DCP1 is essential for human decapping process.

(A) Schematic of the reporters used in tethering assays.

(B) Wild-type or DCP1a/b knockout HEK-293T cells were transfected with a mixture of plasmids. One plasmid expressed the β-globin-6xMS2bs, and 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 MS2-HA or 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, highlighting the importance of DCP1 in decapping by showing the absence of decapping activity without functional DCP2. All experimental results were independently repeated at least three times.

(C) Complementation assays with GFP-DCP1a or GFP-DCP1b constructs in HEK-293T DCP1a/b-null cells were performed similarly to panel (A). All experimental results were independently repeated at least three times. This experiment demonstrates whether the reintroduction of DCP1a or DCP1b can restore the decapping activity in cells lacking both DCP1 isoforms.

(D) 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). This quantification provides a comparative measure of the mRNA stability under different conditions.

(E) The domain organization of human DCP1a.

(F) Complementation assays with GFP-DCP1a deletion constructs in HEK-293T DCP1a/b-null cells performed similarly to panel (A). All experimental results were independently repeated at least three times. This experiment helps identify which domains of DCP1a are crucial for its function in the decapping process.

(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). This data provides insight into the functional importance of different DCP1a domains.

(H) A western blot demonstrating equivalent expression of the GFP-tagged proteins in panel (F). Tubulin served as a loading control, confirming that the expression levels of the deletion constructs were comparable.

(I) Complementation assays with GFP-DCP1a fragment constructs in HEK-293T DCP1a/b-null cells performed similarly to panel (A). All experimental results were independently repeated at least three times. This set of experiments further delineates the specific regions of DCP1a necessary for its decapping activity.

(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). This helps validate the findings regarding the essential regions of DCP1a.

(K) A western blot demonstrating equivalent expression of the GFP-tagged proteins in panel (I). Tubulin served as a loading control, ensuring that the variations in mRNA levels were due to functional differences in the DCP1a fragments rather than differences in protein expression.

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), and then counterstained with DAPI to visualize the nucleus (blue). In the merged image, colocalization of DCP2 and EDC4 appears yellow, indicating their interaction in P-bodies. This staining helps to visualize the cellular localization of DCP2 in the presence or absence of DCP1a/b. Scale bar = 10 μm.

(B) Quantification of P-body size in wild-type and DCP1a/b-null HEK-293T cells. The average granule size was measured across at least three different fields of view. The middle line represents the mean of these measurements, and P-values were calculated using unpaired t-tests (ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

(C) GFP-tagged DCP2 proteins were expressed in human HEK-293T wild-type or DCP1a/b-null cells. Following expression, the GFP-tagged DCP2 proteins were purified using GFP antibody and IgG beads. In vitro decapping activity was then tested, with the catalytically inactive DCP2 E148Q mutant serving as a negative control to demonstrate the specificity of the decapping activity. All experimental results were independently repeated at least three times.

(D) Decapping assays in vitro were conducted to measure the fraction of decapped mRNA substrate by detecting the release of m7GDP over time. The results are plotted as a function of time, with error bars representing the standard deviations (SD) from three independent experiments. An unpaired t-test was used to evaluate the statistical difference between samples (ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). This panel demonstrates the decapping efficiency of DCP2 in the presence or absence of DCP1a/b.

(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 Streptavidin beads. This experiment examines the interaction of V5-SBP-tagged DCP2 with endogenous decapping factors in wild-type or DCP1a/b knockout HEK-293T cells. The bound proteins were detected via Western blot, with V5-SBP-MBP employed as a negative control. All experimental results were independently repeated at least three times. This panel highlights the role of DCP1a/b in facilitating or stabilizing interactions between DCP2 and other decapping factors.

(F-G) The interaction of GFP-tagged DCP2 with HA-tagged PNRC1 (F) or V5-tagged PNRC2 (G) was assessed. The proteins were immunoprecipitated using anti-GFP antibodies and analyzed by Western blotting with the indicated antibodies. All experimental results were independently repeated at least three times. These panels demonstrate the interaction between DCP2 and specific decapping co-factors, indicating how DCP1a/b may influence these interactions.

DCP1 facilitates DCP2 interactions with RNA molecules in human cells.

(A-D) The interaction of GFP-tagged DCP2 with various decapping factors was examined. Proteins were immunoprecipitated using anti-GFP antibodies and analyzed by Western blotting as described in Figures 2D and 2E. All experimental results were independently repeated at least three times. These panels demonstrate the binding affinity and specificity of DCP2 for different decapping co-factors, highlighting the importance of these interactions in the decapping process.

(E) Tethering assays were performed in HEK-293T cells using control, shPNRC1, or shPNRC2 plasmids, following a procedure similar to that described in Figure 1A. All experimental results were independently repeated at least three times. This panel investigates the effect of knocking down PNRC1 or PNRC2 on the decapping activity, showing how these factors contribute to the stability and regulation of the β-globin-6xMS2bs mRNA.

(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). This quantification provides a comparative measure of the mRNA stability under different experimental conditions.

(G) Schematic representation of the experimental procedure used in panels H and J. This diagram outlines the steps and components involved in the transfection and subsequent analysis, providing a visual aid for understanding the experimental setup.

(H) DCP1a/b-null HEK-293T cells were transfected with a mixture of plasmids, including β-globin-6xMS2bs, MS2-HA-Strep-SMG7, V5-SBP-DCP2 E148Q, and either full-length or the EVH1 domain of GFP-DCP1a. GFP-MBP was used as a control. The levels of β-globin-6xMS2bs mRNA bound to V5-SBP-DCP2 E148Q were immunoprecipitated using streptavidin beads and quantified by RT-PCR with GAPDH as a reference. The results represent three biological replicates’ mean values ± standard deviations (SD). An unpaired t-test was used to evaluate the statistical difference between samples (ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). This experiment assesses the role of different DCP1a constructs in the interaction and binding efficiency of DCP2 to the target mRNA.

(I) The V5-SBP-DCP2 E148Q immunoprecipitated samples corresponding to panel H were analyzed by Western blotting using the indicated antibodies. This panel confirms the tagged proteins’ expression and proper immunoprecipitation, ensuring the RT-PCR results’ validity in panel H.

(J) The plasmid mixture described in panel H was transfected into DCP1a/b-null HEK-293T cells. 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 either full-length GFP-DCP1a or its EVH1 domain. GFP-MBP served as a control. All experimental results were independently repeated at least three times. This panel explores how different domains of DCP1a influence the interaction between DCP2 and its RNA targets, providing insights into the functional domains required for effective decapping.

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 indicate the number of genes significantly altered in multiple cell lines, highlighting shared and unique gene expression changes across different knockouts.

(B) The plot shows the distribution of fold changes in gene expression when comparing DCP1a_KO or DCP1b_KO to wild-type (WT) cells. The points are colored according to the fold changes observed when comparing DCP1a/b_KO to WT, visually representing how the loss of DCP1a and DCP1b individually and combined affects gene expression.

(C) Gene Set Enrichment Analysis (GSEA) results for hallmark pathways comparing DCP1a knockout and WT cells (left panel) and DCP1b knockout and WT cells (right panel). Activated and suppressed pathways are shown, with dots colored based on the q-value, indicating the statistical significance of the enrichment.

(D) GSEA results for KEGG pathways comparing DCP1a knockout and WT cells (left panel) and DCP1b knockout and WT cells (right panel). Like panel (C), activated and suppressed pathways are shown, with dots colored according to the q-value. This analysis helps identify the biological pathways most affected by the loss of DCP1a or DCP1b.

(E) Boxplots of the mRNA expression levels of DCP1a and DCP1b in various cancer types from The Cancer Genome Atlas (TCGA). Statistically significant differences in expression levels are labeled on top of each pair, with significance determined by the Wilcoxon test (ns: not significant; *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). This panel provides insights into the potential roles of DCP1a and DCP1b in different cancers, highlighting their differential expression patterns.

Metabolome profiling unveils unique functions of DCP1 paralogs in human cells.

(A) Partial Least Squares Discriminant Analysis (PLS-DA) plot illustrating the metabolome profiles of 12 samples, with 3 technical replicates for each knockout cell line. This plot shows how the metabolomic profiles of DCP1a_KO, DCP1b_KO, and DCP1a/b_KO cells differ from each other and wild-type (WT) cells, indicating distinct metabolic alterations resulting from the knockouts.

(B-D) Volcano plots comparing metabolite levels between (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. These plots highlight the metabolites significantly upregulated or downregulated in each knockout compared to WT, providing insight into the metabolic impact of DCP1a and DCP1b loss.

(E-F) Box plots of (E) UDP-GlcNAc and (F) GlcNAc levels between different cell lines. These plots show the relative abundance of specific metabolites in the different knockout and WT cells, indicating how the disruption of DCP1a and DCP1b affects specific metabolic pathways.

(G) Normalized expression levels, abundance of transcripts and metabolites, and fold changes in the 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. This panel integrates transcriptomic and metabolomic data to provide a comprehensive view of how the amino sugar and nucleotide sugar metabolism pathway is altered in DCP1 knockout cells.