DCP1 is essential for human decapping process

(A) Wild-type or DCP1a/b knockout HEK-293T cells were transfected with a mixture of plasmids. One plasmid expressed the β-globin-6xMS2bs, another plasmid expressed the transfection control which contained the β-globin gene fused to the GAPDH 3’ UTR, but it lacked the MS2 binding sites (β-globin-GAP, Control). The third plasmid expressed a MS2-HA or the MS2-tagged SMG7 proteins, and a fourth plasmid encoding a GFP-tagged protein was included in the transfection mixtures where indicated. A northern blot of representative RNA samples is shown. The positions of the polyadenylated (An) and the deadenylated (A0) forms of the β-globin-6xMS2bs reporters are indicated on the right. A red dotted line additionally marks the fast migrating deadenylated (A0) form. The DCP2 inactive mutant (E148Q) serves as a negative control. (B) Complementation assays with GFP-DCP1a or GFP-DCP1b constructs in HEK-293T DCP1a/b-null cells performed essentially as in panel (A). (C) The β-globin-6xMS2bs mRNA levels were normalized to those of the control mRNA. These normalized values were set to 100 in cells expressing MS2-HA (white bars). The mean values for relative mRNA levels in cells expressing MS2-SMG7 were estimated with standard deviations (SD) from three independent experiments (black bars). (D) A western blot demonstrating equivalent expression of the GFP-tagged proteins in panel (A) and (B). Tubulin served as a loading control. (E) The domain organization of human DCP1a. (F) Complementation assays with GFP-DCP1a deletion constructs in HEK-293T DCP1a/b-null cells performed essentially as in panel (A). (G) The β-globin-6xMS2bs mRNA levels were normalized to those of the control mRNA. These normalized values were set to 100 in cells expressing MS2-HA (white bars). The mean values for relative mRNA levels in cells expressing MS2-SMG7 were estimated with standard deviations (SD) from three independent experiments (black bars). (H) A western blot demonstrating equivalent expression of the GFP-tagged proteins in panel (F). Tubulin served as a loading control. (I) Complementation assays with GFP-DCP1a fragment constructs in HEK-293T DCP1a/b-null cells performed essentially as in panel (A). (J) The β-globin-6xMS2bs mRNA levels were normalized to those of the control mRNA. These normalized values were set to 100 in cells expressing MS2-HA (white bars). The mean values for relative mRNA levels in cells expressing MS2-SMG7 were estimated with standard deviations (SD) from three independent experiments (black bars). (K) A western blot demonstrating equivalent expression of the GFP-tagged proteins in panel (I). Tubulin served as a loading control.

DCP1 serves as a bridging factor facilitating the interaction between multiple decapping factors and DCP2.

(A) HEK-293T wild-type or DCP1a/b-null cells were stained with antibodies detecting DCP2 (red) and a P-body marker, EDC4 (green), then counterstained with DAPI to visualize the nucleus (blue). In the merged image, colocalization of DCP2 and EDC4 localization appears yellow. Scale bar=10 μm. (B) GFP-tagged DCP2 proteins were expressed in human HEK-293T wild-type or DCP1a/b-null cells. Subsequently, purification was performed using GFP antibody and IgG beads. In vitro decapping activity was then tested, with the catalytically inactive DCP2 E148Q mutant served as a negative control. (C) Decapping assays in vitro. The fraction of decapped mRNA substrate, measured by the release of m7GDP (panel B), is plotted as a function of time. Error bars, standard deviations (SD) from three independent experiments. (D) The GFP-DCP2 WT or E148Q immunoprecipitated samples corresponding to panel (B) were analyzed by Western blotting using the indicated antibodies. (E) V5-Streptavidin-Binding Peptide (SBP)-DCP2 proteins were expressed in human HEK-293T wild-type or DCP1a/b-null cells, followed by purification using Strepavidin beads. The interaction of V5-SBP-tagged DCP2 with endogenous decapping factors in wild-type or DCP1a/b knockout HEK-293T cells. Strepavidin resin was used for immunoprecipitate the V5-SBP-DCP2 in the presence of RNase A. Bound proteins were detected via Western blot. V5-SBP-MBP employed as a negative control. (F-G) The interaction of GFP-tagged DCP2 with HA-tagged PNRC1 (F) or V5-tagged PNRC2 (G). The proteins were immunoprecipitated using anti-GFP antibodies and analyzed by Western blotting using the indicated antibodies.

DCP1 facilitates DCP2 interactions with RNA molecules in human cells

(A-D) The interaction of GFP-tagged DCP2 with indicated decapping factors. The proteins were immunoprecipitated using anti-GFP antibodies and analyzed as described in Figure 2D and E. (E) The tethering assays with control, shPNRC1 or shPNRN2 plasmids in HEK-293T cells performed essentially as in Figure 1A. (F) The β-globin-6xMS2bs mRNA levels were normalized to those of the control mRNA. These normalized values were set to 100 in cells expressing MS2-HA (white bars). The mean values for relative mRNA levels in cells expressing MS2-SMG7 were estimated with standard deviations (SD) from three independent experiments (black bars). (G) Schematic representation of the experimental procedure in panel H and J. (H) DCP1a/b-null HEK-293T cells were transfected with a plasmid mixture containing the β-globin-6xMS2bs, MS2-HA-Strep-SMG7, V5-SBP-DCP2 E148Q, and either full-length or EVH1 domain of GFP-DCP1a. GFP-MBP serves as a control. The levels of β-globin-6xMS2bs mRNA bound to V5-SBP-DCP2 E148Q were then immunoprecipitated using streptavidin beads and quantified by RT-PCR with GAPDH as a reference. The results presented in each panel represent the mean values ± standard deviations (SD) of three biological replicates. (I) The V5-SBP-DCP2 E148Q immunoprecipitated samples corresponding to Figure 3H were analyzed by Western blotting using the indicated antibodies. (J) The plasmid mixture was transfected to DCP1a/b-null HEK-293T cells as detailed in panel H. Subsequently, Strep-tag beads were used to immunoprecipitate the MS2-HA-Strep-SMG7 bound β-globin-6xMS2bs mRNA to study the in vivo interaction levels between RNA molecules and DCP2 E148Q in the presence of GFP-DCP1a full-length or EVH1 domain. GFP-MBP serves as a control.

Gene expression analysis and pathway enrichment reveal distinct roles of DCP1a and DCP1b in human cells

(A) The upper and lower panels display Venn diagrams illustrating the number of genes that were significantly up- and down-regulated in DCP1a, DCP1b, and DCP1a/b knockout cells (referred to as DCP1a_KO, DCP1b_KO, and DCP1a/b_KO), respectively. The overlapping regions between the diagrams indicate the number of genes that were significantly altered in multiple cell lines. (B) The plot shows the distribution of fold changes in genes when comparing DCP1a_KO or DCP1b_KO to WT. The points on the plot are colored according to the fold changes observed when comparing DCP1a/b_KO to WT. GSEA results for the comparison of (C) hallmark and (D) KEGG pathways between DCP1a knockout and WT and DCP1b knockout and WT. The left and right panels show activated and suppressed pathways, respectively. The dots were colored based on the q-value. (E) Boxplots of the mRNA expression levels of DCP1a and DCP1b in various cancer types in TCGA. Statistically significant differences in the expression levels were labeled on top of each pair (Wilcoxon test, *: p <= 0.05; **: p <= 0.01; ***: p <= 0.001; ****: p <= 0.0001).

Metabolome profiling unveils unique functions of DCP1 paralogs in human cells

(A) PLS-DA plot illustrating the metabolome profiles of the 12 samples, with 3 technical replicates for each knockout cell-line. Volcano plots for comparing metabolite levels of (B) DCP1a_KO and WT, (C) DCP1b_KO and WT, and (D) DCP1a/b_KO and WT. Metabolites with an absolute fold change greater than 1.5 and a false discovery rate (FDR)-adjusted p-value less than 0.01 were considered significant. The box plots of (E) UDP-GlcNAc and (F) GlcNAc between cell-lines. (G) Normalized expression levels/abundance of transcripts and metabolites, and the fold changes in amino sugar and nucleotide sugar metabolism pathway. Significantly upregulated and downregulated genes are colored in pink and blue, respectively. Upregulated and downregulated metabolites are colored in red and blue, respectively.