Fig. 3: The C-terminal unstructured region of TTP is essential for interaction with the NOT module and efficient deadenylation.

a Alignment of sixteen out of twenty-five AlphaFold2 predictions reveals a potential interaction surface between TTP (orange) and the NOT module (NOT1 in grey, NOT2 in light green, NOT3 in dark green). A short region within the C-terminal IDR of TTP converges around NOT3. b Domain organization and summary of activity for the C-terminal truncations of TTP used in deadenylation assays (labeled ‘dA’; panel c) and protein production assays (labeled ‘Repres.’; panel e). c Deadenylation assays assessing the effect of gradual C-terminal truncations of TTP on targeted deadenylation. All TTP variants were added at 100 nM, in twofold excess over CCR4–NOT (50 nM) and substrate RNAs (50 nM). Control reactions included reactions with RNA and the indicated TTP variant (C1-C6) alone. d Pull-down assays with the NOT module and the indicated TTP fragments (colored circles). MBP (black circle) was included as a negative control. e Effect of C-terminal IDR truncations of TTP on the repression of IL-3 protein production in co-transfection assays, measured by relative luminescence. Statistical significance was determined using a two-tailed Student’s t-test followed by Holm-Šidák correction for multiple comparison and shown as exact p values. Each data point represents one biological replicate (n = 5).