A Zebrafish Model for Selenoprotein Synthesis and Function (OR11-01-19).

Abstract

Objectives\nWe have established a zebrafish model system that will allow unprecedented access to the role of selenoprotein function during development. The work described here focuses on a poorly characterized RNA binding protein that is similar to SECIS binding protein 2 (SBP2), which is required for the co-translational insertion of selenocysteine at select UGA codons in selenoprotein mRNAs. This protein, SECISBP2L, shares many features with SBP2 but is has no known function. We hypothesize that the zebrafish model system will reveal a selenoprotein-synthesis related function for SECISBP2L.\n\n\nMethods\nUsing CRISPR/Cas9, we generated zebrafish with a disruption in one of the conserved domains in SECISBP2L. When bred out to a homozygous mutant animal, we verified that SECISBP2L protein expression was eliminated. To analyze selenoprotein synthesis, we metabolically labeled wild-type and mutant embryos with radioactive selenium (Se-75).\n\n\nResults\nIn terms of overt phenotypes in SECISBP2L null fish, we observed no defects in growth, mobility or fertility. However, we noticed a significant sensitivity to oxidative stress as measured by lethality associated with peroxide exposure. In order to detect changes in selenoprotein expression that may have resulted from SECISBP2L loss, we performed Se-75 labeling in embryos. We also began an investigation of the effect of oxidative stress on selenoprotein expression during development. As such, one set of embryos was treated for 24 hours with 100 nM Se-75 and the other with Se-75 plus 200 μM H2O2. In general, we did not observe an overall alteration of selenoprotein expression as a result of SECISBP2L loss. We did, however, observe a significant spike of expression for a 50 kDa selenoprotein that did not occur in the SECISBP2L null animals. Based on this molecular weight, we predict that this band corresponds to selenophosphate synthase (SEPHS2). In addition, subsequent labeling at later time points revealed a general reduction of selenoprotein expression that may result from reduced SEPHS2 expression because it is essential for selenocysteine-tRNA synthesis.\n\n\nConclusions\nWe conclude that SECISBP2L is required for optimal selenoprotein expression and its function may be induced by oxidative stress. We have also demonstrated the value of a zebrafish model system for studying the mechanism of selenoprotein synthesis.\n\n\nFunding Sources\nFunded by the National Institutes of Health.