Evidence that polyadenylation factor CPSF-73 is the mRNA 3' processing endonuclease.
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.
Ryan
Kevin
K
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Calvo
Olga
O
Manley
James L
JL
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
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