THE FIP1 GENE ENCODES A COMPONENT OF A YEAST PRE-MESSENGER-RNA POLYADENYLATION FACTOR THAT DIRECTLY INTERACTS WITH POLY(A) POLYMERASE

We have identified an essential gene, called FIP1, encoding a 327 amino acid protein interacting with yeast poly(A) polymerase (PAP1) in the two-hybrid assay, Recombinant FIP1 protein forms a 1:1 complex with PAP1 in vitro. At 37 degrees C, a thermosensitive allele of FIP1 shows a shortening of poly(A) tails and a decrease in the steady-state level of actin transcripts. When assayed for 3'-end processing in vitro, fip1 mutant extracts exhibit normal cleavage activity, but fail to polyadenylate the upstream cleavage product. Polyadenylation activity is restored by adding polyadenylation factor I (PF I). Antibodies directed against FIP1 specifically recognize a polypeptide in these fractions. Coimmunoprecipitation experiments reveal that RNA14, a subunit of cleavage factor I (CF I), directly interacts with FIP1, but not with PAP1, We propose a model in which PF I tethers PAP1 to CF I, thereby conferring specificity to poly(A) polymerase for pre-mRNA substrates.