Letter | Published:

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′-end1,2,3,4,5,6,7,8. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known4,9,10. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions10,11,12,13,14,15, 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-β-lactamase domain and a novel β-CASP (named for metallo-β-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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Acknowledgements

We thank K. Ryan for discussions; B. Tweel for characterizing the fungus; R. Abramowitz, J. Schwanof and X. Yang for setting up the X4A beamline at the NSLS; and J. Khan and Y. Shen for help with data collection at the synchrotron. This research is supported in part by grants from the National Institutes of Health.

Author information

Competing interests

The atomic coordinates have been deposited at the Protein Data Bank (accession numbers 2I7T, 2I7V and 2I7X). Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Correspondence to Liang Tong.

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Supplementary Notes

This file contains Supplementary Results, Supplementary Methods, Supplementary Tables 1 and 1 and Supplementary Figures 1–9. (PDF 3416 kb)

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Further reading

Figure 1: Structures of human CPSF-73 and yeast CPSF-100 (Ydh1).
Figure 2: The β-CASP domain of CPSF-73 and CPSF-100.
Figure 3: The active site of CPSF-73.
Figure 4: CPSF-73 possesses endoribonuclease activity.

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