Unique pathway of expression of an opal suppressor phosphoserine tRNA.

Abstract

An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. We have shown that the product of this gene functions as a suppressor in an in vitro assay, and we have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, we show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of any known eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells.

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene

“…Selenium supplementation can increase the levels of tRNA [Ser]Sec isoacceptors (Hatfield et al+, 1991;Baek et al+, 1997)+ Furthermore, as little as 30 nM sodium selenite administered to CHO cells in culture for 3 days can stimulate the levels of the selenoprotein, GPx (Diamond et al+, 1996)+ To determine whether the increased amount of tRNA [Ser]Sec in transfected cells could stimulate GPx levels, transfectants and control cells were assayed for GPx activity, measured in a coupled spectrophotometric assay that quantitates the levels of this enzyme with all other components of the reaction in excess+ As shown in Table 2, elevated levels of tRNA [Ser]Sec did not significantly increase the baseline levels of GPx+ However, CHO cells transfected with a GPx expression construct (Diamond et al+, 1997) produced significantly higher levels of GPx only when the culture media was supplemented with selenium ( Figure 3)+ It remained possible that the levels of selenoproteins other than GPx were being influenced by elevated tRNA [Ser]Sec levels+ To assess this possibility, a CHO transfectant expressing approximately eightfold higher levels of the mcm 5 U isoacceptor was incubated with 75 Se and the labeled selenoproteins were examined by gel electrophoresis and compared to similarly prepared proteins from control cells+ As shown in Figure 5, there is no evidence of elevated steady-state levels of any selenoprotein due to increased levels of tRNA [Ser]Sec + It is unlikely that the reason that elevated levels of tRNA [Ser]Sec obtained following transfection did not alter selenoprotein biosynthesis was a functional defect in the tRNA [Ser]Sec generated from the transfected mouse gene+ As shown in Figure 3, tRNA [Ser]Sec transcribed from the mouse gene was clearly capable of being aminoacylated with serine+ To obtain additional data indicating the functionality of the mouse tRNA [Ser]Sec , tRNA from cells producing the highest levels of tRNA [Ser]Sec (Table 1) were aminoacylated with 3 Hserine and the labeled seryl-tRNAs isolated following RPC-5 chromatography+ The mcm 5 U and mcm 5 Um isoacceptors were independently tested for their ability to respond to UGA in a ribosome binding assay+ As shown in Table 3 (Nirenberg & Leder, 1964) Transcription of tRNA [Ser]Sec is highly atypical in that the primary transcript lacks a 59 leader sequence, that is, transcription begins at the first nucleotide within the gene (Lee et al+, 1987)+ Another unique feature of tRNA [Ser]Sec compared to other tRNAs is that the transcription of tRNA …”

Selenoproteins: Molecular Pathways and Physiological Roles

“…, which begins at the first nucleotide within the coding sequence, whereas all canonical tRNAs have a 5= leader sequence that must be processed (208). The regulation of Trsp transcription is also novel among tRNAs.…”

Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA[Ser]Sec is governed by both primary and tertiary structure

“…Oocytes were obtained from mature Xenopus females+ Stage VI oocytes were selected and maintained in OR-2 medium (82+5 mM NaCl, 2+5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM NaHCO 3 , 5 mM HEPES, pH 7+8) at room temperature+ Forty nanoliters of the mixture containing 2-3 ϫ 10 5 cpm of 32 P-labeled tRNAs (prepared as described above) were injected into the cytoplasm of oocytes and the oocytes incubated at 20 8C for 16 h+ After incubation, tRNAs were extracted as described (Tobian et al+, 1985;Lee et al+, 1987), loaded on a RPC-5 column (Kelmers & Heatherly, 1971), and the column developed in a 0+525-0+70 M NaCl linear gradient as described (Diamond et al+, 1993;Choi et al+, 1994)+ Column fractions were counted in a liquid scintillation counter (Pharmacia), the peaks representing isoacceptors pooled, precipitated in ethanol, collected, and prepared for nuclease digestion as described (Choi et al+, 1994)+ …”