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. 2001 Nov;12(11):3428-38.
doi: 10.1091/mbc.12.11.3428.

The target of rapamycin signaling pathway regulates mRNA turnover in the yeast Saccharomyces cerevisiae

A R Albig  1 , C J Decker
Affiliations
Free PMC article

The target of rapamycin signaling pathway regulates mRNA turnover in the yeast Saccharomyces cerevisiae

A R Albig et al. Mol Biol Cell. 2001 Nov.
Free PMC article

Abstract

The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes. We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae. During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected. Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion. The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway. Of the destabilized mRNAs, two different responses to rapamycin were observed. Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure. Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation. In contrast, the mRNAs that respond slowly are destabilized by rapid decapping. In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms.

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Figures

Figure 1
Figure 1
Multiple mRNAs are destabilized at diauxic shift. (A) mRNA half-life analysis of MFA2pG and MFA2M3pG mRNA from either log-phase or diauxic shift cultures. 5′ end-labeled oRP127 was used to detect mRNAs (upper panel). The numbers above the lanes indicate the number of minutes after transcription repression. As a loading control, blots were stripped and reprobed with oRP100 to detect the RNA polymerase III 7S transcript (lower panel). (B) mRNA half-life measurements of six different mRNAs at log phase (LP) or diauxic shift (DS). The numbers above the lanes indicate the number of minutes after transcription repression. The ARO4, CRY1, GRC5, CYS3, and TIF51A mRNAs were detected with the use of 5′ end-labeled oligonucleotides complementary to their coding regions (see MATERIALS AND METHODS). The PGK1 mRNA was detected with a random prime-labeled probe that is complementary to the 5′ UTR and 171 nucleotides of the coding region. The oligonucleotide used to detect CRY1 mRNA is also complementary to CRY2 mRNA, which is much lower in abundance (Paulovich et al., 1993). The oligonucleotide used to detect TIF51A (HYP2) is also complementary to the TIF51B (ANB1, HYP1) mRNA, which is not expressed under these aerobic conditions (Schnier et al., 1991). 7S RNA again was used as a loading control (7S). In both A and B, mRNA half-life values were calculated by normalization to the 7S RNA and are the mean and SD of at least two experiments.
Figure 2
Figure 2
Rapamycin effects on mRNA turnover are similar to the turnover effects caused by diauxic shift. (A) Half-life analysis of the MFA2pG and MFA2M3pG after 210 min of exposure to vehicle (− rapamycin) or rapamycin (+ rapamycin). The number of minutes after transcription inhibition are indicated at the top of each panel. 7S RNA was used for a loading control and was detected with oRP100. Half-life values are the mean and SD of at least two independent experiments. (B) Yeast cultures were grown to early log phase then were treated with rapamycin for 10, 30, 60, or 210 min or alternatively with vehicle (V) for 210 min followed by mRNA half-life analysis. RNA analysis was performed with the same probes that were used in Figure 1B and were normalized by comparison to the 7S RNA. mRNA half-lives at each rapamycin time point are presented as a percentage of the half-life in the vehicle-treated control for that mRNA. The control half-lives were as follows: ARO4, 17.3 ± 1.8 min; CRY1, 26.5 ± 0.7; GRC5, 16.7 ± 2.1; MFA2M3pG, 16 ± 2.5 min; and PGK1, >25 min. The results for all time points are the mean and SD of at least two independent experiments.
Figure 3
Figure 3
Rapamycin does not destabilize mRNAs in the presence of a rapamycin-resistant allele of the TOR1 kinase. The stability of the MFA2M3pG mRNA in a tor1Δ strain carrying either a plasmid bearing the WT TOR1 gene (TOR1) or the rapamycin-resistant TOR1–1 allele (TOR1–1) after treatment with rapamycin (+ rapamycin) or with vehicle alone (− rapamycin) for 210 min. The numbers above each lane indicate the number of minutes after transcription was repressed. The half-life values are the mean and SD of at least three experiments for each strain.
Figure 4
Figure 4
Rapamycin destabilizes the MFA2M3pG mRNA by the acceleration of decapping. Yeast cells expressing MFA2M3pG were treated with vehicle alone (− rapamycin) or rapamycin (+ rapamycin) for 210 min followed by transcriptional pulse-chase analysis of the MFA2M3pG mRNA. The dT lane contains RNA from the 0 min time point, hybridized to oligo(dT) and treated with RNaseH to remove the poly(A) tail. Time points (in minutes) taken after the repression of transcription are indicated at the top. The full-length and fragment species were detected with the use of oRP127 as a probe. The locations of the fragment and full-length species are indicated to the right of the figure, and the poly(A) tail length is marked by A70 and A10 indicators. The marker (M) lane contains 5′ end-labeled HinfI ϕχ174 DNA, and sizes are indicated to the left of the figure.
Figure 5
Figure 5
The ARO4pG mRNA is destabilized after rapamycin treatment by having a prematurely short poly(A) tail. Transcriptional pulse-chase analysis of the ARO4pG mRNA after treatment with either vehicle (− rapamycin) or rapamycin (+ rapamycin) for 60 min is shown. In each sample, the ARO4pG mRNA was cleaved with oCD163 and RNAse H to resolve the length of its poly(A) tail. The poly(A) tail was removed by treatment with oCD163, oligo dT, and RNAse H (dT lane). Full-length and fragment species were detected with oRP127 and are indicated at the right of the figure. Poly(A) tail length is marked by A50 and A0 indicators. The marker (M) lane contains 5′ end-labeled HinfI ϕχ174 DNA, and sizes are indicated to the left of the figure.
Figure 6
Figure 6
Diauxic shift and rapamycin treatment cause the accumulation of a 5′-to-3′ decay intermediate without changing the rate of 3′-to-5′ decay. Analysis of the full-length MFA2M3pG mRNA and its decay fragment at log phase, diauxic shift, or in a tor1Δ strain carrying either the WT TOR1 gene or the rapamycin-resistant TOR1–1 allele after treatment with rapamycin (+ R) or vehicle alone (− R) for 210 min is shown. The MFA2M3pG mRNA and the decay fragment were detected with oRP127. Full-length and fragment species are indicated on the right. The ratio of the fragment to the full-length mRNA (F/FL) is indicated below each lane. The membrane was stripped and rehybridized with oRP100 to detect the 7S RNA as a loading control (bottom panel, 7S).
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