The yeast translation factor eIF4G associates with both the cap-binding protein eIF4E and the poly(A)-binding protein Pab1p. Here we report that the two yeast eIF4G homologs, Tif4631p and Tif4632p, share a conserved Pab1p-binding site. This site is required for Pab1p and poly(A) tails to stimulate the in vitro translation of uncapped polyadenylylated mRNA, and the region encompassing it is required for the cap and the poly(A) tail to synergistically stimulate translation. This region on Tif4631p becomes essential for cell growth when the eIF4E binding site on Tif4631p is mutated. Pab1p mutations also show synthetic lethal interactions with eIF4E mutations. These data suggest that eIF4G mediates poly(A) tail stimulated translation in vitro, and that Pab1p and the domain encompassing the Pab1p-binding site on eIF4G can compensate for partial loss of eIF4E function in vivo.

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The removal of the mRNA poly(A) tail in the yeast Saccharomyces cerevisiae is stimulated by the poly(A)-binding protein (Pab1p). A large scale purification of the Pab1p-stimulated poly(A) ribonuclease (PAN) identifies a 76-kDa and two 135-Da polypeptides as candidate enzyme subunits. Antibodies against the Pan1p protein, which is the minor 135-kDa protein in the preparation, can immunodeplete Pan1p but not PAN activity. The protein sequence of the major 135-kDa protein, Pan2p, reveals a novel protein that was also found in the previously reported PAN purification (Sachs, A. B., and Deardorff, J. A. (1992) Cell 70, 961-973). Deletion of the non-essential PAN2 gene results in an increase of the average length of mRNA poly(A) tails in vivo, and a loss of Pab1p-stimulated PAN activity in crude extracts. These data confirm that Pan2p and not Pan1p is required for PAN activity, and they suggest that ribonucleases other than the Pab1p-stimulated PAN are capable of shortening poly(A) tails in vivo.

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Interaction of the poly(A) binding protein, Pab1p, with mRNA plays an important role in gene expression. This work describes an analysis of pab1 mutants in Saccharomyces cerevisiae. Yeast pab1 mutants were found to be sensitive to elevated concentrations of copper (Cu) and 3-aminotriazole (3-AT) in the growth medium. This phenotype arises because these pab1 mutants underaccumulate mRNA, including the CUP1 and HIS3 mRNAs, the products of which are required for Cu and 3-AT resistance, respectively. To determine the cause of the mRNA underaccumulation, mRNA turnover and production were examined in the pab1-53 mutant. It was found that although the pattern of mRNA decay was altered, and substantial decapping of polyadenylated mRNA could be detected, mRNA was not destabilized in this strain. It was also found that the pab1 mutant was impaired in the production of mRNA. These data show that the decreased level of mRNA in the pab1-53 mutant arises from poor production, and they suggest that yeast Pab1p is involved in an aspect of nuclear mRNA metabolism. They also indicate that deadenylation can be uncoupled from decapping without significant changes in an mRNA's stability, and that this uncoupling can be tolerated by yeast.