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...

The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3′-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5′-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.