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MicroRNAs (miRNAs) recruit the CCR4–NOT complex, which contains two deadenylases, CCR4 and CAF1, to promote shortening of the poly(A) tail. Although both CCR4 and CAF1 generally have a strong preference for poly(A) RNA substrates, it has been reported from yeast to humans that they can also remove non-A residues in vitro to various degrees. However, it remains unknown how CCR4 and CAF1 remove non-A sequences. Herein we show that  Drosophila  miRNAs can promote the removal of 3′-terminal non-A residues in an exonucleolytic manner, but only if an upstream poly(A) sequence exists. This non-A removing reaction is directly catalyzed by both CCR4 and CAF1 and depends on the balance between the length of the internal poly(A) sequence and that of the downstream non-A sequence. These results suggest that the CCR4–NOT complex has an intrinsic activity to remove the 3′-terminal non-A modifications downstream from the poly(A) tail.

CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence