Fig. 5: DCP1 interaction with and effect on DCP2 in human and yeast homologs.

a StrepII-tagged Sp Dcp2(1–504), Sp Dcp2(1–242), or maltose-binding protein (MBP) were immobilized on StrepTactin resin and incubated with purified Sp Dcp1. Bound material was eluted, resolved by SDS-PAGE, and visualized with Coomassie blue. The input lane shows Sp Dcp1 used as prey (representative gel from n = 3). b Pulldowns were performed as in (a) using StrepII-tagged full-length Hs DCP2, the catalytic core Hs DCP2(1–245), MBP, or MBP-Hs PNRC2 as bait and purified Hs DCP1 as prey (representative gel, n = 3). c Mass photometry of Sp Dcp2(1–504) alone (top, orange) reveals a major monomer peak (101 kDa) and a minor dimer shoulder. Addition of equimolar Sp Dcp1 (bottom, navy) shifts the distribution to slightly higher masses (119 kDa, 240 kDa), consistent with formation of a heterodimer of one Dcp2 and one Dcp1. Shaded regions mark expected monomer and dimer windows (representative trace, n = 3). d Mass photometry histograms of Hs DCP1 alone (top, red) show a single trimeric species (~184 kDa). Hs DCP2 alone (middle, orange) is monomeric (~91 kDa). A 1:1 mixture (bottom, navy) yields two populations: the DCP2 monomer and a species at ~180 kDa (pink shading) that matches the DCP1 trimer; no discrete DCP1–DCP2 complex is detected (representative trace, n = 3). Because each trimeric DCP1 particle contains three polypeptides, its particle count is lower than the equimolar DCP2 monomer. e Decapping assays were performed with full-length Hs DCP2 in the absence (blue) or presence (magenta) of equimolar Hs DCP1. Reactions were quenched at the indicated times (0–60 min), products were resolved by TLC, and the percentage of mRNA decapped was quantified. Data are mean ± SEM of three independent experiments (n = 3) with single-exponential fits. f Decapping kinetics of Sp Dcp2(1–242) (cyan) and Sp Dcp2(1–504) (light red) were measured with or without equimolar Sp Dcp1 (dotted lines). Plots show mean ± SEM of three independent experiments (n = 3) with single-exponential fits.