Capped mRNA degradation intermediates accumulate in the yeast spb8-2 mutant

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 5′ end of the mRNA (23, 39–41). 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 5′-3′ 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 5′-3′ 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 3′ 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 and the unidentified gene products of the MRT1 and MRT3 genes, which also appear to regulate mRNA decapping rates, can allow yeast cells to survive in the absence of Pab1p (17). These mutations were therefore suggested to allow for cell viability in the absence of Pab1p by stabilizing the mRNA to an extent that allowed for sufficient expression in the absence of Pab1p. We originally chose to further explore the essential roles of Pab1p in yeast in order to define the mechanistic roles of Pab1p and poly(A) in mRNA metabolism (33, 34). A series of genetic suppression experiments identified mutations in other yeast genes which allowed for cell growth in the absence of Pab1p. Most of these mutations resulted in aberrant 60S ribosomal subunit production. Other laboratories have also identified similar types of pab1Δ bypass suppressors (43). To identify proteins involved in mRNA degradation, including those that functionally interact with Pab1p and the poly(A) tail during this process, we chose to focus our newer studies on those pab1Δ bypass suppressor mutations that did not alter the levels of the ribosomal subunits. Based on published data (17), we reasoned that such mutations could lead to viability in the absence of Pab1p by stabilizing the mRNA. The current lack of sequence information on genes other than DCP1 and XRN1 that are known to alter the pathway of 5′-3′ mRNA degradation in yeast provided us with further incentive to pursue this avenue of investigation. Here we report that a null mutation in the nonessential yeast SPB8 (YJL124c) gene leads to bypass suppression of a PAB1 deletion without altering ribosomal subunit levels. Spb8p was found to contain an Sm-like domain. Mutations within Spb8p led to the accumulation of capped, deadenylated degradation intermediates and in some cases the stabilization of mRNA. Cap stabilization was so complete in the spb8-2 strain that 3′-5′ mRNA degradation intermediates also became readily detectable. These data support the hypotheses that Spb8p is needed for normal rates of mRNA decapping in yeast and that Spb8p allows for rates of decapping and 5′-3′ degradation that preclude the detection of 3′-5′ degradative intermediates.