. C, thermal instability of Secisbp2R543Q in vitro. Recombinant Secisbp2 protein at 160 nm concentration was incubated for 30 min on ice, at 37 °C, and at 40.5 °C and then used for in vitro translation with the luciferase/Gpx4 SECIS construct. The luciferase activity values of the respective Secisbp2WT incubated on ice was defined as 100%. The experiment was done twice in triplicates. Error bars, S.D.
Figure 4.
Figure 4.
Selenoprotein expression in neuron-specific Secisbp2 mutant mice. A, Western blotting against Secisbp2. 100 μg of total cortical protein from two individual mice of each genotype. B, Western blotting against selenoproteins in the cortex of CamK-Cre; Secisbp2C696R/fl (CR) and littermate control mice. C, semiquantitative RT-PCR analysis of selected selenoprotein mRNAs. n = 2; ***, p < 0.001, Student's t test. D, Western blotting against selenoproteins in the cortex of CamK-Cre; Secisbp2R543Q/fl (RQ) and litter mate control mice. E, semiquantitative RT-PCR analysis of selected selenoprotein mRNAs. n = 3; ***, p < 0.001, Student's t test. Images were copied directly from the camera output files and displayed without modifications. The complete image files are available as Fig. S3. Error bars, S.E.
Figure 5.
Figure 5.
Selenoprotein expression in neuron-specific Secisbp2R543Q mice assessed by ribosomal profiling and RNA-Seq in cerebral cortex. A, relative abundance of selenoprotein-related reads in Ribo-Seq (RPF) and RNA-Seq (RNA) in RQ mice compared with controls. n = 2 per genotype. *, q < 0.05, BH correction. Significant changes are highlighted in red. B, RPF coverage of selected selenoprotein mRNAs. The position of the Sec/UGA codon is indicated by a red bar. Reads are plotted in blue for controls (Ctl) and in orange for CamK-Cre; Secisbp2R543Q/fl (RQ) mice. C, URE calculated for selenoproteins with UGA/Sec far from the termination codon. URE is calculated as (3′RPFmutant/5′RPFmutant)/(3′RPFcontrol/5′RPFcontrol). URE is not a good measure if mRNA levels or initiation rates change. D, 3′ RPM (reads 3′ of UGA/Sec per million mapped reads) calculated for selenoproteins. This measure gives a measure for the actual translation of full-length selenoproteins. E, 3′ RPM/mRNA is the same measure as in D, but normalized to mRNA abundance. C–E, *, p < 0.05, Student's t test. Error bars, S.D.
Figure 6.
Figure 6.
Mild inflammatory response in neuron-specific Secisbp2R543Q mice. A and B, brain transcriptomic analysis reveals induction of immunity-related genes (red) on mRNA (A) and RPF levels (B). Selenoproteins are labeled in green and neuronal genes in blue. Only significant regulated genes are shown. n = 2 animals/genotype. *, q < 0.05, BH correction. C, pathway analysis shows that immunity-related pathways were induced on the transcriptional level. D, astrogliosis assessed by GFAP staining in the somatosensory cortex. E, Iba1 staining in the somatosensory cortex. Mean cell density was 375 and 378 cells/mm2 for control and CamK-RQ mice, respectively.
Figure 7.
Figure 7.
Ribosomal coverage of Secisbp2 in cortex. The site of the R543Q mutation is marked in red. Although the ribosomal coverage pattern is similar in mutant and control, it is obvious that the mutant is translated at a lower level, in particular 3′ of the site of the mutation indicated in red.
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