Activation of the Endonuclease that Defines mRNA 3′ Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex

Summary Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3ʹ ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3ʹ end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit “CPFcore” complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3ʹ end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.
Graphical Abstract
Highlights • Crystallography and cryo-EM reveal a Ysh1-Mpe1 interface • Specific pre-mRNA 3ʹ end cleavage is reconstituted from recombinant proteins • Electron microscopy shows that CPFcore assembles around a central scaffold • A model for activation of the 3ʹ endonuclease on substrate RNAs is proposed
The 3ʹ ends of eukaryotic mRNAs are formed by endonucleolytic cleavage by Ysh1/CPSF73. Hill et al. define a minimal machinery for cleavage and polyadenylation in vitro using recombinant yeast proteins and short substrates. They elucidate the architecture of an ∼500 kDa eight-protein assembly, including the atomic details of a Ysh1-Mpe1 interface.