title: Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: Corey R
authors.initials: CR
authors.lastname: Mandel
authors.forename: Syuzo
authors.initials: S
authors.lastname: Kaneko
authors.forename: Hailong
authors.initials: H
authors.lastname: Zhang
authors.forename: Damara
authors.initials: D
authors.lastname: Gebauer
authors.forename: Vasupradha
authors.initials: V
authors.lastname: Vethantham
authors.forename: James L
authors.initials: JL
authors.lastname: Manley
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
mesh_terms: D001665:Binding Sites; D039223:Cleavage And Polyadenylation Specificity Factor / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C450560:CFT2 protein, S cerevisiae; D039223:Cleavage And Polyadenylation Specificity Factor; D012322:RNA Precursors; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D004720:Endonucleases