The utilization of selenocysteine-tRNA^[Ser]Sec isoforms is regulated in part at the level of translation in vitro

Figures & data

Figure 1. Analysis of Sec-tRNA^[Ser]Sec isoforms by RPC-5 chromatography. (A) Purified [3H]Ser-tRNA^[Ser]Sec was resolved into the 2 characteristic isoform peaks by RPC-5 column chromatography. Fractions were analyzed by scintillation counting. (B) Sec-tRNA^[Ser]Sec purified by EEFSEC affinity was aminoacylated in vitro with [3H]Ser as described in the experimental procedures. Total liver tRNA (C) or tRNA eluted from anti-FLAG beads loaded with FLAG-EEFSEC (D) was applied to an RPC-5 column and tRNA^[Ser]Sec was detected in fractions by dot blot hybridization with a [³²P] labeled probe.

Figure 2. In vitro translation of GPX1 and SELENOP mRNAs in rabbit reticulocyte lysate using purified ⁷⁵Se-labeled Sec-tRNA^[Ser]Sec. (A) Large scale purification of the mcm⁵U and mcm⁵Um isoforms of Sec-tRNA^[Ser]Sec by RPC-5 chromatography. The shaded areas represent the fractions that were pooled for each isoform. (B) 100 ng each of GPX1 and SELENOP mRNAs were translated in rabbit reticulocyte lysate in the presence of inorganic ⁷⁵Se or the Sec-tRNA^[Ser]Sec isoforms as indicated. The fold increase in SELENOP and GPX1 translation as a function of the presence of mcm⁵Um Sec-tRNA^Sec is indicated below.

Figure 3. In vitro translation of GPX1 and TXNRD1 in wheat germ lysate using purified ⁷⁵Se-labeled Sec-tRNA^[Ser]Sec. (A) A range of mcm⁵U and mcm⁵Um Sec-tRNA^[Ser]Sec amounts from 2000–6000 cpm were added to wheat germ in vitro translation reactions programmed with 4 nM of either GPX1 (lanes 1–7) or TXNRD1 mRNA (lanes 8–14). The fold increase in GPX1 and TXNRD1 translation as a function of the presence of mcm⁵Um Sec-tRNA^[Ser]Sec is indicated below. (B) 1 and 4 nM GPX1 or TXNRD1 mRNAs were translated in rabbit reticulocyte lysate and wheat germ lysate. The fold increase of GPX1 and TXNRD1 in reticulocyte lysate vs. wheat germ lysate is indicated below.

Figure 4. In vitro translation of GPX1 and TXNRD1 together in wheat germ lysate. (A) GPX1 and TXNRD1 mRNAs were translated in the presence of mcm⁵U and mcm⁵Um Sec-tRNA^[Ser]Sec isoforms as described in Figure 3 but in lanes 3 and 6, equal amounts of mRNA were combined in the same reaction. Phosphorimage quantitation of bands plotting the percent increase as a function of using the mcm⁵Um Sec-tRNA^[Ser]Sec isoform is shown below. (B) Phosphorimage quantitation of multiple experiments comparing the amount of GPX1 or TXNRD1 translation in the presence of mcm⁵U vs. mcm⁵Um Sec-tRNA^[Ser]Sec. A total of 19 experiments are included in this analysis, which is an unpaired 2-tailed t-test.