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Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.

Anderson BR, Karikó K, Weissman D - Gene Ther. (2012)

Bottom Line: A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance.Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA.Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6073, USA.

ABSTRACT
Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.

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Phosphorylation of eIF2α is mediated by both GCN2 and PERK following nucleofectionGCN2−/−/PERK−/− MEF cells were nucleofected without nucleic acid (Nucleofected), mock-treated, or nucleofected with poly(I:C), and then lysed at the indicated times. Phosphorylation of eIF2α was assessed by western blotting. Representative data from one of three independent experiments is shown.
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Figure 5: Phosphorylation of eIF2α is mediated by both GCN2 and PERK following nucleofectionGCN2−/−/PERK−/− MEF cells were nucleofected without nucleic acid (Nucleofected), mock-treated, or nucleofected with poly(I:C), and then lysed at the indicated times. Phosphorylation of eIF2α was assessed by western blotting. Representative data from one of three independent experiments is shown.

Mentions: The removal of a single eIF2α kinase did not fully prevent nucleofection-induced eIF2α phosphorylation, suggesting the possibility that multiple kinases are involved. Therefore, we assessed phosphorylation of eIF2α following nucleofection of MEF cells derived from a dual-knockout GCN2−/−/PERK−/− mouse on a C57Bl/6 background. No eIF2α phosphorylation was observed in nucleofected GCN2−/−/PERK−/− MEF cells (Figure 5). An apparent lack of any baseline eIF2α phosphorylation that was seen in WT or single knockout MEFs was observed in these cells. The dual knockout cells responded to poly(I:C) demonstrating functional PKR and the ability of their eIF2α to be phosphorylated (Figure 5). These data demonstrated that both GCN2 and PERK were required for nucleofection-induced phosphorylation of eIF2α.


Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.

Anderson BR, Karikó K, Weissman D - Gene Ther. (2012)

Phosphorylation of eIF2α is mediated by both GCN2 and PERK following nucleofectionGCN2−/−/PERK−/− MEF cells were nucleofected without nucleic acid (Nucleofected), mock-treated, or nucleofected with poly(I:C), and then lysed at the indicated times. Phosphorylation of eIF2α was assessed by western blotting. Representative data from one of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3345295&req=5

Figure 5: Phosphorylation of eIF2α is mediated by both GCN2 and PERK following nucleofectionGCN2−/−/PERK−/− MEF cells were nucleofected without nucleic acid (Nucleofected), mock-treated, or nucleofected with poly(I:C), and then lysed at the indicated times. Phosphorylation of eIF2α was assessed by western blotting. Representative data from one of three independent experiments is shown.
Mentions: The removal of a single eIF2α kinase did not fully prevent nucleofection-induced eIF2α phosphorylation, suggesting the possibility that multiple kinases are involved. Therefore, we assessed phosphorylation of eIF2α following nucleofection of MEF cells derived from a dual-knockout GCN2−/−/PERK−/− mouse on a C57Bl/6 background. No eIF2α phosphorylation was observed in nucleofected GCN2−/−/PERK−/− MEF cells (Figure 5). An apparent lack of any baseline eIF2α phosphorylation that was seen in WT or single knockout MEFs was observed in these cells. The dual knockout cells responded to poly(I:C) demonstrating functional PKR and the ability of their eIF2α to be phosphorylated (Figure 5). These data demonstrated that both GCN2 and PERK were required for nucleofection-induced phosphorylation of eIF2α.

Bottom Line: A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance.Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA.Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6073, USA.

ABSTRACT
Nucleofection permits efficient transfection even with difficult cell types such as primary and non-dividing cells, and is used to deliver various nucleic acids, including DNA, mRNA, and small interfering RNA. Unlike DNA and small interfering RNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined the factors that are important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of the delivered nucleic acid. We studied the involvement of three ubiquitous kinases capable of phosphorylating eIF2α in mammalian cells and identified that nucleofection-mediated phosphorylation of eIF2α was dependent on general control non-derepressible 2 (GCN2) and RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) but not PKR. A reduction in translation due to eIF2α phosphorylation was observed post nucleofection, demonstrating functional significance. Understanding the impact of nucleofection on translational machinery has important implications for therapeutics currently under development based on the delivery of mRNA, DNA, and small interfering RNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.

Show MeSH
Related in: MedlinePlus