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

Mol Cell. 2019 Mar 21;73(6):1217-1231.e11. doi: 10.1016/j.molcel.2018.12.023. Epub 2019 Feb 5.


Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' 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 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.

Keywords: X-ray crystallography; baculovirus; cleavage; cryo-EM; hydrogen-deuterium exchange; mRNA; mass spectrometry; nuclease; polyadenylation; pre-mRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy
  • Crystallography, X-Ray
  • Cytochromes c / genetics
  • Cytochromes c / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Enzyme Activation
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • Molecular Docking Simulation
  • Multiprotein Complexes
  • Polyadenylation*
  • Polynucleotide Adenylyltransferase / genetics
  • Polynucleotide Adenylyltransferase / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • RNA Precursors / genetics
  • RNA Precursors / metabolism*
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / ultrastructure
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spectrometry, Mass, Electrospray Ionization
  • Structure-Activity Relationship
  • Tandem Mass Spectrometry
  • mRNA Cleavage and Polyadenylation Factors / genetics
  • mRNA Cleavage and Polyadenylation Factors / metabolism*


  • CFT2 protein, S cerevisiae
  • CYC1 protein, S cerevisiae
  • DNA-Binding Proteins
  • MGM101 protein, S cerevisiae
  • Mitochondrial Proteins
  • Mpe1 protein, S cerevisiae
  • Multiprotein Complexes
  • RNA Precursors
  • RNA, Fungal
  • RNA, Messenger
  • Saccharomyces cerevisiae Proteins
  • YSH1 protein, S cerevisiae
  • mRNA Cleavage and Polyadenylation Factors
  • Cytochromes c
  • PAP1 protein, S cerevisiae
  • Polynucleotide Adenylyltransferase
  • Endonucleases