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Recombinant hDcp2 utilizes Mn2+ for optimal decapping activity. In vitro decay assays were carried out by incubating 0.5 μg of histidine-tagged recombinant hDcp2 protein at 37°C for 30 min with cap-labeled pcP-G16 RNA. Reactions were carried out in IVDA-2 buffer lacking divalent cation and supplemented with 2 mM of the indicated cations. Products were resolved on PEI-TLC in 0.75 M LiCl. Migration of unlabeled m7GDP standard was visualized by UV-shadowing and is indicated at right.
