Evidence that polyadenylation factor CPSF-73 is the mRNA 3' processing endonuclease

Area of Science:

• Molecular Biology
• RNA Processing
• Gene Expression

Background:

• mRNA 3' end polyadenylation involves endonucleolytic cleavage and poly(A) tail synthesis.
• This complex process requires over a dozen polypeptides, with the endonuclease identity remaining unknown.
• The cleavage-polyadenylation specificity factor (CPSF) complex plays a crucial role.

Purpose of the Study:

• To identify the endonuclease responsible for mRNA 3' end cleavage.
• To investigate the role of CPSF-73 in this process.
• To elucidate the catalytic mechanism and metal dependence of the cleavage reaction.

Main Methods:

• Site-specific labeling and UV-cross-linking experiments in HeLa nuclear extract.
• Analysis of mutations in the yeast CPSF-73 homolog.
• Biochemical assays to determine metal dependence of 3' cleavage.

Main Results:

• CPSF-73 was identified as one of only two polypeptides contacting the cleavage site in an AAUAAA-dependent manner.
• Mutations in the putative metallo-beta-lactamase domain of yeast CPSF-73 resulted in lethality.
• 3' cleavage was shown to be metal-dependent, specifically requiring Zn(2+).

Conclusions:

• CPSF-73 is the long-sought endonuclease responsible for 3' end processing of mRNA.
• CPSF-73 possesses hydrolytic enzyme activity, likely belonging to the metallo-beta-lactamase family.
• The 3' cleavage reaction is metal-dependent, highlighting the role of tightly bound Zn(2+).