Human homologs of S. cerevisiae decapping enzymes. (A) Schematics of S. cerevisiae Dcp1p (yDcp1) and the human homologs hDcp1a and hDcp1b. (B) Schematics of S. cerevisiae Dcp2p (yDcp2) and the human homolog hDcp2. Gray bars indicate regions of similarity (percent identity given below) between human and S. cerevisiae Dcps. The black bars in yDcp2 and hDcp2 indicate the conserved mutT domains. Residues critical for decapping activity are indicated (see Fig. 4). aa, amino acids.

Decapping assays with immunopurified and recombinant hDcp1a and hDcp2. α-³²P-^m7cap-labeled RNA was incubated with immunopurified FLAG-tagged hDcp1a (lanes 4, 5, 9, and 10; corresponding to 0.2 × 10⁶, 1 × 10⁶, 1 × 10⁶, and 1 × 10⁶ cells' worth of protein, respectively), hDcp2 (lanes 6 and 7; 1 × 10⁶ and 5 × 10⁶ cells' worth, respectively), mock-purified cell extract (lanes 2 and 3; 1 × 10⁶ and 5 × 10⁶ cells' worth, respectively), or no cell extract (lanes 1, 8, and 11) or with purified bacterially expressed GST (lanes 12 and 13; 10 and 100 ng, respectively), GST-hDcp1a (lanes 14 and 15; 10 and 100 ng, respectively), GST-hDcp2 (lanes 16 and 17; 10 and 100 ng, respectively), or GST-hDcp1a and GST-hDcp2 (lanes 18 and 19; 10 and 100 ng each, respectively), and products were separated by thin-layer chromatography. The reaction mixture in lane 10 was treated with nucleoside diphosphate kinase (NDPK) and ATP. The migration of unlabeled GMP, GDP, ^m7GMP, ^m7GDP, and ^m7GTP (25 μg) is indicated on the left.

Transiently expressed hDcp1a and hDcp2 proteins localize primarily in the cytoplasm. The figure shows the results of immunocytochemical staining of fixed, permeabilized HeLa cells, transiently expressing FLAG-tagged hDcp1a (panels 1 to 3) or hDcp2 (panels 4 to 6). Cells were stained with anti-FLAG M2 monoclonal antibody and visualized with Texas red-conjugated anti-mouse immunoglobulin G antibody (panels 1 and 4). Nuclei are visualized with 4′,6′-diamidino-2-phenylindole (DAPI) (panels 2 and 5). Panels 1 to 6 show one cell, representative of at least 100 observed transfected cells.

Model for how the interaction between hUpf1 and hDcp proteins may play a role in NMD. The EJC is deposited onto the mRNA upstream of exon-exon junctions after pre-mRNA splicing. hUpf3 associates with the EJC, followed by hUpf2 and hUpf1. A translation termination event upstream of the EJC/hUpf complex may allow hUpf1 to recruit the hDcp proteins to trigger mRNA decapping. RF, translation release factor.

hDcp1a and hDcp2 interact in cell extracts. (A) Western blot for Myc-tagged hDcp1a and hnRNPA1 proteins, which were transiently expressed in HEK293 cells together with an empty vector (lanes 1 and 2) or a plasmid encoding FLAG-tagged hDcp2 (lane 3) and subjected to anti-FLAG immunoprecipitation after RNase treatment. (B) Western blot for Myc-tagged hDcp2 and hnRNPA1 proteins, transiently expressed in HEK293 cells together with an empty vector (lanes 1 and 2) or a plasmid encoding FLAG-tagged hDcp1a (lane 3) and subjected to anti-FLAG immunoprecipitation after RNase treatment. Immunoprecipitates (P; lanes 2 and 3) are compared to 5% of total extract (T; lane 1).

Mutation of critical residues in hDcp1a and hDcp2 disrupts decapping activity. The figure shows the results of decapping assays with immunopurified wild-type and mutant hDcp1a and hDcp2 proteins as indicated. The amount of protein corresponds to 5 × 10⁶, 0.5 × 10⁶, 5 × 10⁶, 5 × 10⁶, 1 × 10⁶, 0.5 × 10⁶, 0.5 × 10⁶, 5 × 10⁶, and 5 × 10⁶ cells in lanes 2 to 10, respectively. The relative amount of ^m7GDP generated by each hDcp mutant was quantified with a phosphorimager and is given below relative to that of wild-type hDcps, which was set to 100%. A fraction of each assay (lanes 3 to 10) was subjected to Western blotting with anti-FLAG antibodies, shown in the lower panel. wt, wild type.

hDcp1a and hDcp2 interact with the NMD protein hUpf1. (A) Western blot for Myc-tagged hDcp1a, hDcp2, or hnRNPA1, transiently expressed in HEK293 cells together with an empty vector (lanes 1 and 2) or a plasmid encoding FLAG-tagged hUpf1 (lane 3) and subjected to anti-FLAG immunoprecipitation after RNase treatment. (B) Western blot for Myc-tagged hUpf1 or hnRNPA1, transiently expressed in HEK293 cells together with an empty vector (lanes 1, 2, 4, and 5) or a plasmid encoding FLAG-tagged hDcp1a (lane 3) or hDcp2 (lane 6) and subjected to anti-FLAG immunoprecipitation after RNase treatment. Immunoprecipitates (P; lanes 2, 3, 5, and 6) are compared to 5% of the total extract (T; lanes 1 and 4) in panels A and B. (C) RNase-treated extracts from HEK293 cells transiently expressing Myc-hDcp1a, Myc-hnRNPA1, and FLAG-hUpf1 were immunodepleted with preimmune serum (pi; lanes 1, 2, and 3) or anti-hUpf1 (α1; lane 4), anti-hUpf2 (α2; lane 5), or anti-hUpf3b (α3b; lane 6) serum and subsequently immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (upper two panels) were probed for the presence of Myc-hDcp1a and Myc-hnRNPA1 by Western blotting. Five percent of the total extract (T; lane 1) was compared to the pellets (P; lanes 2 to 5). Depleted extracts (D; lower five panels, lanes 3 to 6) were probed with monoclonal anti-Myc antibody or polyclonal rabbit antibodies against hUpf1, hUpf2, hUpf3a, and hUpf3b as indicated.