mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure. Here we report that a mutation in the SPB8 (YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure. Specifically, we find that the spb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs. The presence of this mutation also allows for the detection of mRNA species trimmed from the 3 end. These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3-5 mRNA degradation intermediates in yeast.The function of the poly(A) tail on mRNA in eucaryotes is the subject of much research. It has been shown that in the yeast Saccharomyces cerevisiae, the poly(A) tail acts to enhance the translation of the mRNA (reviewed in reference 36). This activity requires the poly(A)-binding protein Pab1p, which, through an interaction with a protein complex recognizing the cap structure, is thought to stimulate the binding of ribosomes to the 5Ј end of the mRNA (23,(39)(40)(41). Another potential function of the poly(A) tail is to stabilize mRNA. Several independent observations support this hypothesis. A series of detailed studies of both yeast and mammalian cells has documented that mRNA deadenylation usually precedes mRNA degradation (reviewed in reference 8). More specifically, it has been shown that in yeast, mRNA decapping, the initiating event in the degradation of the majority of mRNAs, occurs after deadenylation (9,26,27). Following decapping, these mRNAs are destroyed by the 5Ј-3Ј exoribonuclease Xrn1p (20,26). It has also been shown that decreasing the rate of poly(A) tail removal by mutagenizing mRNA (for example, see reference 28) results in lower rates of mRNA degradation.Degradation through the pathway of decapping and then digestion by the 5Ј-3Ј exonuclease Xrn1p is not the sole means by which mRNA is degraded in yeast. Three key observations form the basis for this conclusion. First, targeted disruption of either the decapping enzyme gene DCP1 or the exonuclease gene XRN1 does not lead to cell inviability or greater than four-to fivefold stabilization of mRNAs that are normally unstable (4,20). Second, the disruption of these genes does not change the stability of the most stable yeast mRNAs, such as PGK1 or ACT1, by more than a factor of 2 (4, 27). Finally, for yeast strains containing a disruption of the XRN1 gene or a mutation in the Dcp1 protein, the existence of mRNA species trimmed from the 3Ј end has been reported (27).A common feature of both the mRNA translation and degradation reactions is that the cap structure and the poly(A) tail appear to be involved in their regulation (reviewed in reference 42). The possibility that the roles of these two structures in the degradation reaction are functionally linked was supported by the recent report that mutations in the yeast decapping enzyme Dcp1p...

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We report here the characterization of a bypass suppressor of pab1⌬ which leads to a fourfold stabilization of the unstable MFA2 mRNA. Cloning of the wild-type gene for that suppressor reveals that it is identical to PAT1 (YCR077c), a gene whose product was reported to interact with Top2p. PAT1 is not an essential gene, but its deletion leads to a thermosensitive phenotype. Further analysis has shown that PAT1 is allelic with mrt1-3, a mutation previously reported to affect decapping and to bypass suppress pab1⌬, as is also the case for dcp1, spb8, and mrt3. Coimmunoprecipitation experiments show that Pat1p is associated with Spb8p. On sucrose gradients, the two proteins cosediment with fractions containing the polysomes. In the absence of Pat1p, however, Spb8p no longer cofractionates with the polysomes, while the removal of Spb8p leads to a sharp decrease in the level of Pat1p. Our results suggest that some of the factors involved in mRNA degradation could be associated with the mRNA that is still being translated, awaiting a specific signal to commit the mRNA to the degradation pathway.A general pathway of mRNA degradation in the yeast Saccharomyces cerevisiae has been described in which mRNAs are deadenylated prior to decapping and then degraded in the 5Ј-to-3Ј direction by the exonuclease Xrn1p (4, 12). The decapping enzyme has been identified in yeast and named Dcp1p (5). In most yeast strains, depletion of Dcp1p leads to a slowgrowth phenotype and to stabilization of mRNAs that are accumulated in a capped and oligoadenylated form (5). However, to become functional, Dcp1p requires the activity of Dcp2p, a putative pyrophosphatase (14). Recently, a polypeptide (M r 70,000) copurifying with Dcp1p has been identified as Ssa1p or Ssa2p, Hsp70 family members (50). Interestingly, two mutations that inhibit decapping, vps16 and mrt1, enhance the interaction of Ssa1p or Ssa2p with Dcp1p. This observation suggests that Ssa1p or Ssa2p could be involved in regulating the activity of Dcp1p.In addition to Dcp1p and Dcp2p, other factors might regulate the decapping activity, since several mutations have been isolated that lead to stabilization of capped, oligoadenylated mRNAs. The best characterized of these factors is Spb8p (6), a protein containing an Sm-like domain that has also been referred to as Lsm1p (40). The Sm motif is found in a set of proteins that interact with the small nuclear RNAs involved in mRNA splicing. However, Spb8p is distinguished from other Sm or Lsm proteins by not being associated with any known small nuclear RNA (29, 38). Mutations within three other loci named mrt1, mrt3, and vps16 lead to an accumulation of capped, oligoadenylated mRNAs (21, 50). While VPS16 has been characterized (22, 50), the genes for mrt1 and mrt3 have not yet been cloned.Several lines of evidence indicate the existence of a link between mRNA translation and degradation (23). It is now well established that the two structures present on the ends of an mRNA, the cap at the 5Ј end and the poly(A) tail at the 3Ј end, act s...

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.