The WD-repeat protein Pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex

Abstract

In the yeast Saccharomyces cerevisiae, pre‐mRNA 3′‐end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)‐binding protein I (Pab1p). We report the characterization of Pfs2p, a WD‐repeat protein previously identified in a multiprotein complex carrying PF I–Pap1p activity. The 3′‐end‐processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one‐step affinity purification method that exploits protein A‐tagged Pfs2p, we showed that this protein is part of a CF II–PF I complex. Pull‐down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II–PF I and CF IA. These results show that Pfs2p plays an essential role in 3′‐end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.