Nucleofection Induces Transient EIF2α Phosphorylation by GCN2 and PERK

Overview

Journal Gene Ther

Date 2012 Feb 4

PMID 22301437

Citations 12

Authors

B R Anderson

K Kariko

D Weissman

Affiliations

Soon will be listed here.

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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References

Kariko K, Buckstein M, Ni H, Weissman D . Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005; 23(2):165-75. DOI: 10.1016/j.immuni.2005.06.008. View

Kariko K, Muramatsu H, Welsh F, Ludwig J, Kato H, Akira S . Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008; 16(11):1833-40. PMC: 2775451. DOI: 10.1038/mt.2008.200. View

Pindel A, Sadler A . The role of protein kinase R in the interferon response. J Interferon Cytokine Res. 2010; 31(1):59-70. DOI: 10.1089/jir.2010.0099. View

Nallagatla S, Toroney R, Bevilacqua P . Regulation of innate immunity through RNA structure and the protein kinase PKR. Curr Opin Struct Biol. 2010; 21(1):119-27. PMC: 3075410. DOI: 10.1016/j.sbi.2010.11.003. View

Gilboa E, Vieweg J . Cancer immunotherapy with mRNA-transfected dendritic cells. Immunol Rev. 2004; 199:251-63. DOI: 10.1111/j.0105-2896.2004.00139.x. View

Van Tendeloo V, Velde A, Van Driessche A, Cools N, Anguille S, Ladell K . Induction of complete and molecular remissions in acute myeloid leukemia by Wilms' tumor 1 antigen-targeted dendritic cell vaccination. Proc Natl Acad Sci U S A. 2010; 107(31):13824-9. PMC: 2922237. DOI: 10.1073/pnas.1008051107. View

Warren L, Manos P, Ahfeldt T, Loh Y, Li H, Lau F . Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010; 7(5):618-30. PMC: 3656821. DOI: 10.1016/j.stem.2010.08.012. View

Silverman J, Ung T, Dever T . Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha. Mol Cell Biol. 1997; 17(7):4146-58. PMC: 232268. DOI: 10.1128/MCB.17.7.4146. View

Vo N, Klein M, Varlamova O, Keller D, Yamamoto T, Goodman R . A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis. Proc Natl Acad Sci U S A. 2005; 102(45):16426-31. PMC: 1283476. DOI: 10.1073/pnas.0508448102. View

10.

Deng J, Harding H, Raught B, Gingras A, Berlanga J, Scheuner D . Activation of GCN2 in UV-irradiated cells inhibits translation. Curr Biol. 2002; 12(15):1279-86. DOI: 10.1016/s0960-9822(02)01037-0. View

11.

Ponsaerts P, van der Sar S, Van Tendeloo V, Jorens P, Berneman Z, Singh P . Highly efficient mRNA-based gene transfer in feeder-free cultured H9 human embryonic stem cells. Cloning Stem Cells. 2005; 6(3):211-6. DOI: 10.1089/clo.2004.6.211. View

12.

Schacht V, Ramirez M, Hong Y, Hirakawa S, Feng D, Harvey N . T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema. EMBO J. 2003; 22(14):3546-56. PMC: 165612. DOI: 10.1093/emboj/cdg342. View

13.

Duncan R, Hershey J . Regulation of initiation factors during translational repression caused by serum depletion. Abundance, synthesis, and turnover rates. J Biol Chem. 1985; 260(9):5486-92. View

14.

Jiang H, Wek R . Phosphorylation of the alpha-subunit of the eukaryotic initiation factor-2 (eIF2alpha) reduces protein synthesis and enhances apoptosis in response to proteasome inhibition. J Biol Chem. 2005; 280(14):14189-202. DOI: 10.1074/jbc.M413660200. View

15.

Hinnebusch A . Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol. 2005; 59:407-50. DOI: 10.1146/annurev.micro.59.031805.133833. View

16.

Yakubov E, Rechavi G, Rozenblatt S, Givol D . Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. Biochem Biophys Res Commun. 2010; 394(1):189-93. DOI: 10.1016/j.bbrc.2010.02.150. View

17.

Perche F, Benvegnu T, Berchel M, Lebegue L, Pichon C, Jaffres P . Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA. Nanomedicine. 2011; 7(4):445-53. DOI: 10.1016/j.nano.2010.12.010. View

18.

Wu S, Hu Y, Wang J, Chatterjee M, Shi Y, Kaufman R . Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum. J Biol Chem. 2002; 277(20):18077-83. DOI: 10.1074/jbc.M110164200. View

19.

Zeenko V, Wang C, Majumder M, Komar A, Snider M, Merrick W . An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation. RNA. 2008; 14(3):593-602. PMC: 2248251. DOI: 10.1261/rna.825008. View

20.

Jousse C, Oyadomari S, Novoa I, Lu P, Zhang Y, Harding H . Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells. J Cell Biol. 2003; 163(4):767-75. PMC: 2173671. DOI: 10.1083/jcb.200308075. View