Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo extensive maturational processing, including 5'-end capping, splicing, and 3'-end cleavage and polyadenylation. The addition of a poly(A) tail is important for mRNA stability, and enhances mRNA transport to the cytoplasm and mRNA translation.

The 3'-end processing events include cleavage at a specific site in the pre-mRNA followed by the addition of the poly(A) tail. In mammals, the cleavage site is defined by an upstream AAUAAA motif and a downstream U or G/U-rich element.

A large number of protein factors have been identified that are crucial for pre-mRNA 3'-end processing. These proteins form several sub-complexes, such as the cleavage and polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF). CPSF contains 5 subunits, CPSF-30, -73, -100, -160, and Fip1, and CstF contains 3 subunits, Cstf-50, -64, and -77. CPSF-160 recognizes the upstream AAUAAA motif, and CstF-64 recognizes the downstream U- or G/U-rich element.

• The crystal structures of human CPSF-73, and its weak sequence homolog yeast Ydh1p (CPSF-100), have been determined.


• The structures of CPSF-73 and CPSF-100 contain an N-terminal metallo-b-lactamase domain and a novel b-CASP domain. A segment of 60 residues after the b-CASP domain contributes to the N-terminal metallo-b-lactamase domain.


• CPSF-73 binds two zinc ions, each in an octahedral coordination. A sulfate ion in the structure is a good mimic of the phosphate group of the pre-mRNA substrate.


• The active site in CPSF-73 is located deep in the interface between the metallo-b-lactamase domain and the b-CASP domain.


• Despite having a similar overall structure, the zinc ligands are absent in CPSF-100. This subunit cannot bind zinc and is inactive.


• RNA cleavage assays show that CPSF-73 possesses predominantly non-specific endoribonuclease activity.


• The structural and biochemical studies provide direct experimental evidence that CPSF-73 is the nuclease for the cleavage reaction of pre-mRNA 3'-end processing.


• The crystal structures of the HAT domain of murine CstF-77 as well as its HAT-C subdomain have been determined.


• The HAT domain contains two subdomains, a HAT-N domain with 5 HAT repeats and a HAT-C domain with 7 repeats.


• The HAT domain structure is a highly extended dimer, spanning about 165 A. The dimer interface is extensive, and the residues in the interface are mostly conserved.


• Analytical ultracentrifugation and yeast two-hybrid studies confirm that HAT domain can dimerize in solution.


• The structural, biochemical and biophysical studies suggest CstF-77 may dimerize during its function in pre-mRNA 3'-end processing.

Publications from this project

• Mandel CR, Kaneko S, Zhang H, Gebauer D, Vethantham V, Manley JL, Tong L. (2006). Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease. Nature, 444, 953-956. Reprint(PDF)
• Bai Y, Auperin TC, Chou C-Y, Chang G-G, Manley JL, Tong L. (2007). Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors. Mol. Cell. 25, 863-875. Reprint(PDF)
• Mandel CR, Gebauer D, Zhang H, Tong L. (2006). A serendipitous discovery that in situ proteolysis is essential for the crystallization of yeast CPSF-100 (Ydh1p). Acta Cryst. F62, 1041-1045. Reprint(PDF)
• Bai Y, Auperin TC, Tong L. (2007). The use of in situ proteolysis in the crystallization of murine CstF-77. Acta Cryst. F63, 135-138. Reprint(PDF)
• Mandel CR, Tong L. (2007). How to get all "A"s in polyadenylation. Structure. 15, 1024-1026. Reprint(PDF)
• Mandel CR, Bai Y, Tong L. (2008). Protein factors in pre-mRNA 3'-end processing Cell. Mol. Life Sci.. 65, 1099-1122. Reprint(PDF)

Funding for this project

• NIH GM077175