RNA-binding Proteins That Specifically Recognize the Selenocysteine Insertion Sequence of Human Cellular Glutathione Peroxidase mRNA

Qichang Shen,Patricia A Mcquilkin,Peter E Newburger

doi:10.1074/jbc.270.51.30448

Abstract

Translational incorporation of the unusual amino acid selenocysteine in eukaryotes requires a coding region UGA codon (which otherwise serves as a termination signal), a selenocysteine insertion sequence (SECIS) in the 3'-untranslated region of the mRNA, and selenocysteyl-tRNA. The mechanisms involved in SECIS recognition by the eukaryotic translational machinery remain unknown. We report the detection of RNA-binding proteins that specifically recognize the SECIS from human cellular glutathione peroxidase (GPX1) transcripts. RNA gel shift assays showed three retarded bands after incubation with COS-1 whole cell lysate or S-100 cytosol fraction or with extracts from hepatoma cell lines HepG2 and Hep3B. The specificity of the binding was demonstrated by competition by cold unlabeled SECIS RNA and by lack of competition by other RNA species with similar stem-loop secondary structures, such as the human immunodeficiency virus (HIV) transactivation-response region of HIV mRNA element, and mutated SECIS constructs. UV cross-linking and SDS-polyacrylamide gel electrophoresis revealed at least two proteins, with estimated molecular masses of 55,000 and 65,000 Da, that bind to the SECIS. Examination of a series of insertion and deletion SECIS mutants indicated recognition of the SECIS primarily through the basal stem region, although the upper stem, loop, and two of three short conserved sequences also appear to contribute to the affinity of the binding.

Highlights

A wide variety of sequence elements have recently been identified in the 5Ј- and 3Ј-untranslated regions (UTR)1 of eukaryotic mRNAs
We have shown that this selenocysteine insertion sequence (SECIS) is necessary for translation of GPX1 mRNA as well as sufficient to direct the translation of an opal (UGA) mutation as selenocysteine in heterologous proteins [9]
In order to identify RNA-binding protein(s) that recognize the SECIS, we examined the effect of incubation with COS-1 cell extract on the migration, in non-denaturing PAGE, of a 32P-labeled RNA probe containing the 87-nt GPX1 SECIS (Fig. 1A)

Summary

Introduction

A wide variety of sequence elements have recently been identified in the 5Ј- and 3Ј-untranslated regions (UTR)1 of eukaryotic mRNAs. UV Cross-linking and SDS-PAGE—100,000 cpm of radiolabeled wild type or 6-nt deletion mutant GPX1 SECIS probes were incubated with 80 ␮g of COS-1 extract in a total of 50 ␮l under the same conditions as for the gel shift assay.

Results

Conclusion