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Translation of 5' leaders is pervasive in genes resistant to eIF2 repression.

Andreev DE, O'Connor PB, Fahey C, Kenny EM, Terenin IM, Dmitriev SE, Cormican P, Morris DW, Shatsky IN, Baranov PV - Elife (2015)

Bottom Line: However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response.Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition.Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

View Article: PubMed Central - PubMed

Affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

ABSTRACT
Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

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Features of the IFRD1 5′ leader required for resistance.(A) Firefly luciferase (Fluc) activity produced by expressionof mRNA containing pGL3 and IRFD1 leaders (outlined on the left) with andwithout an additional stem loop at the 5′ end under differentconditions. Blue (normal conditions) and red (stress conditions) barscorrespond to leaders lacking the stem loop while light blue (normal) andyellow (stress) correspond to leaders with the stem loop. The fold change ofFluc activity in response to stress is indicated above by green arrows.(B) Effect of (CAA)6 addition to the 5′leader of IFRD1 on Fluc activity in response to stress.DOI:http://dx.doi.org/10.7554/eLife.03971.019
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fig5: Features of the IFRD1 5′ leader required for resistance.(A) Firefly luciferase (Fluc) activity produced by expressionof mRNA containing pGL3 and IRFD1 leaders (outlined on the left) with andwithout an additional stem loop at the 5′ end under differentconditions. Blue (normal conditions) and red (stress conditions) barscorrespond to leaders lacking the stem loop while light blue (normal) andyellow (stress) correspond to leaders with the stem loop. The fold change ofFluc activity in response to stress is indicated above by green arrows.(B) Effect of (CAA)6 addition to the 5′leader of IFRD1 on Fluc activity in response to stress.DOI:http://dx.doi.org/10.7554/eLife.03971.019

Mentions: We observed earlier that most stress resistant mRNAs possess efficiently translateduORFs. We hypothesized that some features of the 5′ leaders upstream of uORFsmay be important for resistance. To address this issue we created two additionalreporters based on control pGL3 and IFRD1, where we added a 5′ terminal stemloop of intermediate stability (Figure 5). Asexpected, the addition of this stem loop resulted in a threefold to fourfold decreasein the activity of both reporters under normal conditions. Interestingly, whenarsenite stress was induced, the SL-IFRD1 construct did not exhibit resistance whiletranslation of SL-PGL3 was downregulated as much as the pGL3 construct. Therefore wepropose that efficient loading of pre-initiator complexes to the uORF is necessaryfor stress resistance in IFRD1. Next we addressed the question of whether thespecific sequence upstream of the IFRD1 uORF is required for regulation. Wesubstituted it with an artificial single stranded (CAA)6 sequence of thesame length. This mutation did not alter stress resistance. Thus, we hypothesizethat, for resistant translation, the uORF has to be preceded with a leader allowing ahigh initiation rate at the uORF.10.7554/eLife.03971.019Figure 5.Features of the IFRD1 5′ leader required for resistance.


Translation of 5' leaders is pervasive in genes resistant to eIF2 repression.

Andreev DE, O'Connor PB, Fahey C, Kenny EM, Terenin IM, Dmitriev SE, Cormican P, Morris DW, Shatsky IN, Baranov PV - Elife (2015)

Features of the IFRD1 5′ leader required for resistance.(A) Firefly luciferase (Fluc) activity produced by expressionof mRNA containing pGL3 and IRFD1 leaders (outlined on the left) with andwithout an additional stem loop at the 5′ end under differentconditions. Blue (normal conditions) and red (stress conditions) barscorrespond to leaders lacking the stem loop while light blue (normal) andyellow (stress) correspond to leaders with the stem loop. The fold change ofFluc activity in response to stress is indicated above by green arrows.(B) Effect of (CAA)6 addition to the 5′leader of IFRD1 on Fluc activity in response to stress.DOI:http://dx.doi.org/10.7554/eLife.03971.019
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383229&req=5

fig5: Features of the IFRD1 5′ leader required for resistance.(A) Firefly luciferase (Fluc) activity produced by expressionof mRNA containing pGL3 and IRFD1 leaders (outlined on the left) with andwithout an additional stem loop at the 5′ end under differentconditions. Blue (normal conditions) and red (stress conditions) barscorrespond to leaders lacking the stem loop while light blue (normal) andyellow (stress) correspond to leaders with the stem loop. The fold change ofFluc activity in response to stress is indicated above by green arrows.(B) Effect of (CAA)6 addition to the 5′leader of IFRD1 on Fluc activity in response to stress.DOI:http://dx.doi.org/10.7554/eLife.03971.019
Mentions: We observed earlier that most stress resistant mRNAs possess efficiently translateduORFs. We hypothesized that some features of the 5′ leaders upstream of uORFsmay be important for resistance. To address this issue we created two additionalreporters based on control pGL3 and IFRD1, where we added a 5′ terminal stemloop of intermediate stability (Figure 5). Asexpected, the addition of this stem loop resulted in a threefold to fourfold decreasein the activity of both reporters under normal conditions. Interestingly, whenarsenite stress was induced, the SL-IFRD1 construct did not exhibit resistance whiletranslation of SL-PGL3 was downregulated as much as the pGL3 construct. Therefore wepropose that efficient loading of pre-initiator complexes to the uORF is necessaryfor stress resistance in IFRD1. Next we addressed the question of whether thespecific sequence upstream of the IFRD1 uORF is required for regulation. Wesubstituted it with an artificial single stranded (CAA)6 sequence of thesame length. This mutation did not alter stress resistance. Thus, we hypothesizethat, for resistant translation, the uORF has to be preceded with a leader allowing ahigh initiation rate at the uORF.10.7554/eLife.03971.019Figure 5.Features of the IFRD1 5′ leader required for resistance.

Bottom Line: However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response.Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition.Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

View Article: PubMed Central - PubMed

Affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

ABSTRACT
Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

Show MeSH
Related in: MedlinePlus