Fig. 6: Phosphorylation-dependent interaction between TTP and PABPC1 promotes CCR4–NOT-mediated shortening of PABPC1-coated poly(A) tails.

a, b Pull-down assays testing the interaction between the cytoplasmic poly(A)-binding protein (PABPC1) and either phosphorylated TTP (pTTP) expressed in HEK-293, Sf21, or E. coli cells, or phosphorylated human family members. Proteins were incubated with or without protein phosphatase (black circles) to assess the effect of dephosphorylation on the interaction. MBP was included as a negative control. c Domain architecture of PABPC1 and a summary of observed interactions between PABPC1 fragments and pTTP (d, e). RRM: RNA recognition motif; MLLE: mademoiselle protein–protein interaction domain. d Pull-down assays showing the interaction between pTTP and various fragments of PABPC1, as indicated (black circles). MBP was included as a negative control. e Characterization of the interaction between the RRMs of PABPC1 and pTTP, pZFP36L1, and pZFP36L2, via pull-down assays. f Assessment of PABPC’s ability to interact simultaneously with a poly(A) tail and pTTP. Complexes of PABPC1 with a poly(A) tail of 30 adenosine residues (PABPC1/A30) were preformed before pTTP addition, or complexes of PABPC1 with pTTP (PABPC1/pTTP) were formed before adding A30 oligo. To confirm the poly(A) specificity, incubations were carried out with a C30 oligo. MBP was included as a negative control. g Deadenylation assay testing the effect of PABPC1 on targeted deadenylation by pTTP. A PABPC1:RNA complex was preformed first by addition of PABPC (150 nM) in threefold excess over substrate RNA (50 nM) to saturate the poly(A) tail. pTTP protein (100 nM) was added in twofold excess over CCR4–NOT (50 nM) and substrate RNA (50 nM). Controls included reactions lacking pTTP or PABPC1(-RBP), and reactions with only RNA and PABPC1 (C2) or pTTP (C3) or PABPC1 and pTTP (C4).