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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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(A) Resistance of additional reporters with 5′leaders of ATF5, UCP2, PTP4A1, and SLC35A4 to arsenite treatment.(B and C) Resistance of different 5′leaders to arsenite treatment in HEK293T cells (B) and toarsenite or dithiothreitol (DTT) treatment in Huh7 cells(C). Western blot on panel B demonstrates ubiquitousphosphorylation of eukaryotic initiation factor 2 (eIF2) upon DTTtreatment.DOI:http://dx.doi.org/10.7554/eLife.03971.015
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fig4s1: (A) Resistance of additional reporters with 5′leaders of ATF5, UCP2, PTP4A1, and SLC35A4 to arsenite treatment.(B and C) Resistance of different 5′leaders to arsenite treatment in HEK293T cells (B) and toarsenite or dithiothreitol (DTT) treatment in Huh7 cells(C). Western blot on panel B demonstrates ubiquitousphosphorylation of eukaryotic initiation factor 2 (eIF2) upon DTTtreatment.DOI:http://dx.doi.org/10.7554/eLife.03971.015

Mentions: Under normal conditions the translation of mRNAs bearing the 5′ leaders ofIFRD1, PPP1R15B, UCP2, andPTP4A1 was about sevenfold lower than that of the control mRNAwith the simple non-specific leader (pGL3), whereas SLC35A4 was evenlower (Figure 4A and Figure 4—figure supplement 1A). Arsenite treatmentresulted in significant inhibition of pGL3 and control Rluc translation, whiletranslation of other mRNAs did not change considerably and even slightly increasedfor SLC35A4 and HCV IRES. Similar results were observed in the Huh7 hepatocarcinomacell line (Figure 4—figure supplement1C). To address the effect of arsenite treatment on ongoing translation,which may be more relevant than conditions applied for ribosome profiling, reportermRNAs were transfected and 1 hr later cells were treated with either non-specifictranslational inhibitor cycloheximide or arsenite. As expected, both inhibitorsefficiently blocked translation of control Rluc mRNA but only cycloheximide was ableto arrest translation driven by leaders of resistant mRNAs. Surprisingly, duringarsenite treatment the reporter mRNA with the SLC35A4 5′ leader was able toproduce 15 times more luciferase than after treatment with cycloheximide (Figure 4—figure supplement 2).10.7554/eLife.03971.014Figure 4.Upstream open reading frame (uORF) involvement in modulation of IFRD1and PPP1R15B mRNAs stress 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)

(A) Resistance of additional reporters with 5′leaders of ATF5, UCP2, PTP4A1, and SLC35A4 to arsenite treatment.(B and C) Resistance of different 5′leaders to arsenite treatment in HEK293T cells (B) and toarsenite or dithiothreitol (DTT) treatment in Huh7 cells(C). Western blot on panel B demonstrates ubiquitousphosphorylation of eukaryotic initiation factor 2 (eIF2) upon DTTtreatment.DOI:http://dx.doi.org/10.7554/eLife.03971.015
© Copyright Policy
Related In: Results  -  Collection

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

fig4s1: (A) Resistance of additional reporters with 5′leaders of ATF5, UCP2, PTP4A1, and SLC35A4 to arsenite treatment.(B and C) Resistance of different 5′leaders to arsenite treatment in HEK293T cells (B) and toarsenite or dithiothreitol (DTT) treatment in Huh7 cells(C). Western blot on panel B demonstrates ubiquitousphosphorylation of eukaryotic initiation factor 2 (eIF2) upon DTTtreatment.DOI:http://dx.doi.org/10.7554/eLife.03971.015
Mentions: Under normal conditions the translation of mRNAs bearing the 5′ leaders ofIFRD1, PPP1R15B, UCP2, andPTP4A1 was about sevenfold lower than that of the control mRNAwith the simple non-specific leader (pGL3), whereas SLC35A4 was evenlower (Figure 4A and Figure 4—figure supplement 1A). Arsenite treatmentresulted in significant inhibition of pGL3 and control Rluc translation, whiletranslation of other mRNAs did not change considerably and even slightly increasedfor SLC35A4 and HCV IRES. Similar results were observed in the Huh7 hepatocarcinomacell line (Figure 4—figure supplement1C). To address the effect of arsenite treatment on ongoing translation,which may be more relevant than conditions applied for ribosome profiling, reportermRNAs were transfected and 1 hr later cells were treated with either non-specifictranslational inhibitor cycloheximide or arsenite. As expected, both inhibitorsefficiently blocked translation of control Rluc mRNA but only cycloheximide was ableto arrest translation driven by leaders of resistant mRNAs. Surprisingly, duringarsenite treatment the reporter mRNA with the SLC35A4 5′ leader was able toproduce 15 times more luciferase than after treatment with cycloheximide (Figure 4—figure supplement 2).10.7554/eLife.03971.014Figure 4.Upstream open reading frame (uORF) involvement in modulation of IFRD1and PPP1R15B mRNAs stress 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