1. Activation of the Endonuclease that Defines mRNA 3' Ends Requires Incorporation into an 8-Subunit Core Cleavage and Polyadenylation Factor Complex.
- Author
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Hill CH, Boreikaitė V, Kumar A, Casañal A, Kubík P, Degliesposti G, Maslen S, Mariani A, von Loeffelholz O, Girbig M, Skehel M, and Passmore LA
- Subjects
- Cryoelectron Microscopy, Crystallography, X-Ray, Cytochromes c genetics, Cytochromes c metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Endonucleases genetics, Enzyme Activation, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Molecular Docking Simulation, Multiprotein Complexes, Polynucleotide Adenylyltransferase genetics, Polynucleotide Adenylyltransferase metabolism, Protein Binding, Protein Interaction Domains and Motifs, RNA Precursors genetics, RNA, Fungal genetics, RNA, Messenger genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins genetics, Spectrometry, Mass, Electrospray Ionization, Structure-Activity Relationship, Tandem Mass Spectrometry, mRNA Cleavage and Polyadenylation Factors genetics, Endonucleases metabolism, Polyadenylation, RNA Precursors metabolism, RNA, Fungal metabolism, RNA, Messenger metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins metabolism, mRNA Cleavage and Polyadenylation Factors metabolism
- Abstract
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 "CPF
core " 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., (Copyright © 2019 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.)- Published
- 2019
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