Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK
Identifieur interne : 004710 ( Pmc/Curation ); précédent : 004709; suivant : 004711Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK
Auteurs : Bart R. Anderson [États-Unis] ; Katalin Karik [États-Unis] ; Drew Weissman [États-Unis]Source :
- Gene therapy [ 0969-7128 ] ; 2012.
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 siRNA. Unlike DNA and siRNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined factors important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of delivered nucleic acid. We studied the involvement of 3 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 protein kinase RNA-activated (PKR)-like ER 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 siRNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.
Url:
DOI: 10.1038/gt.2012.5
PubMed: 22301437
PubMed Central: 3345295
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK</title><author><name sortKey="Anderson, Bart R" sort="Anderson, Bart R" uniqKey="Anderson B" first="Bart R." last="Anderson">Bart R. Anderson</name><affiliation wicri:level="1"><nlm:aff id="A1">Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Medicine, University of Pennsylvania, Philadelphia, PA</wicri:regionArea></affiliation></author><author><name sortKey="Karik, Katalin" sort="Karik, Katalin" uniqKey="Karik K" first="Katalin" last="Karik">Katalin Karik</name><affiliation wicri:level="1"><nlm:aff id="A2">Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA</wicri:regionArea></affiliation></author><author><name sortKey="Weissman, Drew" sort="Weissman, Drew" uniqKey="Weissman D" first="Drew" last="Weissman">Drew Weissman</name><affiliation wicri:level="1"><nlm:aff id="A1">Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Medicine, University of Pennsylvania, Philadelphia, PA</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22301437</idno><idno type="pmc">3345295</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345295</idno><idno type="RBID">PMC:3345295</idno><idno type="doi">10.1038/gt.2012.5</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">004711</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">004711</idno><idno type="wicri:Area/Pmc/Curation">004710</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">004710</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK</title><author><name sortKey="Anderson, Bart R" sort="Anderson, Bart R" uniqKey="Anderson B" first="Bart R." last="Anderson">Bart R. Anderson</name><affiliation wicri:level="1"><nlm:aff id="A1">Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Medicine, University of Pennsylvania, Philadelphia, PA</wicri:regionArea></affiliation></author><author><name sortKey="Karik, Katalin" sort="Karik, Katalin" uniqKey="Karik K" first="Katalin" last="Karik">Katalin Karik</name><affiliation wicri:level="1"><nlm:aff id="A2">Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA</wicri:regionArea></affiliation></author><author><name sortKey="Weissman, Drew" sort="Weissman, Drew" uniqKey="Weissman D" first="Drew" last="Weissman">Drew Weissman</name><affiliation wicri:level="1"><nlm:aff id="A1">Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Medicine, University of Pennsylvania, Philadelphia, PA</wicri:regionArea></affiliation></author></analytic><series><title level="j">Gene therapy</title><idno type="ISSN">0969-7128</idno><idno type="eISSN">1476-5462</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="P1">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 siRNA. Unlike DNA and siRNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined factors important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of delivered nucleic acid. We studied the involvement of 3 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 protein kinase RNA-activated (PKR)-like ER 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 siRNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Gresch, O" uniqKey="Gresch O">O Gresch</name></author><author><name sortKey="Engel, Fb" uniqKey="Engel F">FB Engel</name></author><author><name sortKey="Nesic, D" uniqKey="Nesic D">D Nesic</name></author><author><name sortKey="Tran, Tt" uniqKey="Tran T">TT Tran</name></author><author><name sortKey="England, Hm" uniqKey="England H">HM England</name></author><author><name sortKey="Hickman, Es" uniqKey="Hickman E">ES Hickman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hamm, A" uniqKey="Hamm A">A Hamm</name></author><author><name sortKey="Krott, N" uniqKey="Krott N">N Krott</name></author><author><name sortKey="Breibach, I" uniqKey="Breibach I">I 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<pmc-dir>properties manuscript</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-journal-id">9421525</journal-id><journal-id journal-id-type="pubmed-jr-id">8603</journal-id><journal-id journal-id-type="nlm-ta">Gene Ther</journal-id><journal-id journal-id-type="iso-abbrev">Gene Ther.</journal-id><journal-title-group><journal-title>Gene therapy</journal-title></journal-title-group><issn pub-type="ppub">0969-7128</issn><issn pub-type="epub">1476-5462</issn></journal-meta><article-meta><article-id pub-id-type="pmid">22301437</article-id><article-id pub-id-type="pmc">3345295</article-id><article-id pub-id-type="doi">10.1038/gt.2012.5</article-id><article-id pub-id-type="manuscript">NIHMS332400</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Nucleofection induces transient eIF2α phosphorylation by GCN2 and PERK</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Anderson</surname><given-names>Bart R.</given-names></name><degrees>Ph.D.</degrees><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Karikó</surname><given-names>Katalin</given-names></name><degrees>Ph.D.</degrees><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Weissman</surname><given-names>Drew</given-names></name><degrees>M.D., Ph.D.</degrees><xref ref-type="aff" rid="A1">1</xref><xref rid="FN1" ref-type="author-notes">*</xref></contrib></contrib-group><aff id="A1"><label>1</label>Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA</aff><aff id="A2"><label>2</label>Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA</aff><author-notes><corresp id="FN1"><label>*</label>To whom correspondence should be addressed. 522 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6073, <email>dreww@mail.med.upenn.edu</email>, Tel: (215) 614-0291; Fax: (215) 349-5111</corresp></author-notes><pub-date pub-type="nihms-submitted"><day>19</day><month>10</month><year>2011</year></pub-date><pub-date pub-type="epub"><day>02</day><month>2</month><year>2012</year></pub-date><pub-date pub-type="ppub"><month>2</month><year>2013</year></pub-date><pub-date pub-type="pmc-release"><day>01</day><month>8</month><year>2013</year></pub-date><volume>20</volume><issue>2</issue><fpage>136</fpage><lpage>142</lpage><pmc-comment>elocation-id from pubmed: 10.1038/gt.2012.5</pmc-comment>
<permissions><license><license-p>Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: <ext-link ext-link-type="uri" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</ext-link></license-p></license></permissions><abstract><p id="P1">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 siRNA. Unlike DNA and siRNA, mRNA is subject to rapid degradation, which necessitates instant early translation following mRNA delivery. We examined factors important in translation following nucleofection and observed rapid phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) following nucleofection, which occurred in the absence of delivered nucleic acid. We studied the involvement of 3 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 protein kinase RNA-activated (PKR)-like ER 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 siRNA. Strategies to circumvent eIF2α phosphorylation and other downstream effects of activating GCN2 and PERK will facilitate further advancement of nucleic acid-based therapies.</p></abstract><kwd-group><kwd>Nucleofection</kwd><kwd>mRNA therapy</kwd><kwd>eIF2α</kwd><kwd>PERK</kwd><kwd>GCN2</kwd></kwd-group></article-meta></front><floats-group><fig id="F1" orientation="portrait" position="float"><label>Figure 1</label><caption><title>Nucleofection causes phosphorylation of eIF2α in wild-type cells</title><p>Wild-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 <italic>n</italic> = 3 experiments. Asterisks indicate P-value <0.05 compared to mock treatment.</p></caption><graphic xlink:href="nihms332400f1"></graphic></fig><fig id="F2" orientation="portrait" position="float"><label>Figure 2</label><caption><title>Cationic polymer and lipid transfection reagents do not induce phosphorylation of eIF2α in wild-type cells</title><p>Wild-type MEF cells were treated with the indicated transfection reagent or mock-treated and lysed at indicated time points. (A) Phosphorylation of eIF2α was assessed by western blotting. Representative data from one of two 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 <italic>n</italic> = 2 experiments.</p></caption><graphic xlink:href="nihms332400f2"></graphic></fig><fig id="F3" orientation="portrait" position="float"><label>Figure 3</label><caption><title>The absence of GCN2 reduces phosphorylation of eIF2α following nucleofection</title><p>MEF 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 <italic>n</italic> = 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.</p></caption><graphic xlink:href="nihms332400f3"></graphic></fig><fig id="F4" orientation="portrait" position="float"><label>Figure 4</label><caption><title>Nucleofection induces GCN2 phosphorylation in WT cells</title><p>WT MEF cells were nucleofected or mock-treated and then lysed at the indicated time points. (A) Phosphorylation of GCN2 was assessed by western blotting. Representative data of three independent experiments is shown. (B) Quantitation of western blot band densities. Values were normalized to the values obtained in mock-treated cells at 0.1 hours post-shock. Data displayed is mean ± SEM of <italic>n</italic> = 3 experiments. Asterisk indicates P-value <0.05 compared to mock treatment.</p></caption><graphic xlink:href="nihms332400f4"></graphic></fig><fig id="F5" orientation="portrait" position="float"><label>Figure 5</label><caption><title>Phosphorylation of eIF2α is mediated by both GCN2 and PERK following nucleofection</title><p>GCN2<sup>−/−</sup>/PERK<sup>−/−</sup> MEF cells were nucleofected without nucleic acid (Nucleofected), mock-treated, or nucleofected with poly(I:C), and then lysed at the indicated times. Phosphorylation of eIF2α was assessed by western blotting. Representative data from one of three independent experiments is shown.</p></caption><graphic xlink:href="nihms332400f5"></graphic></fig><fig id="F6" orientation="portrait" position="float"><label>Figure 6</label><caption><title>Nucleofection of primary human dendritic cells induces phosphorylation of eIF2α and GCN2</title><p>Primary 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.</p></caption><graphic xlink:href="nihms332400f6"></graphic></fig><fig id="F7" orientation="portrait" position="float"><label>Figure 7</label><caption><title>Nucleofection reduces translation in a GCN2 and PERK dependent manner</title><p>WT or GCN2<sup>−/−</sup>/PERK<sup>−/−</sup> 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<sup>−/−</sup>/PERK<sup>−/−</sup> 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.</p></caption><graphic xlink:href="nihms332400f7"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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