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Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation - PubMed

Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation

Bart R Anderson et al. Nucleic Acids Res. 2010 Sep.

Abstract

Previous studies have shown that the translation level of in vitro transcribed messenger RNA (mRNA) is enhanced when its uridines are replaced with pseudouridines; however, the reason for this enhancement has not been identified. Here, we demonstrate that in vitro transcripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2α), and inhibits translation. In contrast, in vitro transcribed mRNAs containing pseudouridine activate PKR to a lesser degree, and translation of pseudouridine-containing mRNAs is not repressed. RNA pull-down assays demonstrate that mRNA containing uridine is bound by PKR more efficiently than mRNA with pseudouridine. Finally, the role of PKR is validated by showing that pseudouridine- and uridine-containing RNAs were translated equally in PKR knockout cells. These results indicate that the enhanced translation of mRNAs containing pseudouridine, compared to those containing uridine, is mediated by decreased activation of PKR.

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Figures

Figure 1.
Figure 1.

Translational inhibition by unmodified in vitro transcribed mRNA. (A) In vitro transcribed mRNAs encoding Renilla luciferase (Ren) and firefly luciferase (Luc) were synthesized with and without Ψ modifications then mixed (1 : 1 mass ratio) as indicated. The mixed mRNA was complexed with lipofectin and added to HEK293T cells seeded in 96-well plates (0.25 µg RNA/well). Cells were lysed 4 h after transfection and dual luciferase measurements were performed in aliquots (1/20th) of the lysates. Values presented are normalized to cells transfected with Ren and Luc mRNAs when both contained Ψ modifications. Error bars indicate the standard error of n = 3 samples. (B) Unmodified or pseudouridine-containing RNA was delivered to HEK293T cells by lipofection. Cells were subsequently incubated with 35S-methionine/cysteine supplemented medium, lysed, and proteins were TCA precipitated. Data are presented as percentage of counts obtained from mock transfected cells. Data shown are mean values from three independent experiments ± SEM.

Figure 2.
Figure 2.

Activation of purified PKR by in vitro transcribed RNA. Purified PKR was incubated with γ-32P-ATP and in vitro transcribed mRNA for 10 min. Reaction products were separated by SDS–PAGE and imaged using phosphor storage radiography. Unmodified or Ψ-containing mRNAs encoding firefly luciferase contained triphosphates (ppp) or cap at their 5′-ends. Complete capping of RNA was achieved post-transcriptionally using vaccinia capping enzyme. Concentration of mRNA in reactions was 3.1, 6.2, 12.5 and 25 µg/ml. Quantified phosphorylation is presented as a bar graph below each band. Values were normalized to those obtained with 25 µg/ml uncapped, unmodified RNA. No RNA (−) and 79 bp dsRNA were used as negative and positive controls.

Figure 3.
Figure 3.

PKR activation by in vitro transcribed mRNA in cells. Unmodified or Ψ-containing in vitro transcribed firefly luciferase mRNA was delivered to cells by lipofection. Following RNA transfection, cells were lysed at 4 h (A) or at the indicated time (B), proteins were separated by SDS–PAGE, and assayed for phosphorylation of PKR (A) or eIF-2α (B) by western blotting. No RNA (−), poly(dC) and poly(I:C) were used as controls. Relative phosphorylation is indicated below each gel lane, calculated as phosphorylated band density divided by total band density and then normalized to the phosphorylation induced by unmodified RNA.

Figure 4.
Figure 4.

Translation of in vitro transcribed mRNA in the absence of PKR activity. (A) HEK293T cells were transfected with plasmids encoding protein inhibitors of PKR: swinepox C8L protein, wt vaccinia K3L, hyperactive K3L-H47R, inactive K3L-Y76A, or pG5 empty vector. Twenty-four hours later, unmodified or Ψ-modified in vitro transcribed mRNAs encoding firefly luciferase were delivered by lipofection, and luciferase activity was measured 4 h later. Data were normalized to values obtained when cells were first transfected with empty vector then with unmodified RNA. Presented data are mean values from three replicates ± SEM. (B) MEF cell lines derived from wild-type (WT) or transgenic mice that do not express functional PKR (PKR−/−) were transfected with unmodified or Ψ-containing in vitro transcribed mRNAs encoding firefly luciferase. Data were normalized to values obtained when cells were transfected with unmodified RNA and expressed as fold increase in translation of Ψ-containing mRNA over unmodified RNA. Values are from three replicate wells ± SEM, and are representative of at least three independently performed experiments. (C) WT and PKR−/– MEF cells were transfected with unmodified or Ψ-containing in vitro transcribed mRNAs encoding firefly luciferase, or mock transfected with no RNA (–). Cells were lysed 2 h following RNA transfection; proteins were then separated by SDS–PAGE and assayed for eIF-2α phosphorylation by western blotting. Relative phosphorylation is indicated above each gel lane, calculated as phosphorylated band density divided by total band density and then normalized to the phosphorylation induced by unmodified RNA in wild-type cells. Absence of PKR was also confirmed by western blotting.

Figure 5.
Figure 5.

Ψ-containing mRNA does not inhibit PKR activation. An activating 200 bp dsRNA was mixed with a 5–125-fold mass excess of Ψ-containing in vitro transcribed firefly luciferase mRNA prior to incubation with purified PKR. Reaction products were separated by SDS–PAGE. Relative band densities are presented below each gel lane and normalized to dsRNA only. Data shown are representative of three independent experiments.

Figure 6.
Figure 6.

Ψ-containing mRNA does not pull-down PKR. Biotinylated in vitro transcribed unmodified or Ψ-containing RNAs were incubated with HEK293T cell lysates for 2 h. The RNA and bound proteins were pulled down using streptavidin-agarose beads. An aliquot of lysate that was incubated only with beads but without RNA (−) was also processed. Aliquots of pull-down proteins as well as the supernatants were separated by SDS–PAGE. PKR and PABP were detected by western blotting. Relative band densities of PKR divided by PABP compared to unmodified RNA are presented below each gel lane.

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