Extended Data Fig. 1: Architecture of Ccr4-Not and deadenylation assay setup.
From: Phosphorylation-dependent tuning of mRNA deadenylation rates
(a) Schematic diagram showing known interactions between domains of Ccr4-Not (domain diagrams) and RNA-binding protein (colored text) short linear motifs (SLiMs). In the domain diagrams, structured domains are depicted as rounded rectangles and linkers or disordered regions are shown as connecting lines. All core subunits of the complex assemble around the Not1 scaffold protein (interacting regions indicated with dashed lines) except Ccr4, which is bridged via Caf1. Colored domains contain known interaction surfaces with SLiMs. Interactions between SLiMs and domains of Ccr4-Not that have been studied through X-ray crystal structures are labelled with asterisks. (b) Representative deadenylation assay and densitometry analysis. Wild-type Puf3 (WT) was incubated to form a 1:1 complex with a 5ʹ fluorescently labelled substrate RNA, containing a Pumilio response element (PRE) (UGUAAAUA) in a 20-nt region upstream of a 30-nt poly(A) tail. Puf3-bound RNAs were then incubated with recombinant Ccr4-Not complex and fractions were taken at indicated time points. Products were resolved on denaturing PAGE before imaging (top). Intensity profiles across each lane were integrated (middle) and the fraction of deadenylated RNA was quantified for each time point. Data were then plotted as a function of time (bottom), enabling quantitation of deadenylation rate. Assays were performed in triplicate with quantification shown for all replicates. Straight lines are drawn between data points for easier visualization. M, molecular weight marker. (c) Deadenylation assays with Ccr4-Not alone (top) and indicated Puf3 truncations, showing a representative gel and quantitation in triplicate. Domain diagrams for the Puf3 constructs are shown in Fig. 1a. (d) Summary graph of assays shown in (c) but plotted on a log2 time scale. Open shapes for each construct show the average of 3 replicate assays with error bars indicating standard deviation. The t1/2 values used for plotting deadenylation activities were interpolated from the plot.
