Fig 2: Analysis of the molecular weight distribution of decapping complex subunits.(A) Protein standard was run on a Superose 6 column. (B, C, D) Chromatograms of (B) control, (C) DCP1a KO, and (D) DCP1b KO whole cell lysates run on a Superose 6 column. (E, F, G) Column fractions from (E) control, (F) DCP1a KO, and (G) DCP1 KO cells were examined by Western blotting. Column fraction number is indicated on top. The number adjacent to the protein name indicates the gel number. Note: samples are loaded differently for the EDC3 blot from DCP1a KO cells: the input control sample is loaded at the end instead of at the beginning of the blot. The fractions with the highest content of the relevant protein are enclosed or indicated with a red arrow.Source data are available for this figure.

Fig 3: DCP1a is critical for interactions between known decapping complex members.(A) DDX6 was immunoprecipitated in control cells, DCP1a KO, and DCP1b KO cells with IgG immunoprecipitation as a control. (B) DDX6 was immunoprecipitated in control cells, DCP1a KO, and DCP1b KO cells with IgG immunoprecipitation as a control. IPs were sent for profiling by LC/MS/MS. PCA analysis of the samples is shown. (C) Venn diagram represents the high-confidence proteins found in all conditions of the DDX6 IP. (D) Heatmap reports intensities (sum of peptide peaks as analyzed by mass spectrometry) of the core decapping complex proteins DCP1a, DCP1b, and EDC3 in the DDX6 IP for each condition. Biological replicas 1, 2, and 3 are shown. The heatmap is scaled to DDX6 intensity and ctrl samples (set to 1). The numbers in the heatmap reflect relative scaled protein intensities in these samples. Detailed explanation for normalization is available in the Materials and Methods section. Note: DCP1a and DCP1b proteins in DCP1a KO and DCP1b KO cells are not considered high-confidence by the same definition in the Methods section. The number of their unique razor peptides in those conditions is 0 as their genes are deleted. EDC4 is not considered high-confidence in DCP1a KO cells as its q-value is higher than 0.05. However, its P-value is lower than 0.05, and it was important to include EDC4 in this specific comparison and heatmap. (E) iBAQ analysis shows the relative amounts of DCP1a, DCP1b, EDC3, and EDC4 to DDX6 in the control condition. iBAQ values were used to determine the relative amounts of proteins in the DDX6 IP in the ctrl condition. Three replicas were averaged, and block size corresponds to the relative amount of DCP1a, DCP1b, EDC3, and EDC4 to DDX6 in the ctrl condition. (F) Ontology analysis of the core 52 proteins present in the DDX6 IP (DDX6 protein was excluded from this list). MF, Molecular Function; BP, Biological Process; CC, Cellular Compartment. Raw P-value is shown. Enrichment ratio incorporates the number of overlapping proteins found in the input protein set versus the GO group. (G) Heatmap showing intensities of the core 52 proteins (DDX6 protein was excluded from this list) that do not depend on the presence or absence of DCP1a or DCP1b in the DDX6 IP. Biological replicas 1, 2, and 3 are shown. The heatmap is created and clustered with the pheatmap function in R studio. It is scaled by row. (G, H) Network analysis (WebGestalt) of the clusters identified in panel (G). The input proteins are used as seeds, and the top-ranking/interacting neighbors are identified and used to identify the appropriate network of proteins.Source data are available for this figure.