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

Abstract

Translational control in eukaryotes is exerted by many means, one of which involves a ribosome translating multiple cistrons per mRNA as in bacteria. It is called reinitiation (REI) and occurs on mRNAs where the main ORF is preceded by a short upstream uORF(s). Some uORFs support efficient REI on downstream cistrons, whereas some others do not. The mRNA of yeast transcriptional activator GCN4 contains four uORFs of both types that together compose an intriguing regulatory mechanism of its expression responding to nutrients' availability and various stresses. Here we subjected all GCN4 uORFs to a comprehensive analysis to identify all REI-promoting and inhibiting cis-determinants that contribute either autonomously or in synergy to the overall efficiency of REI on GCN4. We found that the 3 ′ sequences of uORFs 1-3 contain a conserved AU 1-2 A/ UUAU 2 motif that promotes REI in position-specific, autonomous fashion such as the REI-promoting elements occurring in 5 ′ sequences of uORF1 and uORF2. We also identified autonomous and transferable REI-inhibiting elements in the 3 ′ sequences of uORF2 and uORF3, immediately following their AU-rich motif. Furthermore, we analyzed contributions of coding triplets and terminating stop codon tetranucleotides of GCN4 uORFs showing a negative correlation between the efficiency of reinitiation and efficiency of translation termination. Together we provide a complex overview of all cis-determinants of REI with their effects set in the context of the overall GCN4 translational control.

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

“…This is consistent with the observation that the ∆8  truncation impairs reinitiation on GCN4 mRNA following translation of uORF1 and uORF2, an effect attributable to the loss of interaction between the eIF3a NTD and nucleotides 5’ of each uORF stop codon that otherwise stabilize post-termination 40S subunits and allow them to resume scanning in the presence of WT eIF3 (Gunišová et al, 2016; Munzarová et al, 2011; Szamecz et al, 2008). This role of the eIF3a NTD is also consistent with its placement immediately adjacent to the exit-channel pore in several structures of eIF3 bound to the 40S, in both yeast and mammals (Aylett et al, 2015; Georges, des et al, 2015; Llácer et al, 2015).…”

“…Mutations in the C-terminal domain (CTD) of eIF3a, which interacts with the 40S subunit near the mRNA entry channel, impair mRNA recruitment in yeast cells without affecting the integrity of the PIC (Chiu et al, 2010). The eIF3a N-terminal domain (NTD) interacts functionally with mRNA near the exit channel, enhancing reinitiation upon translation of the upstream open reading frame 1 (uORF1) and uORF2 of GCN4 mRNA in a manner dependent on the sequence upstream of these two uORFs (Gunišová et al, 2016; Gunišová and Valášek, 2014; Munzarová et al, 2011; Szamecz et al, 2008). Consistent with this, mammalian eIF3 can be cross-linked to mRNA at positions 8–17 nucleotides upstream of the AUG codon (Pisarev et al, 2008), near the exit channel, and a mutation within the eIF3a NTD, which has been located near the exit channel in structures of the PIC, interferes with mRNA recruitment in yeast cells (Khoshnevis et al, 2014).…”

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

“…The 5' proximal uORF1 and uORF2 are positive, REI-promoting sequences, whose ability to allow efficient REI is determined by five cis-acting REI-promoting elements (RPEs i. to v.) mapping upstream of these uORFs and making contacts with eIF3 ( Figure S2A). By contrast, the 5' distal uORF3 and uORF4 are negative, REI-non-permissive sequences lacking any RPEs (Gunisova et al, 2016;Munzarová et al, 2011).…”

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship

“…GCN4 protein targets most genes required for amino acid biosynthesis (Natarajan et al 2001). Upon starvation, translation re-initiation at the multiple uORFs is down-regulated and the GCN4 protein expression level thus rises (Hinnebusch 2005;Gunišová et al 2016). Multiple examples of uORF-mediated regulation of protein levels have been published; however, numerous studies also highlight the biological role of uORF-encoded peptides Cabrera-Quio et al 2016;Plaza et al 2017).…”