title: A flexible linker region in Fip1 is needed for efficient mRNA polyadenylation.
abstract: Synthesis of the poly(A) tail of mRNA in Saccharomyces cerevisiae requires recruitment of the polymerase Pap1 to the 3' end of cleaved pre-mRNA. This is made possible by the tethering of Pap1 to the Cleavage/Polyadenylation Factor (CPF) by Fip1. We have recently reported that Fip1 is an unstructured protein in solution, and proposed that it might maintain this conformation as part of CPF, when bound to Pap1. However, the role that this feature of Fip1 plays in 3' end processing has not been investigated. We show here that Fip1 has a flexible linker in the middle of the protein, and that removal or replacement of the linker affects the efficiency of polyadenylation. However, the point of tethering is not crucial, as a fusion protein of Pap1 and Fip1 is fully functional in cells lacking genes encoding the essential individual proteins, and directly tethering Pap1 to RNA increases the rate of poly(A) addition. We also find that the linker region of Fip1 provides a platform for critical interactions with other parts of the processing machinery. Our results indicate that the Fip1 linker, through its flexibility and protein/protein interactions, allows Pap1 to reach the 3' end of the cleaved RNA and efficiently initiate poly(A) addition.
authors.affiliation: Department of Biochemistry, Tufts University School of Medicine and the Sackler Graduate School of Biomedical Sciences, Boston, Massachusetts 02111, USA.
authors.forename: Chukwudi
authors.initials: C
authors.lastname: Ezeokonkwo
authors.forename: Alexander
authors.initials: A
authors.lastname: Zhelkovsky
authors.forename: Rosanna
authors.initials: R
authors.lastname: Lee
authors.forename: Andrew
authors.initials: A
authors.lastname: Bohm
authors.forename: Claire L
authors.initials: CL
authors.lastname: Moore
mesh_terms: D026905:Amino Acid Transport Systems / Q000378:metabolism*; D001483:Base Sequence; D000073718:Pancreatitis-Associated Proteins; D026723:Polyadenylation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D026905:Amino Acid Transport Systems; C429560:BAP3 protein, S cerevisiae; C450579:FIP1 protein, S cerevisiae; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors