Mandel Corey R, Kaneko Syuzo, Zhang Hailong, Gebauer Damara, Vethantham Vasupradha, Manley James L, Tong Liang
Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
Nature. 2006 Dec 14;444(7121):953-6. doi: 10.1038/nature05363. Epub 2006 Nov 26.
Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 A resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 A resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-beta-lactamase domain and a novel beta-CASP (named for metallo-beta-lactamase, CPSF, Artemis, Snm1, Pso2) domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.
大多数真核生物信使核糖核酸前体（前体mRNA）都要经历广泛的成熟加工过程，包括在3'端进行切割和聚腺苷酸化。尽管已经鉴定出许多切割反应所需的蛋白质，但核酸内切酶的身份仍不清楚。最近的分析表明，切割和聚腺苷酸化特异性因子（CPSF-73）的73 kDa亚基可能是参与此反应及相关反应的核酸内切酶，不过尚无直接数据证实这一点。在此，我们报告了人CPSF-73与锌离子和可能模拟底物磷酸基团的硫酸盐结合的2.1埃分辨率晶体结构，以及相关酵母蛋白CPSF-100（Ydh1）的2.5埃分辨率晶体结构。CPSF-73和CPSF-100都包含两个结构域，一个金属β-内酰胺酶结构域和一个新的β-CASP（以金属β-内酰胺酶、CPSF、Artemis、Snm1、Pso2命名）结构域。CPSF-73的活性位点有两个锌离子，位于两个结构域的界面处。纯化的重组CPSF-73具有RNA核酸内切酶活性，而破坏活性位点锌结合的突变会消除这种活性。我们的研究提供了首个直接实验证据，证明CPSF-73是前体mRNA 3'端加工核酸内切酶。
