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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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Nucleofection reduces translation in a GCN2 and PERK dependent mannerWT or GCN2−/−/PERK−/− MEF cells were nucleofected and then lysed at the indicated time. Luciferase enzymatic activity was measured as relative light units (RLU). Asterisks indicate P-value <0.05 and daggers indicate P-value <0.001 comparing GCN2−/−/PERK−/− to WT MEFs. Data is representative of 3 independent experiments. (A) Cells were transfected with pCMV-luciferase plasmid 24 hours prior to nucleofection. Luciferase activity was normalized to RLU present in non-nucleofected cells (mock) at the same timepoint. Data displayed is mean ± SEM of four replicate wells. (B) Luciferase mRNA was delivered by nucleofection. Data is mean ± SEM of three replicate wells.
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Figure 7: Nucleofection reduces translation in a GCN2 and PERK dependent mannerWT or GCN2−/−/PERK−/− MEF cells were nucleofected and then lysed at the indicated time. Luciferase enzymatic activity was measured as relative light units (RLU). Asterisks indicate P-value <0.05 and daggers indicate P-value <0.001 comparing GCN2−/−/PERK−/− to WT MEFs. Data is representative of 3 independent experiments. (A) Cells were transfected with pCMV-luciferase plasmid 24 hours prior to nucleofection. Luciferase activity was normalized to RLU present in non-nucleofected cells (mock) at the same timepoint. Data displayed is mean ± SEM of four replicate wells. (B) Luciferase mRNA was delivered by nucleofection. Data is mean ± SEM of three replicate wells.

Mentions: The functional relevance of nucleofection-induced eIF2α phosphorylation was assessed by measuring translation following nucleofection. One day prior to nucleofection, WT and GCN2−/−/PERK−/− MEF cells were transfected with a firefly luciferase expression plasmid. Cells were nucleofected in the absence of a nucleic acid and translation was measured by quantitation of luciferase enzyme activity. In WT cells, translation was decreased following nucleofection corresponding to the timeframe of eIF2α phosphorylation. However, nucleofected GCN2−/−/PERK−/− MEFs had a significantly smaller decrease in translation post nucleofection (Figure 7A). Nucleofection, in general, leads to toxicity and cell death, which was apparent in both cell lines as a reduction in translation that continued at 24 hr and beyond.


Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.

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

Nucleofection reduces translation in a GCN2 and PERK dependent mannerWT or GCN2−/−/PERK−/− MEF cells were nucleofected and then lysed at the indicated time. Luciferase enzymatic activity was measured as relative light units (RLU). Asterisks indicate P-value <0.05 and daggers indicate P-value <0.001 comparing GCN2−/−/PERK−/− to WT MEFs. Data is representative of 3 independent experiments. (A) Cells were transfected with pCMV-luciferase plasmid 24 hours prior to nucleofection. Luciferase activity was normalized to RLU present in non-nucleofected cells (mock) at the same timepoint. Data displayed is mean ± SEM of four replicate wells. (B) Luciferase mRNA was delivered by nucleofection. Data is mean ± SEM of three replicate wells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Nucleofection reduces translation in a GCN2 and PERK dependent mannerWT or GCN2−/−/PERK−/− MEF cells were nucleofected and then lysed at the indicated time. Luciferase enzymatic activity was measured as relative light units (RLU). Asterisks indicate P-value <0.05 and daggers indicate P-value <0.001 comparing GCN2−/−/PERK−/− to WT MEFs. Data is representative of 3 independent experiments. (A) Cells were transfected with pCMV-luciferase plasmid 24 hours prior to nucleofection. Luciferase activity was normalized to RLU present in non-nucleofected cells (mock) at the same timepoint. Data displayed is mean ± SEM of four replicate wells. (B) Luciferase mRNA was delivered by nucleofection. Data is mean ± SEM of three replicate wells.
Mentions: The functional relevance of nucleofection-induced eIF2α phosphorylation was assessed by measuring translation following nucleofection. One day prior to nucleofection, WT and GCN2−/−/PERK−/− MEF cells were transfected with a firefly luciferase expression plasmid. Cells were nucleofected in the absence of a nucleic acid and translation was measured by quantitation of luciferase enzyme activity. In WT cells, translation was decreased following nucleofection corresponding to the timeframe of eIF2α phosphorylation. However, nucleofected GCN2−/−/PERK−/− MEFs had a significantly smaller decrease in translation post nucleofection (Figure 7A). Nucleofection, in general, leads to toxicity and cell death, which was apparent in both cell lines as a reduction in translation that continued at 24 hr and beyond.

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