Abstract
Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.
Highlights
Several factors required for the specific incorporation of selenocysteine (Sec)2 as the 21st amino acid in the genetic code have been identified, and their roles have been assessed in mammalian cells and tissues
The SEC insertion sequence (SECIS) elements that have been identified in the known selenoproteins in rodents and known selenoproteins in humans [7] fall into two classes that differ in the occurrence of a ministem that is adjacent to the apical loop of the structure [8]
Because selenoproteins and Sec tRNAm[Secrm]S5eUcm are responsive to selenium status [2, 4] and because the two protein factors were implicated in selenoprotein metabolism [16, 17], the effects of the siRNA constructs on the targeted mRNAs were examined in cells grown in the presence or absence of supplemental selenium
Summary
Several factors required for the specific incorporation of selenocysteine (Sec)2 as the 21st amino acid in the genetic code have been identified, and their roles have been assessed in mammalian cells and tissues (see Refs. 1 and 2 for reviews). Because selenoproteins and Sec tRNAm[Secrm]S5eUcm are responsive to selenium status [2, 4] and because the two protein factors were implicated in selenoprotein metabolism [16, 17], the effects of the siRNA constructs on the targeted mRNAs were examined in cells grown in the presence or absence of supplemental selenium.
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