Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source - PubMed (original) (raw)

Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source

K M Kuhn et al. Mol Cell Biol. 2001 Feb.

Abstract

The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.

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Figures

FIG. 1

Polysomal association of mRNAs in yeast cells grown exclusively in glucose medium or after a 10-min shift to glycerol medium. Absorbance profiles at 254 nm of the collected sucrose gradients are shown above the panels. Fourteen equal-volume fractions were collected from each gradient (fraction 1 is the top of the gradient), and purified RNAs were analyzed by Northern blotting. The levels of ACT1, ADH1, and URA3 mRNAs were visualized by a phosphorimage of the Northern blot. The CUP1 promoter was induced by addition of 10 mM CuSO4 to the glycerol medium (but not to the glucose medium).

FIG. 2

Expression of ribosomal-protein-encoding (RP) mRNAs in yeast cells after the carbon source shift. Each colored square represents the ratio of total mRNA (tot) or high-molecular-weight polysomal mRNA (poly vs. poly; fractions 10 to 14) isolated following incubation in glycerol medium for 5 or 10 min, relative to the amount of the mRNA isolated from cells grown in the presence of glucose. In addition, mRNA abundance in fractions 2 through 6, designated as mRNA protein complexes (mRNP), was compared to the abundance of mRNA present in polysomes (poly vs. mRNP). Black squares denote no significant difference in the amount of RNA isolated from glucose-grown or glycerol-shifted cells; red squares and green squares denote RNAs that are more or less abundant, respectively. The intensity of the color is proportional to the log2 of the fold increase or decrease, with maximal intensity corresponding to an eightfold increase or decrease. For duplicated RP mRNA genes (designated A and B), only data for the A gene are presented. The cDNA for RPL29 (YFR032CA) was not included in this batch of microarrays. A gray box indicates an unreadable spot on the microarray. The color intensity scale was adapted from reference . Selected RP mRNAs (highlighted in green) were chosen for further analysis.

FIG. 3

Relative abundances and translational activities of selected RP mRNAs as a function of time following transfer to glycerol medium. (A) Total levels of RP mRNAs (RPL1, RPL2, RPL15, RPL19, and RPS1), as well as a control ADH1 mRNA, after Northern blot hybridization are displayed. (B) Polysome association of RP mRNAs. For details, see the legend to Fig. 1. The Northern blot displaying ADH1 mRNA was taken from Fig. 1. Phosphorimages of the Northern blots are shown. (C) Quantitation of polysomal distribution of averaged RP and ADH1 mRNAs after the shift to glycerol medium (panel B). The data points represent the percent intensity of each fraction relative to the total combined intensity of all fractions for each gradient. The five RP data sets from panel B were combined and averaged to give a general polysomal distribution pattern for the selected RP mRNAs.

FIG. 4

Decreasing the abundance of an mRNA does not decrease its translational efficiency. (A) Total levels of URA3 and ACT1 mRNAs at various time points after addition of the tetracycline analogue doxycycline (2 μg/ml) to yeast cells grown in glycerol medium are shown. (B) Polysomal distribution of URA3 mRNA in yeast cells grown in glycerol medium in the presence (+) or absence (−) of doxycycline (doxy). The plasmid-derived URA mRNA was distinguished from the endogenous URA3 mRNA by using a probe complementary to a V5 six-His extension of the plasmid coding sequence (see Materials and Methods).

FIG. 5

Characterized genes whose relative mRNA abundances increase following a transfer to glycerol medium. Total and polysomal RNA levels were analyzed and displayed as described in the legend to Fig. 2. Genes selected for this figure were induced at least threefold at 10 min after a shift to glycerol medium. Exceptions were MIG1, SNF3, APE3, LAP4, PEP4, ULA1, and UBC8, all of which were induced at least 2.5-fold. The induction of YAK1 and YGP1 mRNAs was confirmed in a separate experiment. Grouping of genes was based on data available from the Yeast Protein Database (

http://www.proteome.com

). Gene names highlighted in red were induced specifically after an abrupt transfer from glucose to glycerol medium, but not during the diauxic shift . Glc7p, glycogen synthase phosphatase; TCA, tricarboxylic acid; GABA, γ-aminobutyric acid.

FIG. 5

http://www.proteome.com

FIG. 6

YPL250C mRNA sediments with polysomes at a time when global translation rates are reduced: time course of YPL250C polysome association from cultures grown exclusively in glucose or shifted to glycerol for 20 min.

FIG. 7

A smaller species of HAC1 RNA associates with polysomes 10 min after transfer from glucose to glycerol medium. Northern analysis with a HAC1 ORF as a hybridization probe detected the full-length _HAC1_u mRNA (arrow) as well as a low-molecular-weight version of _HAC1_i (arrowhead) engaged with polysomes. The polysomal distribution of ADH1, taken from Fig. 1, is shown as a reference. Phosphorimages of the blots are shown below.

FIG. 8

HAC1 mRNA is transiently spliced following glucose depletion. Total RNA was isolated from glycerol-shifted cultures at intervals during a 1-h time course and hybridized either with radiolabeled Hac1 intron, with a small deoxyoligonucleotide that recognizes the Hac1 splice junction (“Splice”), or with a mixture of intron and splice probes (Intron + Splice). The “splice” deoxyoligonucleotide is 18 nucleotides in length, with 9 nucleotides of complementarity to the spliced termini of each exon. 32P-labeled probes are displayed. Phosphorimages of the blots are at top. Full-length HAC1 mRNA (arrow) and the spliced HAC1 (arrowhead) are indicated in the phosphorimage.

FIG. 9

The presence of IRE1 is required for the transient splicing of HAC1 mRNA. Total RNAs were isolated from strain MBS, a ΔIRE1 strain (yCS243a), and the ΔIRE1/p(IRE1) strain transformed with a functional copy of IRE1 (pCS110). Northern blots were hybridized with either radiolabeled full-length IRE1 cDNA or the “splice” oligodeoxynucleotide described in the legend to Fig. 8. Phosphorimages of the blots are shown.

References

1. Ashe M P, De Long S K, Sachs A B. Glucose depletion rapidly inhibits translation initiation in yeast. Mol Biol Cell. 2000;11:833–848. - PMC - PubMed
1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. Current protocols in molecular biology. New York, N.Y: Greene Publishing Associates and John Wiley & Sons; 1994.
1. Belli G, Gari E, Piedrafita L, Aldea M, Herrero E. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. Nucleic Acids Res. 1998;26:942–947. . (Erratum, 26:1855.) - PMC - PubMed
1. Berset C, Trachsel H, Altmann M. The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 1998;95:4264–4269. - PMC - PubMed
1. Boucherie H. Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae. J Bacteriol. 1985;161:385–392. - PMC - PubMed

Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source - PubMed (original) (raw)