The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex.
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.
Ohnacker
M
M
Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Barabino
S M
SM
Preker
P J
PJ
Keller
W
W
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D008969:Molecular Sequence Data; D009154:Mutation; D039102:Poly(A)-Binding Proteins; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors
C512636:HRP1 protein, S cerevisiae; C404156:Pfs2 protein, S cerevisiae; D039102:Poly(A)-Binding Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D011062:Polynucleotide Adenylyltransferase