Abstract
One of the extensively studied mechanisms of gene-specific translational regulation is reinitiation. It takes place on messenger RNAs (mRNAs) where main ORF is preceded by upstream ORF (uORF). Even though uORFs generally down-regulate main ORF expression, specific uORFs exist that allow high level of downstream ORF expression. The key is their ability to retain 40S subunits on mRNA upon termination of their translation to resume scanning for the next AUG. Here, we took advantage of the exemplary model system of reinitiation, the mRNA of yeast transcriptional activator GCN4 containing four short uORFs, and show that contrary to previous reports, not only the first but the first two of its uORFs allow efficient reinitiation. Strikingly, we demonstrate that they utilize a similar molecular mechanism relying on several cis-acting 5′ reinitiation-promoting elements, one of which they share, and the interaction with the a/TIF32 subunit of translation initiation factor eIF3. Since a similar mechanism operates also on YAP1 uORF, our findings strongly suggest that basic principles of reinitiation are conserved. Furthermore, presence of two consecutive reinitiation-permissive uORFs followed by two reinitiation-non-permissive uORFs suggests that tightness of GCN4 translational control is ensured by a fail-safe mechanism that effectively prevents or triggers GCN4 expression under nutrient replete or deplete conditions, respectively.
Highlights
Translational control mechanisms represent one of the critical aspects of the overall regulation of gene expression
We revealed that the REI competence of uORF2 strictly relies on: (i) the structured, eIF3-independent RPE ii. of uORF1, which represents a common REI-promoting element for both of these upstream ORF (uORF), and (ii) a specific, 10-bp long element designated as RPE v., which occurs in the vicinity of the 40S messenger RNAs (mRNAs) exit channel of the 80S ribosome terminating on uORF2 and, not surprisingly, operates in the a/TIF32-N-terminal domain (NTD)-dependent manner
UORF2 from the GCN4 mRNA leader resembles the well-established REI-permissive uORF1 in its high REIpromoting activity that is dependent on the N-terminal domain of the a/TIF32 subunit of eIF3
Summary
Translational control mechanisms represent one of the critical aspects of the overall regulation of gene expression. A functional interaction between these sequences and the N-terminal domain (NTD) of the a/TIF32 subunit of the initiation factor eIF3 was identified and implicated in stabilizing the post-termination 40S subunits on the uORF1 stop codon to enable resumption of scanning for REI downstream [10]. Was identified upstream of the REI-permissive uORF in the mRNA leader of yet another yeast transcriptional activator YAP1 [12] The fact that it likewise operated in the a/TIF32-NTD-dependent manner suggested that at least in yeasts the underlying mechanism of REI on short uORFs might be conserved. Of uORF1, which represents a common REI-promoting element for both of these uORFs, and (ii) a specific, 10-bp long element designated as RPE v., which occurs in the vicinity of the 40S mRNA exit channel of the 80S ribosome terminating on uORF2 and, not surprisingly, operates in the a/TIF32-NTD-dependent manner. Implications of the existence of two consecutive REI-permissive uORFs in the GCN4 mRNA leader are discussed
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