Reconstitution of ATP-dependent 3’-cleavage of mammalian pre-mRNA reveals a central role of RBBP6

The 39 ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction, endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of fourteen polypeptides essential and two stimulatory for RNA cleavage. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, Cleavage Factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.