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.

Figure 1. Structures of human CPSF-73 and yeast CPSF-100 (Ydh1p)

a, Schematic representation of the structure of human CPSF-73. The β-strands and α-helices are labeled, and the two zinc atoms in the active site are shown as gray spheres. The sulfate ion is shown as a stick model. b, Schematic representation of the structure of yeast CPSF-100. The zinc atoms in the CPSF-73 structure are shown for reference.

Figure 2. The β-CASP domain of CPSF-73 and CPSF-100

Schematic drawing of the β-CASP domain of human CPSF-73 (a) and yeast CPSF-100 (b).

Figure 3. The active site of CPSF-73

a, The zinc binding site for human CPSF-73. The motifs are labeled, and the bridging hydroxide ion is shown as a red sphere. Liganding interactions are indicated in thin magenta lines, and hydrogen-bonding interactions in thin red lines. The arrow indicates the nucleophilic attack from the hydroxide ion. b, The zinc binding site in L1 metallo-β-lactamase.

Figure 4. CPSF-73 possesses endoribonuclease activity

a, Cleavage of 5’ capped SV40 late pre-mRNA (SVL) was performed with wild-type (lane 3) or mutant (lane 5) CPSF-73 at 30 °C for 30 min. 0 min incubations are shown in lanes 2 and 4. Cleavage of 5’ capped adenovirus L3 pre-mRNA (Ad-L3) is shown in lanes 7 and 8. Precursor RNAs (Pre) are shown in lanes 1 and 6. M, MspI-digested pBR322DNA markers. b, Time course analysis of RNA cleavage by CPSF-73. Cleavage of 5’ (lanes 2–6) and 3’ (lanes 9–13) end-labeled SVL pre-mRNAs was performed with wild-type CPSF-73 (500 ng); time points were 5, 10, 15, 20, 30 min. Mutant CPSF-73 (500 ng) did not exhibit ribonuclease activity (lanes 7 and 14).