Reconstitution of selenocysteine incorporation reveals intrinsic regulation by SECIS elements

Abstract

Selenoproteins are present in all three domains of life and are responsible for a major part of a cell's antioxidant defense against reactive oxygen species. Synthesis of selenoproteins requires the decoding of a UGA codon as selenocysteine (Sec) instead of translation termination. Sec is incorporated into the growing polypeptide chain during translation elongation and is known to require a set of highly specific factors: the Sec insertion sequence (SECIS) element in the 3' untranslated region, Sec-tRNA(Sec), the Sec-specific elongation factor eEFSec, and SECIS binding protein 2. Since reconstitution has not been reported, whether these factors are sufficient is unknown. Here, we report a novel in vitro translation system in which Sec incorporation has been reconstituted from purified components introduced into a Sec naive system. In addition, we developed a novel method to purify Sec-tRNA(Sec) and active eEFSec/GTP/tRNA ternary complex. We found that the known basal factors are sufficient for Sec incorporation in vitro. Using this highly manipulable system, we have also found that ribosomes from non-Sec-utilizing organisms cannot support Sec incorporation and that some SECIS elements are intrinsically less efficient than others. Having identified the essential set of factors, this work removes a significant barrier to our understanding of the mechanism of Sec incorporation.

Figure 1

Reconstitution of Sec incorporation in wheat germ in vitro translation lysate. In vitro translation of the Sec incorporation reporter mRNA (A) in 50 % of wheat germ lysate in the presence or absence of 160 nM XH-SBP2 and FLAG-eEFSec recombinant proteins, 1.25 μg of total testes aminoacylated tRNA and 80 nM of salt washed rabbit ribosomes (B). Data was normalized for luciferase activity from mutant GPX4 SECIS element. The data represents the mean and standard deviation of three independent experiments (n=3). The asterisk (*) denotes a significant difference vs. no factors by student’s t-test (p < 0.02). (C) In vitro translation of a Sec incorporation reporter mRNA that has a UAA codon instead of the UGA codon shown in (A). Raw luciferase activity (luminescence) was measured by luminometry. The double asterisk (**) denotes a significant difference vs. no SBP2 by student’s t-test (p < 0.02).

Figure 2

Purification of Sec-tRNA^Sec A) In vitro translation of the Sec incorporation reporter mRNAs as described in Figure 1 in the presence of total testis aa-tRNA, 125 ng of Sec-tRNA^Sec, purified FLAG-eEFSec and/or purified FLAG-eEFSec/GTP/tRNA ternary complex (TC) as indicated. The data represents the mean and standard deviation of three independent experiments. B) The presence of Sec-tRNA^Sec was confirmed and quantified by Northern blot. Two-fold serial dilution of in vitro transcribed tRNA^Sec (1.25 – 20ng) and samples derived from tRNA purifications as indicated were analyzed by Northern analysis and hybridized to a probe complementary to the anticodon loop of tRNA^Sec. The amount of Sec-tRNA^Sec from each source was determined by densitometry using the in vitro transcribed tRNA^Sec as a standard curve.

Figure 3

Mammalian ribosomes are required for Sec incorporation. Total translation (A) or Sec incorporation (B) in ribosome-depleted RRL in the presence of 80 nM salt washed ribosomes from rabbit reticulocyte lysate, Saccharomyces cerevisiae, Spodoptera frugiperda Sf21 cells or wheat germ lysate as indicated. The asterisk (*) denotes a significant difference vs. no ribosomes by student’s t-test (p < 0.02).

Figure 4

Differential SECIS element efficiency. (A) In vitro translation of the Sec incorporation reporter mRNA bearing the hGpx4, hTrxR3 and Selenoprotein O SECIS elements in wheat germ lysate with 160 nM XH-SBP2, eEFSec/GTP/tRNA ternary complex and 80 nM of salt washed rabbit ribosomes. The data represents the mean and standard deviation of three independent experiments. (B) Same as in (A) except the translation was performed in rabbit reticulocyte lysate without added ribosomes. The data represents the mean and standard deviation of three independent experiments. The asterisk (*) denotes a significant difference vs. hGPX4 by student’s t-test (p < 0.02).