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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 of primary human dendritic cells induces phosphorylation of eIF2α and GCN2Primary human MDDC were nucleofected or mock-treated and lysed at the indicated times. Phosphorylation of eIF2α and GCN2 was assessed by western blotting. For quantitation of western blot band densities, values were calculated as the ratio of phosphorylated eIF2α or GCN2 to total eIF2α and normalized to the values obtained in mock-treated cells at 0.2 hours post-shock.
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Figure 6: Nucleofection of primary human dendritic cells induces phosphorylation of eIF2α and GCN2Primary human MDDC were nucleofected or mock-treated and lysed at the indicated times. Phosphorylation of eIF2α and GCN2 was assessed by western blotting. For quantitation of western blot band densities, values were calculated as the ratio of phosphorylated eIF2α or GCN2 to total eIF2α and normalized to the values obtained in mock-treated cells at 0.2 hours post-shock.

Mentions: Nucleofection is increasingly used to deliver mRNA to human dendritic cells (DCs) and T cells and has entered clinical trials. Therefore, we assessed the influence of nucleofection on eIF2α phosphorylation in primary human monocyte-derived dendritic cells (hMDDCs). As seen in MEF cell lines, nucleofection induced phosphorylation of eIF2α in hMDDCs (Figure 6). Furthermore, phosphorylation of GCN2 was also induced in hMDDC following nucleofection but not mock treatment (Figure 6).


Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK.

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

Nucleofection of primary human dendritic cells induces phosphorylation of eIF2α and GCN2Primary human MDDC were nucleofected or mock-treated and lysed at the indicated times. Phosphorylation of eIF2α and GCN2 was assessed by western blotting. For quantitation of western blot band densities, values were calculated as the ratio of phosphorylated eIF2α or GCN2 to total eIF2α and normalized to the values obtained in mock-treated cells at 0.2 hours post-shock.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Nucleofection of primary human dendritic cells induces phosphorylation of eIF2α and GCN2Primary human MDDC were nucleofected or mock-treated and lysed at the indicated times. Phosphorylation of eIF2α and GCN2 was assessed by western blotting. For quantitation of western blot band densities, values were calculated as the ratio of phosphorylated eIF2α or GCN2 to total eIF2α and normalized to the values obtained in mock-treated cells at 0.2 hours post-shock.
Mentions: Nucleofection is increasingly used to deliver mRNA to human dendritic cells (DCs) and T cells and has entered clinical trials. Therefore, we assessed the influence of nucleofection on eIF2α phosphorylation in primary human monocyte-derived dendritic cells (hMDDCs). As seen in MEF cell lines, nucleofection induced phosphorylation of eIF2α in hMDDCs (Figure 6). Furthermore, phosphorylation of GCN2 was also induced in hMDDC following nucleofection but not mock treatment (Figure 6).

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