Translational Redefinition of UGA Codons Is Regulated by Selenium Availability

Michael T Howard,Bradley A Carlson,Christine B Anderson,Dolph L Hatfield

doi:10.1074/jbc.m113.481051

Abstract

Incorporation of selenium into ~25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA([Ser]Sec) Um34 methylation. Furthermore, we find evidence for translation in the 5'-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.

Highlights

Selenium is incorporated into selenoproteins as the amino acid, selenocysteine
As previous in vitro studies have shown that UGA redefinition to encode Sec is in competition with termination [34, 35], we postulated that the ability to quantify ribosomes located 5Ј and 3Ј of UGA-Sec codons would provide a means of estimating changes in Sec incorporation efficiency and a more accurate method for measuring changes in full-length selenoprotein synthesis
We find that selenoprotein mRNA levels in WT mouse liver are relatively resistant to changing levels of dietary selenium with the notable exception of glutathione peroxidase 1 (Gpx1), Sepw1, and selenoprotein H (Selh) in both WT and TrspA37G mice as well as Deiodinase 1 (Dio1) in selenium-deficient TrspA37G mice (Fig. 2 and supplemental Fig. S2)

Summary

Background

Selenium is incorporated into selenoproteins as the amino acid, selenocysteine. Results: Dietary selenium supplementation increases ribosome density downstream of selenocysteine-encoding UGA codons. We find evidence for translation in the 5؅-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins. To directly examine the mechanisms of selenium-dependent regulation of selenoprotein expression in vivo, we have applied ribosome profiling, RNA-Seq, and traditional biochemical approaches to examine selenoprotein synthesis in liver Using these approaches, we have defined the changes in mRNA abundance and translational activity that occur in response to altered dietary selenium intake and expression of a mutant SectRNA[Ser]Sec that prevents Um34 synthesis. These studies provide a unique view of selenoprotein translational control and UGA redefinition efficiency in mammalian tissue and its regulation by dietary selenium intake

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION