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Science 30 November 2007:
Vol. 318. no. 5855, pp. 1455 - 1458
DOI: 10.1126/science.1147347
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Reports

5'-Triphosphate-Dependent Activation of PKR by RNAs with Short Stem-Loops

Subba Rao Nallagatla1*, Jungwook Hwang2*, Rebecca Toroney1, Xiaofeng Zheng1,3, Craig E. Cameron2,4{dagger} and Philip C. Bevilacqua1{dagger}

1 Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
2 Integrative Biosciences, Pennsylvania State University, University Park, PA 16802, USA.
3 Department of Biochemistry and Molecular Biology, Life Sciences College, Peking University, Beijing 100871, China.
4 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.


Figure 1 Fig. 1. Activation of PKR by ss-dsRNA is 5'-triphosphate–dependent. (A) PKR has two dsRBMs (the dsRBD) and a kinase domain. (B) Experimental structures of ss-dsRNAs (6). (C) Activation assays using ss-dsRNAs [10% SDS–polyacrylamide gel electrophoresis (PAGE)]. RNAs were transcribed and untreated (5'-ppp) or CIP-treated (5'-OH). A no-RNA lane (–) is provided. Phosphorylation activities are normalized to 0.1 µM dsRNA-79 (no CIP). [View Larger Version of this Image (25K GIF file)]
 

Figure 2 Fig. 2. Activation of PKR by ssRNA is 5'-triphosphate–dependent in vitro. (A) Effects of 5'-OH and 5'-p on PKR activation. ssRNA-47 was synthesized with 5'-OH or 5'-p, and concentrations were 0.1, 0.5, 1, 2, 3, 5, and 10 µM (0.1 µM is omitted for 5'-p). (B) Effects of 5'-pp and 5'-ppp on PKR activation. ssRNA-47 was transcribed with guanosine triphosphate (GTP) only or with a GDP:GTP ratio of 12:1 and tested for PKR activation. RNA concentrations were 0.16, 0.31, 0.63, 1.25, 2.5, and 5 µM. (C) Effect of a 7mG cap on PKR activation. ssRNA-47 was transcribed with a 7mGpppG:GTP ratio of 4:1, 8:1, or 12:1. RNA concentrations were 0.16, 0.31, 0.63, 1.25, 2.5, and 5 µM. (D) PKR phosphorylation data from (C). The ratio of 7mGpppG:GTP was 0 (solid line, black circles); 4 (dashed line, white circles); 8 (solid line, black boxes); and 12 (dashed line, white boxes). Phosphorylation activities are normalized to 0.1 µM dsRNA-79 (no CIP). [View Larger Version of this Image (45K GIF file)]
 

Figure 3 Fig. 3. Activation of PKR by ssRNA is 5'-triphosphate–dependent in vivo. (A) Origin of cell line, capacity to produce IFN-{alpha}/β, and capacity to signal from the IFN-{alpha}/β receptor are indicated. A block of IFN-{alpha}/β production in Huh-7.5 cells is observed only if RIG-I signaling is required (17). The IFN-{alpha}/β gene cluster is deleted in Vero cells (23). (B to D) Cells were plated 24 hours before transfection in the absence (lanes 1 to 4) or presence (lanes 5 to 8) of 1000 units of IFN{alpha} per milliliter. Cells were transfected with RNA (2 µg), prepared as for in vitro experiments. Proteins were denatured in SDS buffer and resolved by 10% SDS-PAGE. Phosphorylated PKR (PKR-p), phosphorylated eIF2{alpha} (eIF2{alpha}-p), and β actin (loading control) were identified by Western blotting. (E) Experiments were performed as in (B) using ssRNA-110. The 5' end was prepared by no treatment (ppp-lane). CIP treatment (HO lane), or priming transcription with a modified guanosine. (F) The 5' ends of dsRNA-79, ssRNA-79TS, and ssRNA-79BS were prepared as in (E). Samples in (E) and (F) were IFN-treated. [View Larger Version of this Image (40K GIF file)]
 

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