4.4 Article

Evidence that polyadenylation factor CPSF-73 is the rnRNA 3′ processing endonuclease

Journal

RNA
Volume 10, Issue 4, Pages 565-573

Publisher

COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.5214404

Keywords

polyadenylation; mRNA; endonuclease; Zn2+-dependent

Funding

  1. NIGMS NIH HHS [GM-28983, R01 GM028983] Funding Source: Medline

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Generation of the polyadenylated 3' end of an mRNA requires an endonucleolytic cleavage followed by synthesis of the poly(A) tail. Despite the seeming simplicity of the reaction, more than a dozen polypeptides are required, and nearly all appear to be necessary for the cleavage reaction. Because of this complexity, the identity of the endonuclease has remained a mystery. Here we present evidence that a component of the cleavage-polyadenylation specificity factor CPSF-73 is the long-sought endonuclease. We first show, using site-specific labeling and UV-cross-linking, that a protein with properties of CPSF-73 is one of only two polypeptides in HeLa nuclear extract to contact the cleavage site in an AAUAAA-dependent manner. The recent identification of CPSF-73 as a possible member of the metallo-beta-lactamase family of Zn2+-dependent hydrolytic enzymes suggests that this contact may identify CPSF-73 as the nuclease. Supporting the significance of the putative hydrolytic lactamase domain in CPSF-73, we show that mutation of key residues predicted to be required for activity in the yeast CPSF-73 homolog result in lethality. Furthermore, in contrast to long held belief, but consistent with properties of metal lo-beta-lactamases, we show that 3' cleavage is metal-dependent, likely reflecting a requirement for tightly protein-bound Zn2+. Taken together, the available data provide strong evidence that CPSF-73 is the 3' processing endonuclease.

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