Fig. 2. hDcp2 has decapping activity. (A) Analysis of decapping activity from purified His6–GST-tagged recombinant proteins hDcp1ΔC (lanes 2–4), hDcp2 (lanes 5–7) and the control protein ABD (a fragment of human α-actinin) (lanes 8–10) (2 µg each). Cap-labelled RNA was incubated with the indicated proteins as described in Materials and methods. The presence or absence of protein, RNasin (lanes 3, 4, 6, 7, 9 and 10) or tRNA (lanes 4, 7 and 10) in the reactions is indicated by (+) and (–) symbols above the lanes, respectively. Lane 1 contains a control reaction with buffer alone. The reaction products were separated by PEI cellulose TLC along with unlabelled standards; their positions of migration are indicated on the right. The position of the input RNA, which remained at the origin of loading, is also indicated. A Coomassie Blue-stained gel containing His6–GST-tagged hDcp1ΔC and hDcp2 is shown on the right. (B) Decapping activity of purified GST–hDcp2-His6. Cap-labelled RNA was incubated with buffer alone (lane 1) or 100 ng of full-length GST–hDcp2-His6 purified by two successive affinity purification steps (lane 2). The purified GST–hDcp2-His6 protein detected by Coomassie Blue staining is shown on the right. (C) Full-length in vitro translated Dcp1 is not active for decapping. Various amounts of purified His6-tagged hDcp1 protein (30–90 ng) were assayed for decapping (lanes 2–4). hDcp2 was used as a positive control (lane 5) while enzyme storage buffer was used as a negative control (lane 1) for the decapping reaction. An aliquot of the in vitro translated, purified His6-Dcp1 was fractionated by gel electrophoresis and stained with Coomassie Blue. This protein contains only a His6 tag and thus migrates at a position lower than the truncated protein expressed in E.coli (A) that contains in addition a GST tag.