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, and not DCP1b, mediates the interaction of DCP2 with EDC4 and EDC3.(A) Comparison of DCP2 and DCP1(a/b) protein domains in Saccharomyces cerevisiae (Sc) and Homo sapiens (Hs). EVH1, Ena/Vasp homology domain 1; NRD, N-terminus by a helical regulatory domain; TD, trimerization domain, low-complexity regions or short linear motifs. Note: The NRD and NUDIX domain in Hs DCP2 are connected by a flexible hinge (not shown) consisting of 4 amino acids. The low-complexity regions were defined using http://smart.embl-heidelberg.de/. Major protein domains were defined as described previously in Jonas and Izaurralde, 2013. Created with BioRender.com. (B) HCT116 wt cells were infected with lentiviruses carrying shRNAs targeting LUC, DCP1b, or DCP1a. On day two, the cells were selected with puromycin. On day 5, cells were infected with the second virus expressing DCP1b or an empty vector control. Cells were harvested 3 d after the second round of infections, and DCP2 was immunoprecipitated. Western blots (WB) showing inputs, IgG, and DCP2 immunoprecipitation in shLUC, shDCP1a, and shDCP1b cells (n = 2). * Indicates that those protein bands were from a different SDS–PAGE gel. (C) HCT116 cells were transfected with Cas9 protein and no gRNA to create control cells, or Cas9 protein and a mix of sgRNAs targeting either DCP1a or DCP1b. 3 d post-transfection, single-cell clones were selected and cultured for 10 d. Clones were validated and DCP2 was immunoprecipitated. WB showing inputs, IgG immunoprecipitation in control (ctrl), DCP2 IP in ctrl, DCP1a KO, and DCP1b KO cells (n = 3). * Indicates that those protein bands were from a different SDS–PAGE gel. (D) Ctrl, DCP1a KO (polyclonal pool), and DCP1b KO (single-cell clone) cells were infected with a lentivirus expressing DCP1b or the empty vector control. 3 d post-infection, cells were harvested and DCP2 was immunoprecipitated. WB show inputs, IgG, and DCP2 IP (n = 1). * Indicates that those protein bands were from a different SDS–PAGE gel.Source data are available for this figure.