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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.
Molecular Cell ( IF 14.5 ) Pub Date : 2019-02-05 , DOI: 10.1016/j.molcel.2018.12.023
Chris H Hill 1 , Vytautė Boreikaitė 1 , Ananthanarayanan Kumar 1 , Ana Casañal 1 , Peter Kubík 1 , Gianluca Degliesposti 1 , Sarah Maslen 1 , Angelica Mariani 1 , Ottilie von Loeffelholz 2 , Mathias Girbig 1 , Mark Skehel 1 , Lori A Passmore 1
Affiliation  

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.

中文翻译:

定义 mRNA 3' 末端的核酸内切酶的激活需要掺入 8 亚基核心切割和多聚腺苷酸化因子复合物。

切割和多聚腺苷酸化因子 (CPF/CPSF) 是一种多蛋白复合物,对真核 mRNA 3' 末端的形成至关重要。CPF 在特定位点切割前 mRNA 并添加一个 poly(A) 尾。切割反应定义了成熟 mRNA 的 3' 端,因此内切核酸酶的活性受到高度调节。在这里,我们显示重组酵母蛋白重组特定的前 mRNA 切割需要将 Ysh1 核酸内切酶纳入八亚基“CPFcore”复合物。切割还需要辅助切割因子 IA 和 IB,它们结合底物前 mRNA 和 CPF,可能促进活性复合物的组装。使用 X 射线晶体学、电子显微镜和质谱,我们确定了与 Mpe1 结合的 Ysh1 的结构以及 CPFcore 中亚基的排列。一起,
更新日期:2019-02-05
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