? DESCRIPTION (provided by applicant): Selenium is an essential trace element that is incorporated into 25 human proteins as the amino acid selenocysteine (Sec). Interestingly, the co-translational incorporation of selenocysteine occurs at in-frame stop codons (UGA) that are found upstream of RNA structures known as selenocysteine insertion sequence (SECIS) elements, which reside in the 3' UTR of all selenoprotein mRNAs. We have recently demonstrated that the factors known to be essential for Sec incorporation are, in fact, sufficient. These factors include a specialized elongation factor (eEFSec), a SECIS binding protein (SBP2), and a specialized transfer RNA (Sec-tRNASec). Significant progress has been made regarding which protein domains are important for Sec incorporation activity, but the mechanism by which these factors allow Sec incorporation has not been subjected to study in vivo, particularly in the context of embryonic development. In addition, an SBP2 paralogue exists, termed SECISBP2L, but it does not participate in Sec incorporation in vitro and its function remains unknown. As such, the study of selenoprotein synthesis and function is still an emerging field and requires the development of novel and affordable model systems. Here we propose to develop a zebrafish model system in which to study both the mechanism of Sec incorporation as well as selenoprotein function. The use of zebrafish is warranted for four major reasons: 1) the factors required for Sec incorporation are conserved in all vertebrates; 2) the complement of selenoproteins in fish is very similar to that in humans; 3) the cost of developing fish models is vastly lower than that in mice and 4) as a tractable model for vertebrate development, the use of zebrafish will allow unprecedented access to the mechanisms by which selenoprotein function during early development. In the first Aim we will lay the groundwork by investigating expression dynamics and developmental profile for Sec incorporation factors, after which we will determine the function of the SBP2 paralogue, SECISBP2L. Although SECISBP2L was identified at the same time as SBP2, its function has remained elusive. Our preliminary data derived from embryos injected with anti SECISBP2L morpholino oligonucleotides suggests that it may play a role in hematopoeisis and/or vascular development. In the second aim, we propose to model human disease in fish by introducing specific mutations in endogenous SBP2 and determining the effects on selenoprotein production. In addition, although we expect that SBP2 deletion will result in embryonic lethality, we propose to precisely define the developmental process that is altered in the absence of selenoproteins. Importantly, this methodology will allow the resolution of the long-sought function for the N-terminal half of SBP2, which is vertebrate-specific and has no known function. Finally, as an initial foray into studying the basic functions of selenoproteins in zebrafish, we propose to study macrophage function in order to determine the underlying basis for the role that selenoproteins are known to play in macrophage migration.

R21HD083616

2016-04-01

2020-03-31