Functional Analysis of the Interplay between Translation Termination, Selenocysteine Codon Context, and Selenocysteine Insertion Sequence-binding Protein 2

Malavika Gupta,Paul R Copeland

doi:10.1074/jbc.m707061200

Abstract

A selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region and an in-frame UGA codon are the requisite cis-acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA[Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding selenoprotein P (Sel P) unique. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences were dramatically reduced in the presence of excess SBP2. Mutational analysis of the "fourth base" of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency.

Highlights

These are an in-frame UGA codon and a structure called the selenocysteine insertion sequence (SECIS) (Sec insertion sequence) element [3]
selenoprotein P (Sel P) Codon Contexts—To understand the impact of codon context on Sec incorporation, we used each of the 10 rat Sel P UGAs, along with their four flanking codons on both sides, replacing the corresponding sequence in the open reading frame of a firefly luciferase construct (Fig. 1, Sec1–Sec10)
In addition to the Sel P UGA codons and their native codon contexts, our constructs contained the first 770 nucleotides of the rat Sel P 3Ј-UTR including both of its SECIS elements (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Codon Context Plasmid Construction—A firefly luciferase construct containing an in-frame UGA at position 258 was created as previously described [22]. PacI and NotI linkers were used to clone the first 770 nucleotides of the Sel P 3Ј-UTR downstream of the Luc UGA open reading frame. This stretch of 3Ј-UTR contained both SECIS elements. XhoI digestion generated a template with a 3Ј-UTR downstream of the T7 promoter, allowing transcription of the coding region and the 770 nucleotides of the Sel P 3Ј-UTR. 12.5-␮l reactions contained 8 ␮l of rabbit reticulocyte lysate (RRL), 0.25 ␮l of 40 units/␮l of RNasin (Promega), 0.25 ␮l of 1 mM amino acid mixture minus methionine, 0.5 ␮l of [35S]Met at 10 mCi/ml, 100 ng of reporter mRNA and varying amounts of CTSBP2 or 1ϫ phosphate-buffered saline and 2 mM dithiothreitol.

RESULTS

A G C tototo

DISCUSSION