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.
Highlights
Protein synthesis or mRNA translation is a complex and highly conserved process that can be separated into initiation, elongation, termination and ribosome recycling phases
Mutations in eIF3 subunits and hcr1 deletion affect stop codon read-through eIF3 and the eIF3-core-associated factors like HCR1 and RLI1 play a role in ribosome recycling – at least in vitro [5,6], while only RLI1 is to date known to somehow promote the preceding translation termination step [2]
In order to address whether eIF3 itself is likewise functionally involved in translation termination, we first measured the frequency of stop codon read-through in a collection of eIF3 mutants using an established dual-luciferase reporter assay, designed to be independent of mRNA levels [14]
Summary
Protein synthesis or mRNA translation is a complex and highly conserved process that can be separated into initiation, elongation, termination and ribosome recycling phases. These four phases are distinct in time, there is a longstanding notion for some form of communication among them. Several initiation factors and related proteins have been proposed to function in more than one phase These include ABCE1/RLI1 and GLE1, which are believed to promote both the initiation and termination phases by a mechanism that remains to be elucidated [1,2,3], eIF5A proposed to stimulate all three major phases [4], and the bona fide translation initiation factor eIF3, which has been recently suggested to promote the recycling phase, at least in a mammalian in vitro reconstituted system [5,6]. In contrast to the most of eIFs, eIF3 interacts with the solvent-exposed side of the small ribosomal subunit [7] and as such it was proposed to be able to interact with active 80S ribosomes post-initiation [8,9,10]
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