Involvement of Human Release Factors eRF3a and eRF3b in Translation Termination and Regulation of the Termination Complex Formation

Abstract

eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.In eukaryotes, two release factors, eRF1 and eRF3, are required to complete protein synthesis. These translation termination factors associate in a complex which binds to the elongating ribosome when a stop codon enters the A site. eRF1 recognizes all three stop codons by direct interaction at the decoding A site (5, 10) and activates the peptidyltransferase center, which triggers the hydrolysis of the peptidyltRNA, generating a free full-sized polypeptide. eRF3 is a GTPase that stimulates eRF1 activity in a GTP-dependent manner (40). eRF3 alone can bind GTP, but its GTPase activity requires the presence of both eRF1 and ribosomes, which may play the role of a composite GTPase-activating protein (11, 12). The eRF3 GTP-bound form interacts with eRF1 in vitro and in vivo to constitute the active translation termination complex (12,36,40). In the yeast Saccharomyces cerevisiae, eRF1 and eRF3 are encoded by the essential genes SUP45 and SUP35, respectively. Mutations in either of these genes cause an omnipotent suppressor phenotype and the ability to enhance nonsense suppression by a weak suppressor tRNA (reviewed in reference 18). Recently, it has been shown in S. cerevisiae that eRF3 GTPase activity facilitates stop codon decoding by eRF1 (33).The C-terminal regions of eRF3 proteins are highly conserved through evolution and carry the four canonical GTPbinding motifs of the GTPase superfamily (2). This domain is essential for translation termination and interaction with eRF1. The N-terminal region varies in both length and sequence among species. In yeast, it is neither essential for cell viability nor required for termination but is responsible for prion-like [PSI ϩ ] factor formation (29, 31). Otherwise, this domain participates in...

Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation

“…As a starting point for these experiments, there is the surprising observation that eRF1 abundance strongly correlates to that of eRF3a in a wide range of human cell lines (Chauvin et al 2005). This raises the possibility that the relative abundances of eRF1 and eRF3a are tightly adjusted by a mechanism that controls the formation of the translation termination complex and the efficiency of termination.…”

“…We then analyzed the translation termination activity of eRF3a variants by complementation experiments in eRF3a-depleted cells (Chauvin et al 2005). Parallel cultures of 559C cells, a cell line expressing a lacZ reporter gene interrupted by a premature stop codon, were depleted in eRF3a by electroporation of a plasmid expressing a siRNA, si-3a1, specifically targeting eRF3a mRNA.…”

“…This raises the possibility that the relative abundances of eRF1 and eRF3a are tightly adjusted by a mechanism that controls the formation of the translation termination complex and the efficiency of termination. The first clue was provided by the fact that eRF3a depletion induces a decrease in eRF1 stability in human cells, suggesting that eRF1 is proteolytically degraded when not complexed with eRF3a (Chauvin et al 2005). To go further in the understanding of this regulation, we examined the status of eRF3a when not associated with eRF1.…”

“…In mammals, two distinct proteins, eRF3a and eRF3b, are able to function as a translation termination factor in vivo (Chauvin et al 2005). Depletion of eRF3a, which is likely to be the main factor acting in translation termination, causes a significant reduction of 1 the eRF1 level by decreasing its stability (Chauvin et al 2005).…”

“…In mammals, two distinct proteins, eRF3a and eRF3b, are able to function as a translation termination factor in vivo (Chauvin et al 2005). Depletion of eRF3a, which is likely to be the main factor acting in translation termination, causes a significant reduction of 1 the eRF1 level by decreasing its stability (Chauvin et al 2005). In budding yeast, some reports showed that a reduced level of either eRF1 or eRF3 did not affect the level of its partner (Chabelskaya et al 2004;Salas-Marco and Bedwell 2004), whereas Valouev et al (2002) reported that depletion of either of the release factors is accompanied by a reduction in the level of the other.…”