Yeast mRNA poly(A) tail length control can be reconstituted in vitro in the absence of Pab1p-dependent poly(A) nuclease activity

Abstract

Regulation of poly (A) tail length during mRNA 3′-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly (A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3′-end processing than that of cytoplasmic Pablp. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3′-end formation were purified from wild-type and pan mutant cells. In both situations, 3′-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.