Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine

Abstract

The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3′-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5′-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.

Selective up-regulation of human selenoproteins in response to oxidative stress

“…The SECIS element, present in the 3Ј UTRs of selenoprotein mRNAs, is considered the main player in this translational regulation, together with its interacting partners. Indeed, the SECIS serves as a platform for mRNP assembly that subsequently dictates the efficiency of selenoprotein mRNA translation by the ribosome (23,24,39). The stoichiometry and the nature of mRNP complexes present in the nucleus and cytoplasm remain to be determined.…”

“…23, 24. Ctrl and Sup culture media were made according to Refs. 23,24) and the references therein. The same lot number of FCS was used in all experiments because selenium is provided by this source.…”

“…After 30 min of exposure, the media were removed and replaced by the appropriate ones. Cell extracts were harvested 24 h post-treatment in 300 l of lysis buffer (23,24). Cellular fractionations were performed with the ProteoJET cytoplasmic and nuclear protein extraction kit (Fermentas) following the instructions of the manufacturer.…”

“…Gpx1 expression was more affected than Gpx4 by selenium availability (22), essentially via a translational control mechanism (23). We established HEK293 cell lines stably expressing luciferase-SECIS constructs to quantitatively evaluate the translational regulation of selenoprotein expression (23,24). Indeed, we demonstrated that, in response to selenium level variations, selenocysteine insertion was driven by the nature of the SECIS element.…”

“…We used HEK293 cells in our study because selenoproteins are expressed at significant levels in this cell line (23,24) and also because we developed cellular tools to quantitatively study the recoding of UGA as selenocysteine in this particular cell line (23). We found that down-regulation of the antioxidant defense in the Ctrl condition was linked to a selective reduction of several selenoproteins.…”