Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in Fungi and animals.

Thorsten Stehlik,Alina C Stiebler,Julia Ast,Michael Bölker,Johannes Freitag,Britta A M Tillmann,Kay O Schink,Mick F Tuite

doi:10.1371/journal.pgen.1004685

Abstract

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated.

Highlights

Translation of mRNA usually occurs with high fidelity, different recoding mechanisms exist that alter the amino acid sequence of the resulting polypeptide [1,2]
We have recently reported that translational readthrough and alternative splicing are used in fungi to generate peroxisomal isoforms of several glycolytic enzymes [17]
We have previously shown that in the fungus Ustilago maydis, a peroxisomal targeting signal 1 (PTS1) containing isoform of triosephosphate isomerase (Tpi1) is generated by stop codon readthrough (Fig. 1A) [17]

Summary

Introduction

Translation of mRNA usually occurs with high fidelity, different recoding mechanisms exist that alter the amino acid sequence of the resulting polypeptide [1,2]. Bacteria containing suppressor tRNAs and Saccharomyces cerevisiae strains expressing a non-functional prion form of the eukaryotic release factor 3 display significantly enhanced bypass of stop codons [4,5]. This results in C-terminal extension of many proteins and increases phenotypic variability. We report that translational readthrough at a short conserved stop codon context is used in animals and fungi to generate peroxisomal isoforms of important metabolic enzymes

Author Summary

Conclusions

Materials and Methods