Translation initiation factor eIF3 promotes programmed stop codon readthrough

Abstract

Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.

Translational readthrough potential of natural termination codons in eucaryotes – The impact of RNA sequence

“…56 Recently, it has been proposed that eIF3, the eukaryotic translation initiation factor, could be involved in promoting programmed RT of all three STOP codons set in the unfavorable termination context. 63 In this mechanism, which appears to be evolutionary conserved, eIF3 interacts with the pre-termination complex, where it prevents eRF1 from recognizing the third/wobble position of the STOP codon. As a consequence, nc-tRNAs with a mismatch in the same position can decode the STOP codon, allowing the protein synthesis to be continued.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…The eRF1, composed of three domains, functionally mimics a tRNA with the N-terminal domain recognizing the stop codon (Bertram et al 2000), the central domain with its methylated GGQ motif promotes hydrolysis of the peptidyl-tRNA bond (Heurgue-Hamard et al 2005), and the C-terminal domain interacts with eRF3. Recognition of all three stop codons by eRF1 is mediated by the YxCxxxF and TASNIKS motifs, as well as by other binding pockets/cavities in the N domain Namy et al 2001;Namy et al 2002;Namy et al 2003;Beznoskova et al 2015 Genes are grouped by publication/similar mechanism. Many examples given rely on a single publication and detailed mechanisms remain unknown.…”

In-depth analysis of cis-determinants that either promote or inhibit reinitiation on GCN4 mRNA after translation of its four short uORFs

“…We hypothesized that this factor could lie in the identity of the base immediately following the stop codon (the so-called +4 nucleotide), which is known to strongly influence the efficiency of stop codon recognition (see, for example, Beznosková et al 2015b). In particular, cytosine at the +4 position is the leakiest base with respect to efficiency of translation termination among all four bases at all three stop codons (Beznosková et al 2015a;Dabrowski et al 2015).…”

“…7B). As a control, we used the TGA stop codon with the C at the +4 position (TGA-C), which is known to allow relatively high levels of readthrough (Beznosková et al 2015b). Notably, readthrough on uORF4 is even ∼1.5-fold higher than readthrough on this very rare terminating tetranucleotide with the so-called programmed readthrough.…”