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. 2004 Jul;10(7):1142-52.
doi: 10.1261/rna.7370104.

A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis

Ruth R Jameson  1 , Alan M Diamond
Affiliations

A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis

Ruth R Jameson et al. RNA. 2004 Jul.

Abstract

Selenium is biologically active through the functions of selenoproteins that contain the amino acid selenocysteine. This amino acid is translated in response to in-frame UGA codons in mRNAs that include a SECIS element in its 3' untranslated region, and this process requires a unique tRNA, referred to as tRNA([Ser]Sec). The translation of UGA as selenocysteine, rather than its use as a termination signal, is a candidate restriction point for the regulation of selenoprotein synthesis by selenium. A specialized reporter construct was used that permits the evaluation of SECIS-directed UGA translation to examine mechanisms of the regulation of selenoprotein translation. Using SECIS elements from five different selenoprotein mRNAs, UGA translation was quantified in response to selenium supplementation and alterations in tRNA([Ser]Sec) levels and isoform distributions. Although each of the evaluated SECIS elements exhibited differences in their baseline activities, each was stimulated to a similar extent by increased selenium or tRNA([Ser]Sec) levels and was inhibited by diminished levels of the methylated isoform of tRNA([Ser]Sec) achieved using a dominant-negative acting mutant tRNA([Ser]Sec). tRNA([Ser]Sec) was found to be limiting for UGA translation under conditions of high selenoprotein mRNA in both a transient reporter assay and in cells with elevated GPx-1 mRNA. This and data indicating increased amounts of the methylated isoform of tRNA([Ser]Sec) during selenoprotein translation indicate that it is this isoform that is translationally active and that selenium-induced tRNA methylation is a mechanism of regulation of the synthesis of selenoproteins.

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Figures

FIGURE 1.
FIGURE 1.
GPx-1 and TR levels are influenced by selenium and tRNA[Ser]Sec levels. CHO cells overexpressing tRNA[Ser]Sec (ST4) or a dominant-negative A37G tRNA[Ser]Sec mutant whose expression results in reduced levels of mcm5Um (i6A−) were grown in standard medium or medium supplemented with 30 nM selenium for 3 d and the indicated selenoprotein levels were determined. Values are the average of three independent lysates ± standard deviation. (A) Glutathione peroxidase activity was measured by a coupled spectrophotometric assay and activity is expressed as nanomoles NADPH per minute per milligram of protein. (B) Thioredoxin reductase activity was measured from partially purified protein extracts by the reduction of sulfhydryl groups in DNTB. Activity is expressed as micromoles of NTB reduced per micromole of protein.
FIGURE 2.
FIGURE 2.
UGA suppression in CHO cells is affected by tRNA[Ser]Sec levels, but not selenium or GPx-1 mRNA overexpression. CHO cells overexpressing wild-type tRNA[Ser]Sec (ST4), A37G tRNA[Ser]Sec mutant (i6A−), or elevated GPx-1 mRNA were transfected with the pBPLUGA reporter construct. Cells were transiently transfected and incubated for 3 d in standard medium or medium supplemented with 30 nM selenium. UGA suppression was measured as luciferase activity, achieved by the translation of the in-frame UGA codon separating the luc and βgal genes, and normalized by dividing by βgal activity. Values are the average of three independent lysates ± standard deviation.
FIGURE 3.
FIGURE 3.
SECIS elements from different selenoproteins support UGA translation with different efficiencies. CHO cells were transfected with the pBPLUGA reporter constructs containing SECIS sequences from five indicated selenoproteins. UGA translation was measured as luciferase activity achieved by the translation of the in-frame UGA codon separating the luc and βgal genes, and normalized by dividing by βgal activity. Values are the average of three independent lysates ± standard deviation.
FIGURE 4.
FIGURE 4.
Selenium stimulates UGA translational efficiency. CHO cells were transfected with the pBPLUGA reporter constructs containing SECIS sequences from five selenoproteins, followed by incubation for 3 d in standard medium or medium supplemented with 30 nM selenium. UGA translation was measured as luciferase activity achieved by the translation of the in-frame UGA codon separating the luc and βgal genes, and normalized by dividing by βgal activity. Values are the average percent increase over UGA translation in unsupplemented cells of 12 independent lysates ± standard deviation.
FIGURE 5.
FIGURE 5.
Elevated tRNA[Ser]Sec increases UGA translational efficiency to the same degree in selenium-supplemented or -unsupplemented cells. Control (pLNCX) transfected CHO cells and CHO cells overexpressing tRNA[Ser]Sec (ST4) were transfected with the pBPLUGA reporter constructs containing SECIS sequences from the five indicated selenoproteins, followed by incubation for 3 d in standard medium or medium supplemented with 30 nM sodium selenite. UGA translation was measured as luciferase activity achieved by the translation of the in-frame UGA codon separating the luc and βgal genes, and normalized by dividing by βgal activity. Values are the average of nine independent transfections ± standard deviation, expressed as the percent increase over UGA translation in control cells incubated in standard medium or selenium-supplemented medium.
FIGURE 6.
FIGURE 6.
Reduced levels of the methylated tRNA[Ser]Sec isoform reduce UGA translation efficiency. Control-transfected (pLNCX) CHO cells and cells overexpressing an A37G tRNA[Ser]Sec mutant (i6A−), were transfected with the pBPLUGA reporter constructs containing SECIS sequences from the five indicated selenoproteins, followed by incubation for 3 d in standard medium or medium supplemented with 30 nM sodium selenite. UGA translation was measured as luciferase activity achieved by the translation of the in-frame UGA codon separating the luc and βgal genes, and normalized by dividing by βgal activity. Values are the average of three independent transfections ± standard deviation.
FIGURE 7.
FIGURE 7.
Elevated tRNA[Ser]Sec increases GPx-1 activity in GPx-1-overexpressing cells. CHO transfectants that overexpress GPx-1 mRNA (GPx7) were stably transfected to overexpress tRNA[Ser]Sec by 14- and 25-fold. Control cells are CHO cells transfected with the pLNCX vector only. Values are the average of three GPx-1 activity determinations measured by a coupled spectrophotometric assay, and activity is expressed as nanomoles of NADPH per minute per milligram of protein GPx-1 activities.
FIGURE 8.
FIGURE 8.
Elevated selenoprotein mRNA levels reduce UGA translation efficiency. CHO transfectants that overexpress GPx-1 (GPx7) were transfected with the pBPLUGA reporter constructs containing SECIS sequences from the five indicated selenoproteins. UGA translation was measured as luciferase activity and normalized by dividing by βgal activity. Values are the average of three independent transfections ± standard deviation.
FIGURE 9.
FIGURE 9.
Time course of the increase in mcm5Um and GPx-1 acivity following increased selenium. Control CHO cells were incubated in 30 nM selenium for the number of days indicated followed by determination of GPx-1 activity and tRNA[Ser]Sec isoform distribution. GPx-1 values are the average of three cell lysates ± standard deviation. The level of the methylated isoform was measured by RPC-5 chromatography, and is expressed as the percentage of the total of both tRNA[Ser]Sec isoforms.
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