1. Department of Biological Sciences, Columbia University, New York, New York 10027, USA
2. Present addresses: Array BioPharma Inc., Boulder, Colorado 80301, USA (H.Z.); Johnson & Johnson Pharmaceutical Research & Development LLC, San Diego, California 92121, USA (D.G.).
3. These authors contributed equally to this work.

Correspondence to: Liang Tong¹ Correspondence and requests for materials should be addressed to L.T. (Email: ltong@columbia.edu). The atomic coordinates have been deposited at the Protein Data Bank (accession numbers 2I7T, 2I7V and 2I7X).

Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end^{1, 2, 3, 4, 5, 6, 7, 8}. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known^{4, 9, 10}. 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^{10, 11, 12, 13, 14, 15}, 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) domain¹². 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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