title: The interaction of Pcf11 and Clp1 is needed for mRNA 3'-end formation and is modulated by amino acids in the ATP-binding site.
abstract: Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage-Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1-Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3'-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1-Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1.
authors: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors: Mohamed A
authors: MA
authors: Ghazy
authors: James M B
authors: JM
authors: Gordon
authors: Susan D
authors: SD
authors: Lee
authors: Badri Nath
authors: BN
authors: Singh
authors: Andrew
authors: A
authors: Bohm
authors: Michael
authors: M
authors: Hampsey
authors: Claire
authors: C
authors: Moore
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D009154:Mutation; D010641:Phenotype; D026723:Polyadenylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D026721:RNA 3' End Processing; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C517046:Clp1 protein, S cerevisiae; C512637:PCF11 protein, S cerevisiae; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D000255:Adenosine Triphosphate