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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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Top: Effect of GADD34-Flag (PPP1R15A) overexpression on activity offirefly luciferase under control of the IFRD1 mRNA leader (Fluc, greenbars) and on mRNA encoding Renilla luciferase (Rluc,light pink bars).Bottom: Western blots showing the presence of the GADD34-Flag proteinproduct and the phosphorylation level of eukaryotic initiation factor 2(eIF2).DOI:http://dx.doi.org/10.7554/eLife.03971.018
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fig4s4: Top: Effect of GADD34-Flag (PPP1R15A) overexpression on activity offirefly luciferase under control of the IFRD1 mRNA leader (Fluc, greenbars) and on mRNA encoding Renilla luciferase (Rluc,light pink bars).Bottom: Western blots showing the presence of the GADD34-Flag proteinproduct and the phosphorylation level of eukaryotic initiation factor 2(eIF2).DOI:http://dx.doi.org/10.7554/eLife.03971.018

Mentions: It was conceivable that translational resistance was due to side effects of arsenitetreatment rather than its direct effect of eIF2 inactivation. To directly address theimpact of eIF2 phosphorylation on reporter mRNA translation during arsenite stress,we carried out an experiment where cells were pre-transfected with a plasmid encodingthe full length human PPP1R15A (a.k.a. GADD34) phosphatase subunit which is able toreverse eIF2 phosphorylation (Brush et al.,2003). Arsenite-induced eIF2 phosphorylation, as expected, was almostcompletely alleviated in the presence of GADD34 (residual phosphorylation probablyreflects less than 100% efficient plasmid transfection), see Figure 4—figure supplement 4. As a result, thedownregulation of control Rluc mRNA was only twofold in comparison with a more thansixfold reduction in cells not transfected with the GADD34 plasmid. Translation ofthe IFRD1 reporter was not affected under either condition. We therefore concludedthat translational inhibition caused by arsenite treatment is predominantly due tothe phosphorylation of eIF2. Also, we treated cells with 2.5 mM dithiothreitol (DTT),triggers the Unfolded Protein Response and results in eIF2 phosphorylation (Prostko et al., 1993). We found that theleaders of IFRD1 and PPP1R15B provide translational resistance under these conditionsas well (Figure 4—figure supplement1B).


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)

Top: Effect of GADD34-Flag (PPP1R15A) overexpression on activity offirefly luciferase under control of the IFRD1 mRNA leader (Fluc, greenbars) and on mRNA encoding Renilla luciferase (Rluc,light pink bars).Bottom: Western blots showing the presence of the GADD34-Flag proteinproduct and the phosphorylation level of eukaryotic initiation factor 2(eIF2).DOI:http://dx.doi.org/10.7554/eLife.03971.018
© Copyright Policy
Related In: Results  -  Collection

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

fig4s4: Top: Effect of GADD34-Flag (PPP1R15A) overexpression on activity offirefly luciferase under control of the IFRD1 mRNA leader (Fluc, greenbars) and on mRNA encoding Renilla luciferase (Rluc,light pink bars).Bottom: Western blots showing the presence of the GADD34-Flag proteinproduct and the phosphorylation level of eukaryotic initiation factor 2(eIF2).DOI:http://dx.doi.org/10.7554/eLife.03971.018
Mentions: It was conceivable that translational resistance was due to side effects of arsenitetreatment rather than its direct effect of eIF2 inactivation. To directly address theimpact of eIF2 phosphorylation on reporter mRNA translation during arsenite stress,we carried out an experiment where cells were pre-transfected with a plasmid encodingthe full length human PPP1R15A (a.k.a. GADD34) phosphatase subunit which is able toreverse eIF2 phosphorylation (Brush et al.,2003). Arsenite-induced eIF2 phosphorylation, as expected, was almostcompletely alleviated in the presence of GADD34 (residual phosphorylation probablyreflects less than 100% efficient plasmid transfection), see Figure 4—figure supplement 4. As a result, thedownregulation of control Rluc mRNA was only twofold in comparison with a more thansixfold reduction in cells not transfected with the GADD34 plasmid. Translation ofthe IFRD1 reporter was not affected under either condition. We therefore concludedthat translational inhibition caused by arsenite treatment is predominantly due tothe phosphorylation of eIF2. Also, we treated cells with 2.5 mM dithiothreitol (DTT),triggers the Unfolded Protein Response and results in eIF2 phosphorylation (Prostko et al., 1993). We found that theleaders of IFRD1 and PPP1R15B provide translational resistance under these conditionsas well (Figure 4—figure supplement1B).

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