Limits...
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.

Show MeSH

Related in: MedlinePlus

The absence of GCN2 reduces phosphorylation of eIF2α following nucleofectionMEF cells deficient in the eIF2α kinases, PKR (A-B), PERK (C-D), or GCN2 (E-F), were nucleofected or mock-treated and lysed at indicated times. Phosphorylation of eIF2α was assessed by western blotting (A, C, E). Representative data of 2–4 independent experiments is shown. For 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 (B, D, E). Data displayed is mean ± SEM of n = 2 to 4 experiments. Asterisks indicate P-value <0.05 compared to mock treatment. Double asterisks indicate P-value <0.05 compared to nucleofected WT MEFs.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3345295&req=5

Figure 3: The absence of GCN2 reduces phosphorylation of eIF2α following nucleofectionMEF cells deficient in the eIF2α kinases, PKR (A-B), PERK (C-D), or GCN2 (E-F), were nucleofected or mock-treated and lysed at indicated times. Phosphorylation of eIF2α was assessed by western blotting (A, C, E). Representative data of 2–4 independent experiments is shown. For 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 (B, D, E). Data displayed is mean ± SEM of n = 2 to 4 experiments. Asterisks indicate P-value <0.05 compared to mock treatment. Double asterisks indicate P-value <0.05 compared to nucleofected WT MEFs.

Mentions: Of the four mammalian eIF2α kinases, three, PKR, PERK, and GCN2, are widely distributed in all cell types, whereas HRI is reported to function primarily in erythroid cells.19 Therefore, to identify the eIF2α kinase responsible for nucleofection-induced eIF2α phosphorylation, we took advantage of MEF cell lines created from mice deficient in PKR, PERK, and GCN2. As was done with WT MEFs, cells were nucleofected and eIF2α phosphorylation was assessed by western blotting. Nucleofection of PKR−/− MEF cells resulted in eIF2α phosphorylation comparable to that induced in WT MEF cells (Figure 3A&B), as did nucleofection of PERK−/− MEF cells (Figure 3C&D). Nucleofection also induced eIF2α phosphorylation in GCN2−/− MEF cells, but the peak level of phosphorylation was reduced (Figure 3E&F).


Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.

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

The absence of GCN2 reduces phosphorylation of eIF2α following nucleofectionMEF cells deficient in the eIF2α kinases, PKR (A-B), PERK (C-D), or GCN2 (E-F), were nucleofected or mock-treated and lysed at indicated times. Phosphorylation of eIF2α was assessed by western blotting (A, C, E). Representative data of 2–4 independent experiments is shown. For 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 (B, D, E). Data displayed is mean ± SEM of n = 2 to 4 experiments. Asterisks indicate P-value <0.05 compared to mock treatment. Double asterisks indicate P-value <0.05 compared to nucleofected WT MEFs.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: The absence of GCN2 reduces phosphorylation of eIF2α following nucleofectionMEF cells deficient in the eIF2α kinases, PKR (A-B), PERK (C-D), or GCN2 (E-F), were nucleofected or mock-treated and lysed at indicated times. Phosphorylation of eIF2α was assessed by western blotting (A, C, E). Representative data of 2–4 independent experiments is shown. For 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 (B, D, E). Data displayed is mean ± SEM of n = 2 to 4 experiments. Asterisks indicate P-value <0.05 compared to mock treatment. Double asterisks indicate P-value <0.05 compared to nucleofected WT MEFs.
Mentions: Of the four mammalian eIF2α kinases, three, PKR, PERK, and GCN2, are widely distributed in all cell types, whereas HRI is reported to function primarily in erythroid cells.19 Therefore, to identify the eIF2α kinase responsible for nucleofection-induced eIF2α phosphorylation, we took advantage of MEF cell lines created from mice deficient in PKR, PERK, and GCN2. As was done with WT MEFs, cells were nucleofected and eIF2α phosphorylation was assessed by western blotting. Nucleofection of PKR−/− MEF cells resulted in eIF2α phosphorylation comparable to that induced in WT MEF cells (Figure 3A&B), as did nucleofection of PERK−/− MEF cells (Figure 3C&D). Nucleofection also induced eIF2α phosphorylation in GCN2−/− MEF cells, but the peak level of phosphorylation was reduced (Figure 3E&F).

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