Reconstitution of 3′ end processing of mammalian pre-mRNA reveals a central role of RBBP6

The 3 ' 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 14 polypeptides that are essential and two that are stimulatory for RNA processing. 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, with 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. Here, Schmidt et al. reconstituted the endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail reaction from overproduced and purified proteins, and provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing.

Automated summary

In this study, the endonucleolytic cleavage of eukaryotic mRNA 3' ends followed by polyadenylation was reconstituted using overproduced and purified proteins. A minimal list of 14 essential polypeptides and 2 stimulatory polypeptides for RNA processing were identified. Key findings include the stimulation of cleavage by cleavage factor I and the essential requirement of RBBP6 in contacting and activating the endonuclease CPSF73.