Sbp1p Affects Translational Repression and Decapping in Saccharomyces cerevisiae.

Control of mRNA stability plays a crucial role in regulating gene expression. In eukaryotes, there are two major mRNA decay pathways that both require deadenylation before decay occurs. In yeast, the most common mechanism of mRNA turnover is where deadenylation is followed by decapping. Once the mRNA is deadenylated, the 5′ m7G cap structure is removed by the Dcp1p/Dcp2p decapping complex and the mRNA is subsequently degraded 5′ to 3′ (12, 20, 33). In addition to the decapping enzyme complex, there are a set of factors that act as decapping activators, including Dhh1p, Pat1p, and the Lsm1-7p complex (4, 10, 18, 44, 45). Alternatively, once deadenylation occurs, the mRNA can be degraded by the slower 3′-to-5′ pathway, which is mediated by the exosome (1, 5, 34; for a review, see reference 47). Decapping is an important step in 5′-to-3′ decay, as it permits transcript degradation and is a site of regulation. For instance, short-lived mRNAs are decapped more rapidly than long-lived mRNAs (33, 34). In addition, nonsense transcripts, which are targeted for nonsense-mediated decay, can bypass the need for deadenylation and are rapidly decapped (35). Furthermore, in mammalian cells, the AU-rich element binding proteins TTP and BRF-1 have been proposed to recruit the decapping enzyme to their targets (31). Given these observations, to understand differential mRNA stability, it is important to determine the events that contribute to the control of mRNA decapping. Several lines of evidence indicate that a key contribution to the control of mRNA decapping is that translation and mRNA decapping are in competition. First, in yeast, mRNAs poorly translated because of secondary structures in their 5′ untranslated region (UTR) or weak AUG context are decapped much faster than their wild-type counterparts (27, 34). Second, mutations in translation initiation factors such as eIF4E, the cap binding protein, and Prt1p, which is part of the eIF3 complex, also lead to faster degradation of mRNAs in yeast (39). Third, eIF4E inhibits the decapping activity of Dcp2p in vitro (38). Evidence that there is a relationship between translation and mRNA decapping also comes from the analysis of P bodies (also referred to as Dcp1 bodies or GW182 bodies). P bodies are discrete cytoplasmic foci that contain both nontranslating mRNA and mRNA-decapping factors and are sites where decapping can occur (11, 41). P bodies are conserved in yeast and mammals in that both yeast and mammalian P bodies contain both subunits of the decapping enzyme and decapping activators (11, 14, 22, 30, 41, 46). However, mammalian P bodies differ from yeast P bodies in that they can contain translation initiation factors (14, 26). In addition, mammalian P bodies also contain the RNA binding protein GW182 and the Argonaute proteins, which are involved in micro-RNA function (14, 23, 28, 29). When mRNAs exit translation, they accumulate in P bodies to be targeted for degradation, as disruption of translation has an effect on the size and number of P bodies (43). For instance, trapping mRNAs in translation by using the elongation inhibitor cycloheximide results in loss of P bodies, while blocking of translation initiation results in an increase in both the size and number of P bodies, and decapping is accelerated (3, 39, 41, 43). Furthermore, two independent pathways have recently been identified for translational repression of mRNAs in yeast, and simultaneous disruption of these pathways leads to loss of P-body formation (9). Thus, P-body formation correlates with the status of translation in the cell. It is unclear whether the large P bodies visualized by fluorescence microscopy are required for decapping, or if decapping can occur in smaller P-body complexes containing the decapping machinery. Recent work has begun to elucidate the factors and mechanisms by which mRNAs exit translation and assemble into a translationally repressed mRNP containing decapping factors. Several lines of evidence indicate that the decapping activators Dhh1p and Pat1p mediate the transition from translation to mRNA decay (9). First, overexpression of either Dhh1p or Pat1p causes global translational repression, as seen by decreased polysomes and a reciprocal increase in P bodies. Second, Dhh1p and Pat1p are activators of decapping, as loss of their activities leads to an increased mRNA half-life (10, 44). Third, strains lacking Dhh1p and Pat1p are defective for translational repression and subsequent P-body formation under conditions of glucose deprivation (9). It appears that both Dhh1p and Pat1p act independently for translational repression, as strains lacking either Dhh1p or Pat1p can still repress translation. This suggests that there are two potential mechanisms by which an mRNA can exit translation. An important goal is to further identify factors involved in this transition and the nature of their function. In this work, we identify Sbp1p as a high-copy-number suppressor of both dcp1-2 and dcp2-7 mutations. Sbp1p, previously referred to as Ssb1p, was identified as a major single-stranded RNA binding protein containing two RNA recognition motifs that are separated by an RGG box (24, 25). It should be noted that the standard name for this factor in the yeast database is now Sbp1p, and we will refer to it by this name in this work. Sbp1p was initially localized to the nucleolus and can coimmunoprecipitate with snoRNA10 and snoRNA11, suggesting some function in ribosome biogenesis, although no specific defect in rRNA processing or ribosome biogenesis has been reported in sbp1Δ strains (8, 24, 25). We find that overexpression of Sbp1p can suppress mRNA decay defects in some decapping-defective strains. Furthermore, we show that Sbp1p acts to promote translational repression of mRNA in conjunction with Dhh1p and subsequent mRNA localization to P bodies. We show here that Sbp1p plays a role in the transition of mRNA from translation to mRNA degradation.