Fig. 4: Phosphorylation of the Puf3-IDR tunes Ccr4–Not-mediated deadenylation.
From: Phosphorylation-dependent tuning of mRNA deadenylation rates
a, 1H,15N 2D-HSQC spectral overlay of 75 µM unphosphorylated (gray) and phosphorylated (red) Puf3-IDR1–258. Puf3-IDR1–258 was phosphorylated using a mixture of recombinant Sck1 and Pdk1 at a 1:5 molar ratio. Substantial chemical shift perturbations are observed upon phosphorylation, particularly backbone resonances of phosphorylated serines (black arrows). Inset: side-chain resonances of tryptophans. b, Scheme for producing differentially phosphorylated Puf3 for use in downstream experiments. c, Intact MS analysis of samples taken during the Puf3-IDR1–376 phosphorylation time course. Deconvoluted spectra are shown with masses consistent with modal phosphorylation states shown above each time point (colored arrows). The black arrow (+18*) at 270 min shows maximum incorporation of 18 phosphates under these conditions, with a modal number of 13 phosphorylation sites (dark-red arrow). d, Tryptophan indole peaks from 1H,15N 2D-HSQC showing increasing chemical shift perturbation for the W229 side-chain resonance throughout the phosphorylation time course. These data suggest that structural changes occur in the DFW motif upon phosphorylation. e, 15N R2 transverse relaxation rates of unphosphorylated (black bars) and phosphorylated (red bars) Puf3-IDR109–258. This construct contains four of five phosphorylation sites identified by NMR (black arrows show positions of assigned phosphorylated residues). The increase in R2 rates are consistent with a decrease in backbone dynamics upon phosphorylation. f, Deadenylation t1/2 of Ccr4–Not with differentially phosphorylated full-length Puf3 proteins prepared according to b. IDR phosphorylation states were determined using intact MS, indicated as modal states (c). Experiments were performed in triplicate (open circles) and averaged (line).
