Fig. 1: An N-terminal IDR of Puf3 is required for stimulation of deadenylation by Ccr4–Not.
From: Phosphorylation-dependent tuning of mRNA deadenylation rates
a, Domain diagrams of S. pombe Puf3 constructs used in this study. Puf3 contains a 376-aa N-terminal IDR and a C-terminal Pumilio RNA-binding domain (RBD) or PUM. Puf3-IDR1–258 and Puf3-IDR109–258 NMR constructs are indicated above the full-length protein. Truncation constructs used for deadenylation assays in b are indicated. b, Deadenylation assays using recombinant S. pombe Ccr4–Not complex, MBP-tagged full-length or truncated Puf3 constructs and 5ʹ fluorescently labeled substrate RNA containing a Pumilio recognition element upstream of a 30-nt poly(A) tail. The reaction mixture was resolved using denaturing PAGE at the indicated time points. The sizes of substrate RNA with a 30-nt poly(A) tail (A30) or no poly(A) tail (A0) are indicated. Deadenylation rates are plotted as t1/2. Experiments were performed in triplicate (open circles) and averaged (line). c, 1H,15N 2D-HSQC of 50 µM Puf3-IDR1–258 with (orange) or without (gray) 1 µM Ccr4–Not. Addition of Ccr4–Not at this concentration resulted in minimal exchange broadening. Crosspeaks of the DFW motif are indicated. d,e, Peak intensities (d) and transverse relaxation rates (R2) (e) of Puf3-IDR1–258 from 1H,15N 2D-HSQC experiments alone (black) and in the presence of Ccr4–Not at 50:1 (orange) and 100:1 (blue) ratios. The increase in R2 across the whole IDR is consistent with the conformational ensemble sampling bound states. Higher R2 rates in several distinct regions are indicative of additional contributions, from both direct interactions with Ccr4–Not and the exchange regime between free and bound states. Dashed lines indicate two regions with changes in R2 that were mutated in Fig. 2a. Conservation of the DFW motif in Schizosaccharomycetes is shown in the sequence alignment in d. Sp, S. pombe; So, Schizosaccharomyces octosporus; Scr, Schizosaccharomyces cryophilus; Sj, Schizosaccharomyces japonicus.
