Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease

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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?Å 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?Å resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-?-lactamase domain and a novel ?-CASP (named for metallo-?-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.

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