Identification of a protein component of a mammalian tRNASec complex implicated in the decoding of UGA as selenocysteine

Abstract

This report describes a novel RNA-binding protein, SECp43, that associates specifically with mammalian selenocysteine tRNA (tRNA Sec ). SECp43, identified from a degenerate PCR screen, is a highly conserved protein with two ribonucleoprotein-binding domains and a polar/acidic carboxy terminus. The protein and corresponding mRNA are generally expressed in rat tissues and mammalian cell lines. To gain insight into the biological role of SECp43, affinity-purified antibody was employed to identify its molecular partners. Surprisingly, the application of native HeLa cell extracts to a SECp43 antibody column results in the purification of a 90-nt RNA species identified by direct sequencing and Northern blot analysis as tRNA Sec . The purification of tRNA Sec by the antibody column is striking, based on the low abundance of this tRNA species. Using recombinant SECp43 as a probe for interacting protein partners, we also identify a 48-kDa interacting protein, which is a possible component of the mammalian selenocysteine insertion (SECIS) pathway. To our knowledge, SECp43 is the first cloned protein demonstrated to associate specifically with eukaryotic tRNA Sec .

The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element

“…First, a class of tRNA Sec binding proteins is composed of EFsec (a dedicated translation elongation factor), SECp43, the phosphoserine tRNA kinase PSTK and a selenocysteine synthase. [12][13][14][15][16][17] Then, a class of SECIS binding proteins have also been identified: SECIS binding protein 2 (SBP2), ribosomal protein L30 (rpL30), translation initiation factor 4A3 (eIF4A3) and nucleolin. [18][19][20][21] However, despite many efforts, the precise found that the SECIS modulates UGA recoding efficiency, 11 we elaborated different growth media with various selenium amounts mimicking adequate, deficient and supplemented dietary intake.…”

Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

“…Sec incorporation requires a cisacting mRNA stem-loop structure termed the selenocysteine insertion sequence (SECIS) element, at least one if not several of which are located in the 3Ј untranslated region (UTR) of mammalian selenoprotein mRNAs (reviewed in Low and Berry, 1996;Atkins et al, 1999), as well as a number of trans-acting factors that govern generation or function of the specialized selenocysteyl-tRNA Sec (Lee et al, 1989; Low et al, 1995Low et al, , 2000Ding and Grabowski, 1999;Copeland et al, 2000). One important determinant of Sec codon usage as a site for Sec incorporation is the concentration of dietary selenium (Se), an essential trace element required for the synthesis of selenocysteyl-tRNA Sec (reviewed in Stadman, 1996).…”

Evidence for Direct Roles of Two Additional Factors, SECp43 and Soluble Liver Antigen, in the Selenoprotein Synthesis Machinery

“…Protein overlay analysis provided evidence for the interaction of SECp43 with a 48-kDa protein in HeLa cytoplasmic extracts (16), whereas SLA was detected in patients with an autoimmune chronic hepatitis as an autoantibody that precipitated Sec tRNA [Ser]Sec from human cell extracts and recognized a 48-kDa protein (17). However, the identity of the 48-kDa protein, the requirements of the SECp43 complex formation, and the roles of either SECp43 or SLA were not determined.…”