Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells

Abstract

Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1Δ as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.

Translation Elongation Factor 1A Mutants with Altered Actin Bundling Activity Show Reduced Aminoacyl-tRNA Binding and Alter Initiation via eIF2α Phosphorylation

“…1A). The idea that defects at one phase of translation can have a feedback effect on a different phase has been discussed (41). Therefore, we wanted to determine whether a general defect in translation would be enough to cause an increase in eIF2␣ phosphorylation levels.…”

The role of eIF3 and its individual subunits in cancer

“…Its presence on the ribosome following translation of short ORFs or following termination may promote reinitiation on the same mRNA [27]. Another possibility is that eIF3 may function in termination, as has been claimed for yeast eIF3, especially its 3j subunit [28]. eIF3 also has been implicated in post-termination ribosome recycling where it promotes the splitting of the 60S subunit from the 40S subunit still bound to deacylated tRNA and mRNA [29,30].…”

Mechanism of Cytoplasmic mRNA Translation

“…The placement of the eIF3 subunits and their contacts with the ribosome and other initiation factors awaits further structural data, but a picture is beginning to emerge at the molecular level (Wilson and Doudna Cate, 2012;Hashem et al, 2013;Liu et al, 2014). Yeast eIF3 is also implicated in termination of protein synthesis, termination codon read-through and ribosome reinitiaton suggesting that we still have much to learn about this multi-functional factor and its roles during translation in all organisms (Pisarev et al, 2007;Beznosková et al, 2013).…”