Following an abrupt shift from a fermentable to a nonfermentable carbon source, S. cerevisiae
displays a diverse array of adaptive changes in gene expression at both the transcriptional and translational levels. The shift from a fermentable carbon source, glucose, to a nonfermentable carbon source, glycerol, resulted in a marked reduction in overall translation of mRNAs. The accumulation of 80S monosomes and ribosomal subunits after the carbon source shift indicated that the initiation phase of translation was inhibited. Two major mechanisms of downregulating global translation initiation in S. cerevisiae
have been described. In the first case, amino acid starvation induces phosphorylation of initiation factor eIF2α at serine 51 by the Gcn2p kinase, which results in a reduced concentration of 40S subunits, which carry initiator tRNA-eIF2 complexes, and subsequent derepression of GCN4
mRNA translation (20
). In the second case, induction of a diauxic shift by gradual glucose exhaustion leads to degradation of initiation factor eIF4G (4
), resulting in limiting concentrations of the cap-binding protein complexes needed to recruit 40S subunits onto mRNAs. In the case of the rapid shift from glucose to glycerol medium described here, translational inhibition was still observed in a yeast strain that expressed a mutated eIF2α whose phosphorylation site at serine 51 had been changed to alanine. In addition, eIF4G was not degraded after shifting S. arevisiae
from glucose to glycerol medium at a time when translation was markedly depressed (K. Kuhn, unpublished data). Therefore, the mechanism of global translational repression that occurs following a rapid withdrawal of glucose must differ from that described during amino acid starvation or the diauxic shift. Recently, mutants in the cyclic-AMP-dependent kinase pathway have been shown to be resistant to translational inhibition after glucose withdrawal (1
), suggesting that this signaling pathway is involved in translational regulation. We noted an increased abundance of several mRNAs encoding catalytic subunits of the cyclic-AMP-dependent kinase protein kinase A (PKA) upon carbon source shift (TPK1
[3.8-fold], and TPK3
[1.5-fold]). In addition, the relative abundance of the BCY1
mRNA, which encodes the PKA regulatory subunit, was also induced twofold. These findings support the hypothesis by Ashe et al. (1
) that increased concentration and activity of PKA can play a role in the inhibition of translation following a shift from a fermentable to a nonfermentable carbon source.
Loss of polysome-associated RP mRNAs after a shift to glycerol.
Mammalian RP mRNAs contain terminal oligopyrimidine (5′ TOP) sequence elements in their 5′ noncoding regions that can negatively regulate translation initiation, particularly during serum starvation (reviewed in reference 33
). In contrast, yeast RP mRNAs do not contain 5′ TOP elements, nor do they contain any obvious consensus sequences in their 5′ noncoding regions. With the exception of L30 mRNA, whose translation is negatively regulated by its encoded product Rpl30p (11
), yeast RP mRNAs are generally not thought to be under translational control (33
Curiously, all yeast RP mRNAs redistributed from polysomes to monosomes and untranslated mRNPs within 5 min after the shift to glycerol medium, suggesting that RP mRNAs are coordinately regulated at the translational level. Furthermore, we have shown that the reduced rate of ribosomal loading is not due to an inhibition of RP gene transcription. To gain information about the mechanism of the marked translational repression of RP mRNAs after a carbon shift, translation studies using a reporter mRNA containing the noncoding regions of RPL15 were initiated. Preliminary experiments showed that the 3′ noncoding region of RPL15, but not its short 5′ noncoding region, mimicked the overall 80S and polysomal distribution pattern seen with endogenous RPL15 mRNA during growth in glycerol (K. Kuhn, unpublished observation). Thus, signals for translational repression after a carbon shift may reside in the 3′ noncoding regions of RP mRNAs.
Translational regulation of YPL250C mRNA.
YPL250C mRNAs were predominantly associated with an increased number of ribosomes following the transfer from glucose to glycerol medium. The mechanism by which YPL250C mRNAs selectively recruit ribosomes when the overall activity of the translational apparatus is diminished is being investigated. Preliminary results have indicated that the 3′ noncoding region of the YPL250C mRNA is sufficient to mobilize a reporter mRNA into polysomes during glycerol-induced translational inhibition (K. Kuhn, unpublished data). Detailed characterization of the 3′ noncoding region in YPL250C mRNA will likely provide clues to the mechanism by which this mRNA confers preferential polysomal association to YPL250C mRNAs after the carbon source shift. A BY4742 strain with a YPL250C gene knockout mutation (Research Genetics) was viable and, when grown on glucose or glycerol medium, displayed a phenotype similar to that of the parent BY4742 and MBS strains used in this study. However, overexpression of YPL250C protein led to a slow-growth phenotype (K. Kuhn, unpublished data), implying that YPL250C gene expression may be tightly regulated under normal growth conditions. Nevertheless, the predicted 136 ORF-encoded YPL250C gene product is not absolutely essential for adaptation to a nonfermentable carbon source.
Splicing of HAC1u mRNA.
Following the shift from glucose to glycerol medium, HAC1u transcripts were spliced by an Ire1p-dependent mechanism. HAC1i mRNA accumulation was transient, however, and none of the known genes in the UPR pathway was detectably induced (data not shown). Alternatively, the apparent activation of Ire1p following the carbon source shift, with no obvious consequence to the UPR pathway, raises the possibility that the regulatory function of Ire1p, and perhaps Hac1p, may not be solely devoted to the UPR and may influence expression of previously unrecognized target genes. The transient nature of HAC1 mRNA splicing and translation may reflect an adaptive response to environmental stress. Transient translational events may occur in a stressed organism as an intermediate response toward adaptation to a new equilibrium state. Nevertheless, if one considers that the average cell cycle of S. cerevisiae, when grown in minimal medium, lasts approximately 120 min, then a significant amount of protein may have been synthesized within 15 min.
Translation of HAC1u
has been shown to be regulated during the elongation step (reviewed in references 7
). It is interesting, however, that the polysomal distribution of HAC1u
mRNA in Fig. differs from that detailed in previously published reports, in which the majority of HAC1u
mRNA sedimented with polysomes while only a minor fraction (~20%) sedimented with low-density mRNP fractions (8
). In contrast, we have routinely observed an accumulation of HAC1u
mRNA in the mRNP fractions (Fig. , fractions 2 and 3) and very little sedimentation of this unspliced mRNA with polysome fractions. We are uncertain about the origin of this discrepancy. HAC1u
mRNA is known to be distributed throughout the cytoplasm in punctate clusters (8
). Although the composition of these clusters is presently unknown, the HAC1u
mRNA within these clusters may be difficult to extract. Our findings argue that translation of HAC1u
mRNA is blocked at the initiation step, in addition to the previously reported elongation step, and that there may be a cytoplasmic pool of HAC1u
mRNA which is sequestered as a translationally inactive mRNP complex.
An interesting observation has connected the protein secretory pathway with ribosome biosynthesis. The continued functioning of the secretory pathway has been shown to be essential for ribosome biosynthesis, because inhibition of the secretory pathway reduces transcription of genes encoding RP mRNAs (34
). The fact that components of the ER are affected by the carbon source shift is exemplified by the activation of the bifunctional kinase-endonuclease Ire1p and the tRNA ligase Rlg1p.
The genomic response of S. cerevisiae to nutritional change was very rapid. By combining polysomal fractionation with cDNA microarray analysis, we have primarily focused on the translational activity of thousands of individual mRNAs after a rapid depletion of glucose. Identification of individual mRNAs that are translationally controlled has historically relied on cumbersome analyses of suspected mRNA species. The cDNA microarray analysis has uncovered an mRNA species (YPL250C) that can be selectively translated during a global translational inhibition, as well as a coordinate regulation of an entire class of mRNAs (RP mRNAs). Activation of the bifunctional kinase-endonuclease Ire1p and the tRNA ligase Rlg1p after a carbon source shift was confirmed by the appearance of spliced HAC1 mRNAs in polysomal fractions. This latter finding exemplifies the effectiveness of the cDNA microarray, which can allow the detection of multiple levels of regulation that operate in the genomic response of an organism to nutritional change.