Selenium influences the turnover of selenocysteine tRNA[Ser]Sec in Chinese hamster ovary cells

Abstract

Selenocysteine transfer RNA (tRNA[ser]sec) is a central molecule in the production of selenium-containing proteins, and may play a role in the regulation of their biosynthesis. Selenium concentration influences both the levels of tRNA[Ser]Sec and the relative abundance of two isoforms. To study the mechanism by which selenium affects tRNA[Ser]Sec levels, Chinese hamster ovary (CHO) cells were treated with the transcription inhibitor, actinomycin D, and tRNA[Ser]Sec levels were determined by Northern blotting, primer extension and reverse-phase column chromatography. Turnover of tRNA[Ser]Sec in CHO cells was faster than the total tRNA population. Supplementation of the culture media with selenium reduced turnover of tRNA[Ser]Sec, but did not influence turnover of a randomly selected serine tRNA. Inhibition of transcription with actinomycin D resulted in a relative increase in the abundance of the isoform containing methylcarboxymethyl-5′-uridine-2′-O-methylribose in the wobble position of the anticodon. Primer extension studies, which permitted the independent evaluation of the tRNA[Ser]Sec arising from the introduced mouse gene and that derived from the host CHO gene, indicated an accelerated decline in tRNA[Ser]Sec derived from both the transfected and the native gene. These results provide additional insight into the levels of regulation that control the translation of selenium containing proteins in mammalian cells.