Drosophila CCR4/CAF1 Deadenylation Assay

Preparation of recombinant Drosophila CCR4/CAF1 heterodimer and deadenylation assay with the enzyme was previously described (Niinuma et al. 2016 (link)). Deadenylation reaction to estimate the inhibitory effect by AMP, ADP, and ATP contained 4.2 µL of water, 3 µL of 1.5 µM CCR4/CAF1 heterodimer, 3 µL of 5× lysis buffer, 1.5 µL 10 mM DTT, 0.3 µL of 40U/µL RNasin plus (Promega), 1.5 µL of ∼5 nM noC-A₄₀, and 1.5 µL of 10× AMP, ADP, or ATP. The mixture was incubated at 25°C, and 3 µL of it was collected at each time point. The equal volume of formamide loading dye was added to the collected sample immediately. Then, the sample was electrophoresed on 5% denaturing polyacrylamide gel and analyzed by PhosphoImager (Typhoon FLA 7000, GE Healthcare).