Identification and Molecular Cloning of a Human Selenocysteine Insertion Sequence-binding Protein: A BIFUNCTIONAL ROLE FOR DNA-BINDING PROTEIN B

Qichang Shen,Rui Wu,Jack L Leonard,Peter E Newburger

doi:10.1074/jbc.273.10.5443

Abstract

Prokaryotic and eukaryotic cells incorporate the unusual amino acid selenocysteine at a UGA codon, which conventionally serves as a termination signal. Translation of eukaryotic selenoprotein mRNA requires a nucleotide selenocysteine insertion sequence in the 3'-untranslated region. We report the molecular cloning of the binding protein that recognizes the selenocysteine insertion sequence element in human cellular glutathione peroxidase gene (GPX1) transcripts and its identification as DNA-binding protein B, a member of the EFIA/dbpB/YB-1 family. The predicted amino acid sequence contains four arginine-rich RNA-binding motifs, and one segment shows strong homology to the human immunodeficiency virus Tat domain. Recombinant DNA-binding protein B binds the selenocysteine insertion sequence elements from the GPX1 and type I iodothyronine 5'-deiodinase genes in RNA electrophoretic mobility shift assays and competes with endogenous GPX1 selenocysteine insertion sequence binding activity in COS-1 cytosol extracts. Addition of antibody to DNA-binding protein B to COS-1 electromobility shift assays produces a slowly migrating "supershift" band. The molecular cloning and identification of DNA-binding protein B as the first eukaryotic selenocysteine insertion sequence-binding protein opens the way to the elucidation of the entire complex necessary for the alternative reading of the genetic code that permits translation of selenoproteins.

Highlights

Both prokaryotic and eukaryotic cells incorporate the sulfurlike element selenium into polypeptides by the translational insertion of the unique amino acid selenocysteine
Expression Library Screening—Two ␭gt11 cDNA expression libraries from human K562 and HeLa cell lines were screened with a 32P-labeled synthetic RNA transcript from the selenocysteine insertion sequence (SECIS) region of the human cellular glutathione peroxidase gene GPX1 [4]
Two additional radiolabeled RNA probes were used as negative controls: a mutant GPX1 SECIS probe with a deletion of six nucleotides at the 3Ј end and a probe containing the human immunodeficiency virus (HIV) trans-activation response (TAR) element found at the 5Ј ends of all nascent HIV-1 transcripts

Summary

A BIFUNCTIONAL ROLE FOR DNA-BINDING PROTEIN B*

From the Departments of Pediatrics, Physiology, Nuclear Medicine, and Molecular Genetics-Microbiology and the Cancer Center, University of Medical Center, Worcester, Massachusetts 01605. Ety within the active site; examples include the bacterial formate dehydrogenases [1, 2], the mammalian glutathione peroxidase family [3,4,5,6], and the iodothyronine deiodinase family [7] In both prokaryotic and eukaryotic mRNA transcripts, selenocysteine is encoded by a UGA codon, which conventionally serves as one of the three translation termination signals [8, 9]. This protein was originally isolated and characterized based on its DNA-binding properties, which include both affinity for unspecified promoter/enhancer region sites [17] and functional binding to “Y box” transcriptional activation sites [18, 19] It contains four arginine-rich motifs characteristic of a group of RNA-binding proteins [20] and binds to the SECIS element of human cellular glutathione peroxidase mRNA

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION