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Instruments / Technical Area	Publication Details	Published	Actions
I04-1-Macromolecular Crystallography (fixed wavelength)	Activation of the endonuclease that defines mRNA 3′ ends requires incorporation into an 8-subunit core cleavage and polyadenylation factor complex Chris H. Hill , Vytautė Boreikaitė , Ananthanarayanan Kumar , Ana Casanal , Peter Kubík , Gianluca Degliesposti , Sarah Maslen , Angelica Mariani , Ottilie Von Loeffelholz , Mathias Girbig , Mark Skehel , Lori A. Passmore Molecular Cell DOI: 10.1016/j.molcel.2018.12.023 Diamond Proposal Number(s): [15916] Open Access Show Abstract ▼ 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.	Feb 2019
Krios I-Titan Krios I at Diamond	Architecture of eukaryotic mRNA 3′-end processing machinery Ana Casanal , Ananthanarayanan Kumar , Chris H. Hill , Ashley D. Easter , Paul Emsley , Gianluca Degliesposti , Yuliya Gordiyenko , Balaji Santhanam , Jana Wolf , Katrin Wiederhold , Gillian L. Dornan , Mark Skehel , Carol V. Robinson , Lori A. Passmore Science 546 DOI: 10.1126/science.aao6535 Diamond Proposal Number(s): [15622] Open Access Show Abstract ▼ Abstract: Newly transcribed eukaryotic pre-mRNAs are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four beta-propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate messenger RNA 3′-end processing	Oct 2017

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