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Activation of the endonuclease that defines mRNA 3′ ends requires incorporation into an 8-subunit core cleavage and polyadenylation factor complex

DOI: 10.1016/j.molcel.2018.12.023 DOI Help

Authors: Chris H. Hill (MRC Laboratory of Molecular Biology) , Vytautė Boreikaitė (MRC Laboratory of Molecular Biology) , Ananthanarayanan Kumar (MRC Laboratory of Molecular Biology) , Ana Casanal (MRC Laboratory of Molecular Biology) , Peter Kubík (MRC Laboratory of Molecular Biology) , Gianluca Degliesposti (MRC Laboratory of Molecular Biology) , Sarah Maslen (MRC Laboratory of Molecular Biology) , Angelica Mariani (MRC Laboratory of Molecular Biology) , Ottilie Von Loeffelholz (Université de Strasbourg) , Mathias Girbig (MRC Laboratory of Molecular Biology) , Mark Skehel (MRC Laboratory of Molecular Biology) , Lori A. Passmore (MRC Laboratory of Molecular Biology)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Molecular Cell

State: Published (Approved)
Published: February 2019
Diamond Proposal Number(s): 15916

Open Access Open Access

Abstract: 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.

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

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 13/02/2019 10:28


Discipline Tags:

Genetics Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)