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.

Identification of a minimal hDcp2 domain sufficient for cap hydrolysis. (A) The hDcp2 protein and the various truncation proteins are shown schematically. The three regions highly conserved in hDcp2 from diverse organisms are denoted as the 37 amino acid Box A, the 109 amino acid Nudix fold with the central 23 amino acid Nudix motif, and the 19 amino acid Box B segment. The numbers above and on either side of the schematic diagrams represent amino acid positions. The decapping activity for each protein is summarized at right by a + or − for those that can and cannot decap, respectively. (B) Purified recombinant protein was resolved on a 12.5% SDS-PAGE and detected with Coomassie blue. (C) Decapping assays of hDcp2 and hDcp2 mutant proteins. The assays were carried out using 20 pmoles of each protein with cap-labeled pcP-G₁₆ substrate, and the reaction products were resolved by TLC. The quantitation for the decapping efficiency of each protein is presented as the percentage decapping using ImageQuant 5.2 software. Migration of the standards are shown at right. (D) The minimal hDcp2^94–257 protein retains methyl cap specificity and efficiently utilizes different RNA substrates. Decapping assays were carried out as described above using the 3′ UTR sequences of the indicated mRNAs (α-globin, β-globin, c-myc) and pcP. N-7 methyl-containing capped pcP RNA is denoted as m⁷G—RNA, whereas the unmethylated is represented as G—RNA. Migration of the standards are indicated on the right.

hDcp2 does not utilize the cap structure as substrate. (A) hDcp2 is unable to hydrolyze the cap structure in the absence of the RNA body. ³²P-labeled cap analog was incubated with 10 pmole of each hDcp2 protein and the products resolved by TLC. The scavenger decapping activity DcpS, which can hydrolyze the cap structure, was used as the positive control (lane 2). Standards are indicated at left. (B) hDcp2 is unable to cross-link to cap analog. ³²P-labeled cap analog was UV cross-linked with 20 pmole of the indicated proteins and the products resolved by 12.5% SDS-PAGE. GST fusion protein of the cap-binding protein, eIF4E (lane 1), was used as the positive control. Histidine-tagged hDcp2 protein in lane 2, and an unrelated RNA-binding protein control, GST-mDAZL (Jiao et al. 2002), was used in lane 3. Molecular weight standards are indicated at right. The cap analog substrate is shown at bottom, and the star denotes the labeled phosphate.

hDcp2 is an RNA-binding protein. (A) RNA can compete for hDcp2-mediated decapping. Capped and uncapped pcP-G₁₆ RNAs were used as competitor. In vitro decay assays were carried out with 20 pmole of hDcp2 with no competitor (None), 1:1 or 1:200 molar fold excess competitor RNA as indicated. The decapping products were resolved by TLC and the percent decapping is plotted. Both RNAs were found to compete equally for hDcp2 activity. (B) Binding of hDcp2 to RNA by Northwestern analysis. Six μg of each protein was resolved by SDS-PAGE, transferred to nitrocellulose, and probed with uniform-labeled, uncapped pcP RNA. The proteins are shown schematically in Figure 2 ▶ and their size on SDS-PAGE corresponds to the following molecular weights as follows: hDcp2, 48kD; hDcp2^ΔN53, 43 kD; hDcp2^ΔN93, 38 kD; hDcp2^ΔN119, 35 kD; hDcp2^ΔN206, 25 kD; hDcp2^ΔN244, 20 kD; hDcp2^94–257, 19 kD; hDcp2^1–207, 23 kD. (C) Binding of hDcp2 to ssDNA. [³⁵S]methionine in vitro-translated proteins were bound to ssDNA-agarose beads and resolved by SDS-PAGE. The total lanes contain 25% of the input protein and the Bound lanes contain the protein bound to ssDNA. The hDcp2 and hDcp2^94–257 proteins are as shown in Figure 2 ▶ and hDcp2^94–207 contains only the Nudix fold and lacks Box B. The Firefly Luciferase control protein is designated as Luciferase.

Cap binding of hDcp2 is RNA dependent. (A) hDcp2 can be UV cross-linked to cap-labeled RNA. An autoradiograph of an SDS-PAGE showing 10 pmole of the indicated protein cross-linked to ³²P-cap-labeled pcP-G₁₆ RNA. Cross-linking of the eIF4E cap-binding protein (GST–eIF4E, 50 kD; lanes 1,4) was used as a positive control. An unrelated GST–mDAZL RNA-binding protein (68 kD; lane 3), or purification of a His-tag empty vector (lane 9) are shown as negative controls. The sizes of the hDcp2 proteins and truncations are as described in the legend to Figure 4 ▶. The capped RNA substrate is shown at bottom, where the line represents the RNA and the star denotes the labeled phosphate.