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

CR Mandel, S Kaneko, H Zhang, D Gebauer… - Nature, 2006 - nature.com
CR Mandel, S Kaneko, H Zhang, D Gebauer, V Vethantham, JL Manley, L Tong
Nature, 2006nature.com
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 …
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 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-β-lactamase domain and a novel β-CASP (named for metallo-β-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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