The Selenocysteine-Specific Elongation Factor Contains Unique Sequences That Are Required for Both Nuclear Export and Selenocysteine Incorporation.

Aditi Dubey,Paul R Copeland,Alexander F Palazzo

doi:10.1371/journal.pone.0165642

Aditi Dubey, Paul R Copeland + Show 1 more

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https://doi.org/10.1371/journal.pone.0165642

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Abstract

Selenocysteine (Sec) is a critical residue in at least 25 human proteins that are essential for antioxidant defense and redox signaling in cells. Sec is inserted into proteins cotranslationally by the recoding of an in-frame UGA termination codon to a Sec codon. In eukaryotes, this recoding event requires several specialized factors, including a dedicated, Sec-specific elongation factor called eEFSec, which binds Sec-tRNASec with high specificity and delivers it to the ribosome for selenoprotein production. Unlike most translation factors, including the canonical elongation factor eEF1A, eEFSec readily localizes to the nucleus of mammalian cells and shuttles between the cytoplasmic and nuclear compartments. The functional significance of eEFSec’s nuclear localization has remained unclear. In this study, we have examined the subcellular localization of eEFSec in the context of altered Sec incorporation to demonstrate that reduced selenoprotein production does not correlate with changes in the nuclear localization of eEFSec. In addition, we identify several novel sequences of the protein that are essential for localization as well as Sec insertion activity, and show that eEFSec utilizes CRM1-mediated nuclear export pathway. Our findings argue for two distinct pools of eEFSec in the cell, where the cytoplasmic pool participates in Sec incorporation and the nuclear pool may be involved in an as yet unknown function.

Highlights

Humans utilize the essential trace element selenium (Se) to make at least 25 essential proteins
From several lines of evidence, it is currently believed that a complex containing SECIS binding protein 2 (SBP2) and eEFSec/GTP/Sec-tRNASec is assembled on the Sec insertion sequence (SECIS) element, which allows the ribosomal A-site containing the in-frame UGA codon to be interpreted as Sec
This experiment shows that overexpressing eEFSec in McArdle cells does not have a significant positive or negative effect on selenoprotein production when compared to the eEF1A control, suggesting that the endogenous protein is not limiting or that selenoprotein mRNAs are not accessible by the transfected pool of eEFSec

Summary

Introduction

Humans utilize the essential trace element selenium (Se) to make at least 25 essential proteins These proteins are called selenoproteins and perform myriad functions including the maintenance of cellular oxidative homeostasis, thyroid function, and sperm production [1]. First is a key feature shared by all selenoprotein mRNAs—a stem-loop structure in the 3’ untranslated region (UTR) called the Sec insertion sequence (SECIS) element. This is the only known essential cis acting element. The remaining are trans acting factors, including the SECIS binding protein 2 (SBP2), the selenocystyl-tRNASec (Sec-tRNASec), and the selenocysteine-specific elongation factor called eEFSec (reviewed in [2]). Since selenoprotein production is differentially regulated under conditions of oxidative stress or limiting selenium supply, it remains unknown whether coordinated regulation of eEFSec and SBP2 occurs

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