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

RNA. 2004 Apr;10(4):565-73. doi: 10.1261/rna.5214404.

Abstract

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 Zn(2+)-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 metallo-beta-lactamases, we show that 3' cleavage is metal-dependent, likely reflecting a requirement for tightly protein-bound Zn(2+). Taken together, the available data provide strong evidence that CPSF-73 is the 3' processing endonuclease.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Cleavage And Polyadenylation Specificity Factor / metabolism*
  • Endoribonucleases / metabolism*
  • HeLa Cells
  • Humans
  • Precipitin Tests
  • RNA Precursors / metabolism*
  • RNA Processing, Post-Transcriptional / physiology*
  • RNA, Messenger / biosynthesis
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • mRNA Cleavage and Polyadenylation Factors / genetics
  • mRNA Cleavage and Polyadenylation Factors / metabolism

Substances

  • Cleavage And Polyadenylation Specificity Factor
  • RNA Precursors
  • RNA, Messenger
  • Saccharomyces cerevisiae Proteins
  • YSH1 protein, S cerevisiae
  • mRNA Cleavage and Polyadenylation Factors
  • Endoribonucleases