Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pH_c) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pH_c and the central nutrient signalling cAMP‐Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pH_c correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pH_c showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.     

p38 mitogen-activated protein kinase/Hog1p regulates translation of the AU-rich-element-bearing MFA2 transcript

AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3' untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3'-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3'-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3'-UTR in a regulated manner to control MFA2 3'-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3'-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3'-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3'-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.     

Transient inhibition of translation initiation by osmotic stress.

Cells respond and adapt to changes in the environment. In this study, we examined the effect of environmental stresses on protein synthesis in the yeast Saccharomyces cerevisiae. We found that osmotic stress causes irreversible inhibition of methionine uptake, transient inhibition of uracil uptake, transient stimulation of glucose uptake, transient repression of ribosomal protein (RP) genes such as CYH2 and RPS27, and the transient inhibition of translation initiation. Rapid inhibition of translation initiation by osmotic stress requires a novel pathway, different from the amino acid-sensing pathway, the glucose-sensing pathway, and the TOR pathway. The Hog1 MAP kinase pathway is not involved in the inhibition of either methionine uptake or translation initiation but is required for the adaptation of translation initiation after inhibition and the repression of RP genes by osmotic stress. These results suggest that the transient inhibition of translation initiation occurs as a result of a combination of both acute inhibition of translation and the long-term activation of translation by the Hog1 pathway.     

p53- and ERK7-Dependent Ribosome Surveillance Response Regulates Drosophila Insulin-Like Peptide Secretion

Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.     

Deregulation of oncogene‐induced senescence and p53 translational control in X‐linked dyskeratosis congenita

Defects in ribosome biogenesis and function are present in a growing list of human syndromes associated with cancer susceptibility. One example is X‐linked dyskeratosis congenita (X‐DC) in which the DKC1 gene, encoding for an enzyme that modifies ribosomal RNA, is found to be mutated. How ribosome dysfunction leads to cancer remains poorly understood. A critical cellular response that counteracts cellular transformation is oncogene‐induced senescence (OIS). Here, we show that during OIS, a switch between cap‐ and internal ribosome entry site (IRES)‐dependent translation occurs. During this switch, an IRES element positioned in the 5′untranslated region of p53 is engaged and facilitates p53 translation. We further show that in DKC1^m cells, p53 IRES‐dependent translation is impaired during OIS ex vivo and on DNA damage in vivo. This defect in p53 translation perturbs the cellular response that counteracts oncogenic insult. We extend these findings to X‐DC human patient cells in which similar impairments in p53 IRES‐dependent translation are observed. Importantly, re‐introduction of wild‐type DKC1 restores p53 expression in these cells. These results provide insight into the basis for cancer susceptibility in human syndromes associated with ribosome dysfunction.     

Deficiency in mTORC1‐controlled C/EBPβ‐mRNA translation improves metabolic health in mice

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E‐binding proteins (4E‐BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1‐associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPβ) mRNA into the C/EBPβ‐LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E‐BPs is required for suppression of LIP. Intriguingly, mice lacking the cis‐regulatory upstream open reading frame (uORF) in the C/EBPβ‐mRNA, which is required for mTORC1‐stimulated translation into C/EBPβ‐LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPβ‐isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.     

The RNA‐binding protein Hfq is important for ribosome biogenesis and affects translation fidelity

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA‐mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli. Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Hfq assists ribosome assembly and associates with pre‐30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq‐mediated regulation of ribosomes is independent of its function as sRNA‐regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm‐like protein Hfq beyond its function in small RNA‐mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.     

Glucose Tolerance and Skeletal Muscle Gene Expression in Response to Alternate Day Fasting

Objective: Alternate day fasting may extend lifespan in rodents and is feasible for short periods in nonobese humans. The aim of this study was to examine the effects of 3 weeks of alternate day fasting on glucose tolerance and skeletal muscle expression of genes involved in fatty acid transport/oxidation, mitochondrial biogenesis, and stress response. Research Methods and Procedures: Glucose and insulin responses to a standard meal were tested in nonobese subjects (eight men and eight women; BMI, 20 to 30 kg/m²) at baseline and after 22 days of alternate day fasting (36 hour fast). Muscle biopsies were obtained from a subset of subjects (n = 11) at baseline and on day 21 (12‐hour fast). Results: Glucose response to a meal was slightly impaired in women after 3 weeks of treatment (p < 0.01), but insulin response was unchanged. However, men had no change in glucose response and a significant reduction in insulin response (p < 0.03). There were no significant changes in the expression of genes involved in mitochondrial biogenesis or fatty acid transport/oxidation, although a trend toward increased CPT1 expression was observed (p < 0.08). SIRT1 mRNA expression was increased after alternate day fasting (p = 0.01). Discussion: Alternate day fasting may adversely affect glucose tolerance in nonobese women but not in nonobese men. The gene expression results indicate that fatty acid oxidation and mitochondrial biogenesis are unaffected by alternate day fasting. However, the increased expression in SIRT1 suggests that alternate day fasting may improve stress resistance, a commonly observed feature of calorie‐restricted rodents.     

A minimal base‐pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA

Escherichia coli SgrS is an Hfq‐binding small RNA that is induced under glucose‐phosphate stress to cause translational repression and RNase E‐dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31‐nt‐long stretch in the 3′ region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre‐annealed with ptsG mRNA by a high‐temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base‐pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base‐pairing region is sufficient to inhibit ptsG translation in the context of full‐length SgrS in vivo. We conclude that SgrS 168–181 is a minimal base‐pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high‐temperature pre‐treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.