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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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Related in: MedlinePlus

Nucleofection causes phosphorylation of eIF2α in wild-type cellsWild-type MEF cells were nucleofected or mock-treated and lysed at the indicated times. (A) Phosphorylation of eIF2α was assessed by western blotting with an antibody specific for phosphorylated eIF2α (eIF2αP), and then re-probed for total eIF2α. Representative data from one of three independent experiments is shown. (B) Quantitation of western blot band densities. Values were calculated as the ratio of phosphorylated to total eIF2α and normalized to the values obtained in mock-treated cells at 0.1 hours post-shock. Data displayed is mean ± SEM of n = 3 experiments. Asterisks indicate P-value <0.05 compared to mock treatment.
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Figure 1: Nucleofection causes phosphorylation of eIF2α in wild-type cellsWild-type MEF cells were nucleofected or mock-treated and lysed at the indicated times. (A) Phosphorylation of eIF2α was assessed by western blotting with an antibody specific for phosphorylated eIF2α (eIF2αP), and then re-probed for total eIF2α. Representative data from one of three independent experiments is shown. (B) Quantitation of western blot band densities. Values were calculated as the ratio of phosphorylated to total eIF2α and normalized to the values obtained in mock-treated cells at 0.1 hours post-shock. Data displayed is mean ± SEM of n = 3 experiments. Asterisks indicate P-value <0.05 compared to mock treatment.

Mentions: The impact of nucleofection on translation was first studied in a mouse embryonic fibroblast (MEF) cell line derived from wild-type (WT) C57Bl/6 mice. Nucleofection conditions were optimized according to manufacturer's guidelines, and it was determined that program T-020 provided the best cell survival with high transfection efficiency. WT MEFs were then nucleofected without including any nucleic acid in the transfection mix. As a negative control, cells were mock treated by subjecting them to the same manipulation and buffers but without electric shock. Following nucleofection, eIF2α phosphorylation was assessed by western blotting using an antibody specific for eIF2α phosphorylated at serine 51. As shown in Figure 1, nucleofection induced phosphorylation of eIF2α four-fold over the baseline level present in mock-treated cells.


Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.

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

Nucleofection causes phosphorylation of eIF2α in wild-type cellsWild-type MEF cells were nucleofected or mock-treated and lysed at the indicated times. (A) Phosphorylation of eIF2α was assessed by western blotting with an antibody specific for phosphorylated eIF2α (eIF2αP), and then re-probed for total eIF2α. Representative data from one of three independent experiments is shown. (B) Quantitation of western blot band densities. Values were calculated as the ratio of phosphorylated to total eIF2α and normalized to the values obtained in mock-treated cells at 0.1 hours post-shock. Data displayed is mean ± SEM of n = 3 experiments. Asterisks indicate P-value <0.05 compared to mock treatment.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Nucleofection causes phosphorylation of eIF2α in wild-type cellsWild-type MEF cells were nucleofected or mock-treated and lysed at the indicated times. (A) Phosphorylation of eIF2α was assessed by western blotting with an antibody specific for phosphorylated eIF2α (eIF2αP), and then re-probed for total eIF2α. Representative data from one of three independent experiments is shown. (B) Quantitation of western blot band densities. Values were calculated as the ratio of phosphorylated to total eIF2α and normalized to the values obtained in mock-treated cells at 0.1 hours post-shock. Data displayed is mean ± SEM of n = 3 experiments. Asterisks indicate P-value <0.05 compared to mock treatment.
Mentions: The impact of nucleofection on translation was first studied in a mouse embryonic fibroblast (MEF) cell line derived from wild-type (WT) C57Bl/6 mice. Nucleofection conditions were optimized according to manufacturer's guidelines, and it was determined that program T-020 provided the best cell survival with high transfection efficiency. WT MEFs were then nucleofected without including any nucleic acid in the transfection mix. As a negative control, cells were mock treated by subjecting them to the same manipulation and buffers but without electric shock. Following nucleofection, eIF2α phosphorylation was assessed by western blotting using an antibody specific for eIF2α phosphorylated at serine 51. As shown in Figure 1, nucleofection induced phosphorylation of eIF2α four-fold over the baseline level present in mock-treated cells.

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