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The eIF4G homolog DAP5/p97 supports the translation of select mRNAs during endoplasmic reticulum stress.

Lewis SM, Cerquozzi S, Graber TE, Ungureanu NH, Andrews M, Holcik M - Nucleic Acids Res. (2007)

Bottom Line: We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES.Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop.In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis Research Centre, Children's Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario, K1H 8L1 Canada.

ABSTRACT
DAP5/p97 is a member of the eIF4G family of translation initiation factors that has been suggested to play an important role in the translation of select messenger RNA molecules. We have shown previously that the caspase-cleaved form of DAP5/p97, termed p86, is required for the induction of the endoplasmic reticulum (ER)-stress-responsive internal ribosome entry site (IRES) of the caspase inhibitor HIAP2. We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES. Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop. In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97. Significantly, the induction of DAP5/p97 during ER stress is caspase-independent, whereas the induction of HIAP2 requires proteolytic processing of DAP5/p97. Thus, DAP5/p97 is a translational activator that selectively modulates translation of specific mRNAs during conditions of cellular stress in both a caspase-dependent and caspase-independent manner.

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DAP5/p97 and HIAP2 mRNAs remain associated with polysomes during ER stress. (A) Polysome profiles from tunicamycin (Tm; 8.5 μM) or DMSO-treated cells were generated as described in Materials and Methods. (B) The polysomal distribution of actin and BiP mRNAs (shown as percentage of total RNA) was used to confirm the induction of ER stress and validate the qRT-PCR approach. Mean ± SEM of two independent experiments performed in triplicates is shown. The images of agarose gels on the right are representative of results from RT-PCR reactions that were run in parallel. (C) The levels of DAP5/p97 and HIAP2 mRNAs that remain associated with polysomes in Tm-treated cells (relative to actin) were determined as in (B). The polysomal association of ATF4, which is known to be translated during ER stress, was used as positive control; NF90, which is not known to be translated during ER stress, was used as negative control. Average ± SD of two independent experiments performed in triplicate. DMSO treated samples were set as 100.
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Figure 2: DAP5/p97 and HIAP2 mRNAs remain associated with polysomes during ER stress. (A) Polysome profiles from tunicamycin (Tm; 8.5 μM) or DMSO-treated cells were generated as described in Materials and Methods. (B) The polysomal distribution of actin and BiP mRNAs (shown as percentage of total RNA) was used to confirm the induction of ER stress and validate the qRT-PCR approach. Mean ± SEM of two independent experiments performed in triplicates is shown. The images of agarose gels on the right are representative of results from RT-PCR reactions that were run in parallel. (C) The levels of DAP5/p97 and HIAP2 mRNAs that remain associated with polysomes in Tm-treated cells (relative to actin) were determined as in (B). The polysomal association of ATF4, which is known to be translated during ER stress, was used as positive control; NF90, which is not known to be translated during ER stress, was used as negative control. Average ± SD of two independent experiments performed in triplicate. DMSO treated samples were set as 100.

Mentions: Induction of ER stress by various triggers results in enhanced translation of the inhibitor of apoptosis (IAP) protein HIAP2 in various cell types (11,21). In addition, induction of ER stress leads to caspase-dependent cleavage of the DAP5/p97 protein, a member of the eIF4G family of initiation factors, to generate a p86 isoform that specifically enhances translation of HIAP2 via an IRES element located within the 5′ UTR of HIAP2 mRNA (11). While investigating the role of DAP5/p97 in ER stress, we have noticed that the levels of DAP5/p97 are also elevated following induction of ER stress by tunicamycin (an inhibitor of protein glycosylation) or thapsigargin (an inhibitor of ER calcium pump) (Figure 1 and data not shown). The induction of DAP5/p97 expression by tunicamycin was dose-dependent and paralleled the expression pattern of known ER stress-inducible genes GRP78/Bip and HIAP2 (Figure 1A). Examination of DAP5/p97 and HIAP2 mRNA levels from treated and untreated samples by quantitative RT-PCR revealed that the levels of mRNA did not change following drug treatment [Figure 2C and (11)]. These data indicate that the observed changes in DAP5/p97 protein levels are likely due to translational up-regulation.Figure 1.


The eIF4G homolog DAP5/p97 supports the translation of select mRNAs during endoplasmic reticulum stress.

Lewis SM, Cerquozzi S, Graber TE, Ungureanu NH, Andrews M, Holcik M - Nucleic Acids Res. (2007)

DAP5/p97 and HIAP2 mRNAs remain associated with polysomes during ER stress. (A) Polysome profiles from tunicamycin (Tm; 8.5 μM) or DMSO-treated cells were generated as described in Materials and Methods. (B) The polysomal distribution of actin and BiP mRNAs (shown as percentage of total RNA) was used to confirm the induction of ER stress and validate the qRT-PCR approach. Mean ± SEM of two independent experiments performed in triplicates is shown. The images of agarose gels on the right are representative of results from RT-PCR reactions that were run in parallel. (C) The levels of DAP5/p97 and HIAP2 mRNAs that remain associated with polysomes in Tm-treated cells (relative to actin) were determined as in (B). The polysomal association of ATF4, which is known to be translated during ER stress, was used as positive control; NF90, which is not known to be translated during ER stress, was used as negative control. Average ± SD of two independent experiments performed in triplicate. DMSO treated samples were set as 100.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: DAP5/p97 and HIAP2 mRNAs remain associated with polysomes during ER stress. (A) Polysome profiles from tunicamycin (Tm; 8.5 μM) or DMSO-treated cells were generated as described in Materials and Methods. (B) The polysomal distribution of actin and BiP mRNAs (shown as percentage of total RNA) was used to confirm the induction of ER stress and validate the qRT-PCR approach. Mean ± SEM of two independent experiments performed in triplicates is shown. The images of agarose gels on the right are representative of results from RT-PCR reactions that were run in parallel. (C) The levels of DAP5/p97 and HIAP2 mRNAs that remain associated with polysomes in Tm-treated cells (relative to actin) were determined as in (B). The polysomal association of ATF4, which is known to be translated during ER stress, was used as positive control; NF90, which is not known to be translated during ER stress, was used as negative control. Average ± SD of two independent experiments performed in triplicate. DMSO treated samples were set as 100.
Mentions: Induction of ER stress by various triggers results in enhanced translation of the inhibitor of apoptosis (IAP) protein HIAP2 in various cell types (11,21). In addition, induction of ER stress leads to caspase-dependent cleavage of the DAP5/p97 protein, a member of the eIF4G family of initiation factors, to generate a p86 isoform that specifically enhances translation of HIAP2 via an IRES element located within the 5′ UTR of HIAP2 mRNA (11). While investigating the role of DAP5/p97 in ER stress, we have noticed that the levels of DAP5/p97 are also elevated following induction of ER stress by tunicamycin (an inhibitor of protein glycosylation) or thapsigargin (an inhibitor of ER calcium pump) (Figure 1 and data not shown). The induction of DAP5/p97 expression by tunicamycin was dose-dependent and paralleled the expression pattern of known ER stress-inducible genes GRP78/Bip and HIAP2 (Figure 1A). Examination of DAP5/p97 and HIAP2 mRNA levels from treated and untreated samples by quantitative RT-PCR revealed that the levels of mRNA did not change following drug treatment [Figure 2C and (11)]. These data indicate that the observed changes in DAP5/p97 protein levels are likely due to translational up-regulation.Figure 1.

Bottom Line: We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES.Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop.In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis Research Centre, Children's Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario, K1H 8L1 Canada.

ABSTRACT
DAP5/p97 is a member of the eIF4G family of translation initiation factors that has been suggested to play an important role in the translation of select messenger RNA molecules. We have shown previously that the caspase-cleaved form of DAP5/p97, termed p86, is required for the induction of the endoplasmic reticulum (ER)-stress-responsive internal ribosome entry site (IRES) of the caspase inhibitor HIAP2. We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES. Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop. In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97. Significantly, the induction of DAP5/p97 during ER stress is caspase-independent, whereas the induction of HIAP2 requires proteolytic processing of DAP5/p97. Thus, DAP5/p97 is a translational activator that selectively modulates translation of specific mRNAs during conditions of cellular stress in both a caspase-dependent and caspase-independent manner.

Show MeSH
Related in: MedlinePlus