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

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Eukaryotic pre-mRNA processing involves 3'-end cleavage and polyadenylation.
• The specific endonuclease enzyme responsible for this crucial step remained unidentified.
• Previous studies suggested CPSF-73 as a potential candidate, but lacked direct experimental confirmation.

Purpose of the Study:

• To determine the crystal structure of human CPSF-73 and yeast CPSF-100.
• To investigate the enzymatic activity and substrate specificity of CPSF-73.
• To provide direct experimental evidence for the role of CPSF-73 in pre-mRNA processing.

Main Methods:

• X-ray crystallography was used to determine the structures of human CPSF-73 and yeast CPSF-100.
• Purification of recombinant CPSF-73 for in vitro enzymatic assays.
• Site-directed mutagenesis to probe the function of the active site zinc ions.

Main Results:

• Crystal structures revealed CPSF-73 and CPSF-100 possess metallo-beta-lactamase and beta-CASP domains.
• The active site of CPSF-73, containing two zinc ions, is located at the interface of these domains.
• Purified CPSF-73 demonstrated RNA endonuclease activity, which was abolished by mutations affecting zinc binding.

Conclusions:

• CPSF-73 is directly confirmed as the endonuclease responsible for pre-mRNA 3'-end processing.
• The structural and biochemical data elucidate the mechanism of CPSF-73 in RNA maturation.
• This discovery clarifies a fundamental step in eukaryotic gene expression.