title: Evidence that polyadenylation factor CPSF-73 is the mRNA 3' processing endonuclease.
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.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Ryan
authors.forename: Olga
authors.initials: O
authors.lastname: Calvo
authors.forename: James L
authors.initials: JL
authors.lastname: Manley
mesh_terms: D020816:Amino Acid Motifs; D039223:Cleavage And Polyadenylation Specificity Factor / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D011233:Precipitin Tests; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D039223:Cleavage And Polyadenylation Specificity Factor; D012322:RNA Precursors; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C450401:YSH1 protein, S cerevisiae; D039221:mRNA Cleavage and Polyadenylation Factors; D004722:Endoribonucleases