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The effect of eukaryotic release factor depletion on translation termination in human cell lines

Deanna M. Janzen and Adam P. Geballe

ABSTRACT

Two competing events, termination and readthrough (or nonsense suppression), can occur when a stop codon reaches the A-site of a translating ribosome. Translation termination results in hydrolysis of the final peptidyl-tRNA bond and release of the completed nascent polypeptide. Alternatively, readthrough, in which the stop codon is erroneously decoded by a suppressor or near cognate transfer RNA (tRNA), results in translation past the stop codon and production of a protein with a C-terminal extension. The relative frequency of termination versus readthrough is determined by parameters such as the stop codon nucleotide context, the activities of termination factors and the abundance of suppressor tRNAs. Using a sensitive and versatile readthrough assay in conjunction with RNA interference technology, we assessed the effects of depleting eukaryotic releases factors 1 and 3 (eRF1 and eRF3) on the termination reaction in human cell lines. Consistent with the established role of eRF1 in triggering peptidyl-tRNA hydrolysis, we found that depletion of eRF1 enhances readthrough at all three stop codons in 293 cells and HeLa cells. The role of eRF3 in eukarytotic translation termination is less well understood as its overexpression has been shown to have anti-suppressor effects in yeast but not mammalian systems. We found that depletion of eRF3 has little or no effect on readthrough in 293 cells but does increase readthrough at all three stop codons in HeLa cells. These results support a direct role for eRF3 in translation termination in higher eukaryotes and also highlight the potential for differences in the abundance or activity of termination factors to modulate the balance of termination to readthrough reactions in a cell-type-specific manner.

INTRODUCTION

Translation termination is a ubiquitous yet incompletely understood step in gene expression during which the final peptidyl-tRNA bond is hydrolyzed, releasing the nascent polypeptide. In higher eukaryotes, it is believed that this reaction is mediated by two proteins, eukaryotic release factor 1 (eRF1) and eRF3. eRF1, a class I release factor, is responsible for stop codon discrimination through direct interaction with the nucleotides of the stop codon triplet. The eRF1 protein is also directly implicated in triggering peptidyl-tRNA hydrolysis via the highly conserved GGQ motif. Although the hydrolysis event is catalyzed by the peptidyl-transferase center of the ribosome, eRF1 may make contact with the nascent peptide through the GGQ motif. Both in vitro and in vivo, eRF1 is known to interact with the class II release factor eRF3. The eRF3 protein is an eRF1- and ribosome-dependent GTPase that stimulates eRF1 activity in response to GTP hydrolysis.

ERF1
translational termination ; GO:0006415
protein binding ; GO:0005515
translation release factor activity ; GO:0003747

ERF3
protein binding ; GO:0005515
GTPase activity ; GO:0003924
translation release factor activity ; GO:0003747

In contrast to the apparently direct role of eRF1 in termination, the necessity of eRF3 for the termination reaction is uncertain. In in vitro assays, addition of eRF1 alone is sufficient to trigger peptidyl-tRNA hydrolysis, although addition of eRF3 increases the rate of hydrolysis at low stop codon concentrations. The prokaryotic eRF3 homolog is dispensable for bacterial growth, but the yeast and Drosophila eRF3 genes are essential. However, since eRF3 has been implicated in cellular processes other than termination, it is possible that the essential role of eRF3 is not in termination but for an alternative function. It has also been suggested that instead of directly contributing to termination, the eRF3 interaction may instead link termination to other cellular processes. For example, eRF3 binds to the poly(A) binding protein and may be involved in ribosome recycling through this interaction. eRF3 also interacts with the Upf proteins, Upf1, Upf2 and Upf3, and thus may mediate the interplay between termination and the nonsense-mediated decay pathway.

ERF3
protein binding ; GO:0005515
mRNA catabolism, nonsense-mediated decay ; GO:0000184
intracellular ; GO:0005622

Studies of the role of eRF3 in termination in different eukaryotic systems have yielded discrepant results. Since translational readthrough at stop codons occurs at an increased frequency when termination efficiency is reduced, the observation that yeast with reduced levels of functional eRF3 have an omnipotent nonsense suppressor phenotype suggests that eRF3 is needed for efficient termination. Conversely, overexpression of both eRF1 and eRF3 is required to increase termination efficiency in yeast, implicating eRF3 along with eRF1 in the termination reaction. In contrast, both in rabbit reticulocyte lysates and in transfected human cells, increased eRF1 expression alone is sufficient to increase termination efficiency. Increasing eRF3 concentration, either alone or in conjunction with eRF1, produced no further effect on termination efficiency in the mammalian systems. Collectively, these results raise uncertainties about the necessity of eRF3 for termination in higher eukaryotes.

Since the results of augmented release factor expression differed between the yeast and mammalian systems, the effects of decreasing release factor expression in mammalian systems may or may not correspond to those detected in yeast. To examine this question, we used RNA interference technology to deplete release factor pools in human cell lines, and then examined the effect on termination efficiency using a sensitive quantitative readthrough assay. In agreement with and as an extension of observations reported for the effect at a UAG stop codon, we found that depletion of eRF1 results in an omnipotent nonsense suppressor phenotype in human cell lines. Furthermore, we found that depletion of eRF3 also has the potential to decrease termination efficiency at all three stop codons, but in a cell-type-specific manner. Consistent with the observations from studies in yeast, these results imply that eRF3 does play a direct role in translation termination in higher eukaryotes.

Many thanks to Staff Rep Emily Dimmer for supplying the annotations.