Loss of translational control in yeast compromised for the major mRNA decay pathway

Abstract

The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways. In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress. In the past, the paradigm of the eIF2alpha kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2alpha phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.

FIG. 1.

mRNA decapping mutants maintain protein synthesis following glucose starvation. (A) Polysome traces from yMK116 (WT), yMK117 (dcp1Δ), and yMK321 (lsm1Δ). Yeast were grown in SCD medium, harvested, and then resuspended for 10 min in medium containing (+D) or lacking (−D) glucose. Polysomes were analyzed as described in Materials and Methods. The 40S (small ribosomal subunit), 60S (large ribosomal subunit), 80S (monosome), and polysome peaks are labeled. Even though the mutant strains were derived from different backgrounds, only a single wild type is shown for simplicity, as the inhibition of translation is identical in the different wild-type strains (data not shown). (B) [³⁵S]methionine incorporation into proteins over time in the presence (+D) or absence (−D) of glucose for strains yMK23 (WT) and yMK321 (lsm1Δ) (left panel) and strains yMK116 (WT) and yMK117 (dcp1Δ) (right panel). Yeast strains were grown in SCD medium without methionine, harvested, and resuspended in a labeling mixture in the presence or absence of glucose. Aliquots were taken at the indicated times, and the levels of [³⁵S]methionine incorporation into proteins were determined.

FIG. 2.

Decapping activity is required for translational inhibition, but this is not via a global decapping activation in response to stress. (A) Polysome traces from the dcp1Δ strain (yMK117) containing various plasmid-borne (+p) alleles of DCP1. Wild-type DCP1 (yMK279) or mutant alleles of DCP1 (yMK286 [dcp1-7], yMK305 [dcp1-34], yMK283 [dcp1-4], yMK290 [dcp1-13], yMK291 [dcp1-14], and yMK296 [dcp1-21]) were grown in SCD-TRP, harvested, and then resuspended in the same medium in the presence (+D) or absence (−D) of glucose for 10 min. (B) Northern blot stained with methylene blue to visualize rRNA levels (upper panel). The same blot was probed for either PAB1 mRNA or PGK1 mRNA (lower panels). A wild-type strain (yMK23) was grown in rich medium, and RNA was prepared either instantly (−) or following a 10-min incubation in rich medium with (+D) or without (−D) glucose. RNA samples were incubated in vitro with Xrn1p either in the presence (control incubation) or absence (Xrn1p incubation) of EDTA (which inhibits the 5′ to 3′ exoribonuclease). (C) Polysome traces from yMK116 (WT), yMK75 (gcn2Δ), and yMK117 (dcp1Δ) strains. Strains were grown in SCD medium, harvested, and then resuspended in the same medium (+D), medium without amino acids (−AAs), or medium without glucose (−D). Even though the mutant strains are derived from different backgrounds, only a single wild type is shown for simplicity, as the levels of inhibition of translation in the different wild-type strains were identical (data not shown).

FIG. 3.

Major mRNA decay pathway mutants affecting stages downstream of deadenylation are particularly resistant to translational stress. (A) Polysome analyses for a wild-type (WT) strain (yMK116) and various mutant strains (yMK380 [xrn1Δ], yMK704 [dcp2Δ], yMK703 [pat1Δ], yMK711 [ccr4Δ], yMK793 [pan2Δ pan3Δ], yMK705 [ski2Δ], and yMK710 [upf1Δ]). Each strain was grown in SCD medium, harvested, and then incubated for 10 min in SCD medium (+D), for 10 min in medium without amino acids (−AAs), or for 5 min in medium without glucose (−D). (B) A bar chart summarizing polysome analyses for a large number of mutant strains (see Table 1 for strain data). The percentage of polysomes remaining after either amino acid starvation for 10 min or glucose starvation for 5 min was calculated by dividing the area under the polysomal traces following stress (established using National Institutes of Health image J software) by the area found in the absence of stress.

FIG. 4.

dcp1Δ is resistant to many forms of translational stress. (A) Polysome analyses for the wild-type (WT) strain (yMK329) and the dcp1Δ strain (yMK330). Strains were grown in SCD medium and then subjected to a number of stresses. Rapamycin (+ rap) was added to achieve a final concentration of 200 ng/ml for 1 h. A drug vehicle control gave a polysome trace identical to that of unstressed cells (data not shown). Lithium (+Li⁺) was added to achieve a final concentration of 30 mM for 15 min to yeast grown with galactose as the sole carbon source (the nontreated galactose grown control is not shown for simplicity, as the polysome profiles are indistinguishable from those of glucose-grown strains). The removal of amino acids for 10 min (−AAs) or glucose for 5 min (−D) was performed as described in previous figures. (B) Polysome analyses for the BUT^S strain background, yMK36 (WT), and yMK863 (dcp1Δ). Strains were grown in rich medium (YPD), and 1-butanol was added to achieve a final concentration of 0.75% (vol/vol) (+0.75% butanol) or 1% (vol/vol) (+1% butanol) for 10 min.

FIG. 5.

dcp1Δ responds normally to amino acid starvation in terms of eIF2α phosphorylation and GCN4 induction. (A) Protein extracts from strains yMK329 (WT) and yMK330 (dcp1Δ) (top panels) and strains yMK429 (WT) and yMK513 (gcn2Δ) (bottom panels) were blotted and probed with antibodies to eIF2α and phosphospecific antibodies to phosphoserine 51 on eIF2α. C, control. (B) β-Galactosidase assays measured in Miller units from extracts prepared from strains yMK926 and yMK936 (GCN4-lacZ reporter) or strains yMK927 and yMK937 (xxxxGCN4-lacZ reporter). The light-gray bars represent cells which were transferred into complete medium for 1 h, whereas the dark-gray bars represent cells transferred into medium lacking amino acids for 1 h. Error bars represent the standard deviations from three independent experiments. (C) Serial dilution plate assays for yMK513 (gcn2Δ), yMK431 (WT), and yMK861 (dcp1Δ). The SCD medium lacking histidine (SCD-H) plates were incubated for 2 days, whereas the SCD-H plates with 30 mM 3-amino triazole added were incubated for 3 days.

FIG. 6.

Strains that accumulate eIF4G or 40S ribosomal subunits are resistant to translational stress. All strains were grown in SCD medium, selecting for plasmids where appropriate. They were harvested and then resuspended in the same medium (+D), in medium without amino acids (−AAs), or in medium without glucose (−D). (A) Polysome traces from the 60S biogenesis mutants yMK899 (dob1-1) and yMK709 (rrp4-1) as well as from their respective isogenic wild-type strains yMK898 (WT-DOB1) and yMK116 (WT-RRP4). (B) Polysome traces from the 40S biogenesis mutant yMK1046 (fal1-1) and the isogenic wild-type yMK1045 (FAL1). (C) Polysome traces from strains yMK158 and yMK157, which are wild-type (WT) strains bearing either an eIF4G2 overexpression plasmid (↑eIF4G2) or an empty vector (+vector).

FIG. 7.

A model depicting how 5′ to 3′ mRNA decay or 60S biogenesis defects might overcome decreases in ternary complex. Mutants that accumulate either 40S subunits or mRNA/eIF4F complexes drive the assembly of the preinitiation machinery by mass action. TC and MFC represent the ternary complex (eIF2-GTP ) and the multifactor complex (containing ternary complex, eIF3, eIF1, and eIF5), respectively. Arrows represent the effects of the presence of excess 40S ribosomal subunits and eIF4F-bound mRNA on the rate of translation initiation.