Abstract
The dietary requirement for selenium is based on its incorporation into selenoproteins, which contain the amino acid selenocysteine (Sec). The Sec insertion sequence (SECIS) is an RNA structure found in the 3' UTR of all selenoprotein mRNAs, and it is required to convert in-frame UGA codons from termination to Sec-incorporating codons. SECIS-binding protein 2 (Sbp2) is required for Sec incorporation, but its paralogue, SECIS-binding protein 2-like (Secisbp2l), while conserved, has no known function. Here we determined the relative roles of Sbp2 and Secisbp2l by introducing CRISPR mutations in both genes in zebrafish. By monitoring selenoprotein synthesis with 75Se labeling during embryogenesis, we found that sbp2 -/- embryos still make a select subset of selenoproteins but secisbp2l -/- embryos retain the full complement. Abrogation of both genes completely prevents selenoprotein synthesis and juveniles die at 14 days post fertilization. Embryos lacking Sbp2 are sensitive to oxidative stress and express the stress marker Vtg1. We propose a model where Secisbp2l is required to promote essential selenoprotein synthesis when Sbp2 activity is compromised.
Highlights
Selenium is an essential trace element that is incorporated as the amino acid selenocysteine (Sec) into ~20–50 vertebrate proteins known collectively as selenoproteins
Very little is known about the regulation of selenoprotein synthesis during development, so here we have used the zebrafish model system to determine the relative roles of Secisbp2l and SECIS-binding protein 2 (Sbp2), thereby establishing a highly tractable system in which to study the mechanism of Sec incorporation and the consequences of selenoprotein deficiency in vivo
The most notable change was the loss of a diffuse band at ~45 kD and a pair of bands at ~22/20 kD. These results suggest that Secisbp2l may be supporting selenoprotein synthesis, the 68- and 25-kD species, when Sbp2 is absent
Summary
Selenium is an essential trace element that is incorporated as the amino acid selenocysteine (Sec) into ~20–50 vertebrate proteins known collectively as selenoproteins. The muscle-specific selenoprotein Selenon was reported to be required for normal muscle function and calcium flux in zebrafish embryos (Deniziak et al, 2007; Jurynec et al, 2008), and in situ hybridization revealed a complex array of tissue specific expression for 21 selenoprotein mRNAs during zebrafish development (Thisse et al, 2003) Despite these efforts, very little is known about the regulation of selenoprotein synthesis during development, so here we have used the zebrafish model system to determine the relative roles of Secisbp2l and Sbp, thereby establishing a highly tractable system in which to study the mechanism of Sec incorporation and the consequences of selenoprotein deficiency in vivo. Secisbp2l−/− animals showed slightly reduced selenoprotein production, which was completely inhibited and led to death at 14 d post fertilization (dpf) when both genes were ablated
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