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In the fission yeast Schizosaccharomyces pombe, the response to sulfur depletion has been less studied compared to the response to nitrogen depletion. Our study reveals that the fission yeast gene, SPCC417.09c, plays a significant role in the sulfur depletion response. This gene encodes a protein with a Zn 2 Cys 6 fungal-type DNA-binding domain and a transcription factor domain, and we have named it sdr1 + (sulfur depletion response 1). Interestingly, while sulfur depletion typically induces autophagy akin to nitrogen depletion, we found that autophagy was not induced under sulfur depletion in the absence of sdr1 + . This suggests that sdr1 + is necessary for the induction of autophagy under conditions of sulfur depletion. Although sdr1 + is not essential for the growth of fission yeast, its overexpression, driven by the nmt1 promoter, inhibits growth. This implies that Sdr1 may possess cell growth-inhibitory capabilities. In addition, our analysis of Δsdr1 cells revealed that sdr1 + also plays a role in regulating the expression of genes associated with the phosphate depletion response. In conclusion, our study introduces Sdr1 as a novel transcription factor that contributes to an appropriate cellular nutrient starvation response. It does so by inhibiting inappropriate cell growth and inducing autophagy in response to sulfur depletion., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Fission yeast is a good model organism for the study of lifespan. To elucidate the mechanism, we screened for long-lived mutants. We found a nonsense mutation in the ksg1 + gene, which encodes an ortholog of mammalian PDK1 (phosphoinositide-dependent protein kinase). The mutation was in the PH domain of Ksg1 and caused defect in membrane localization and protein stability. Analysis of the ksg1 mutant revealed that the reduced amounts and/or activity of the Ksg1 protein are responsible for the increased lifespan. Ksg1 is essential for growth and known to phosphorylate multiple substrates, but the substrate responsible for the long-lived phenotype of ksg1 mutation is not yet known. Genetic analysis showed that deletion of pck2 suppressed the long-lived phenotype of ksg1 mutant, suggesting that Pck2 might be involved in the lifespan extension caused by ksg1 mutation., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

There are several examples in the nature wherein the mechanism of longevity control of unicellular organisms is evolutionarily conserved with that of higher multicellular organisms. The present microreview focuses on aging and longevity studies, particularly on chronological lifespan (CLS) concerning the unicellular eukaryotic fission yeast Schizosaccharomyces pombe. In S. pombe, >30 compounds, 8 types of nutrient restriction, and >80 genes that extend CLS have been reported. Several CLS control mechanisms are known to be involved in nutritional response, energy utilization, stress responses, translation, autophagy, and sexual differentiation. In unicellular organisms, the control of CLS is directly linked to the mechanism by which cells are maintained in limited-resource environments, and their genetic information is left to posterity. We believe that this important mechanism may have been preserved as a lifespan control mechanism for higher organisms., (© 2021 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Autophagy is an intracellular degradation system widely conserved among various species. Autophagy is induced by the depletion of various nutrients, and this degradation mechanism is essential for adaptation to such conditions. In this study, we demonstrated that sulfur depletion induces autophagy in the fission yeast Schizosaccharomyces pombe. Based on the finding that autophagy induced by sulfur depletion was completely abolished in a mutant in which the ecl1, ecl2 and ecl3 genes were deleted (Δecls), we report that these three genes are essential for the induction of autophagy by sulfur depletion. Furthermore, autophagy-defective mutant cells exhibited poor growth and short lifespan (compared with wild-type cells) under the sulfur-depleted condition. These results indicated that the mechanism of autophagy is necessary for the appropriate adaptation to sulfur depletion., (© 2020 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Most antiaging factors or life span extenders are associated with calorie restriction (CR). Very few of these factors function independently of, or additively with, CR. In this study, we focused on tschimganine, a compound that was reported to extend chronological life span (CLS). Although tschimganine led to the extension of CLS, it also inhibited yeast cell growth. We acquired a Schizosaccharomyces pombe mutant with a tolerance for tschimganine due to the gene crm1. The resulting Crm1 protein appears to export the stress-activated protein kinase Sty1 from the nucleus to the cytosol even under stressful conditions. Furthermore, we synthesized two derivative compounds of tschimganine, α-hibitakanine and β-hibitakanine; these derivatives did not inhibit cell growth, as seen with tschimganine. α-hibitakanine extended the CLS, not only in S. pombe but also in Saccharomyces cerevisiae, indicating the possibility that life span regulation by tschimganine derivative may be conserved across various yeast species. We found that the longevity induced by tschimganine was dependent on the Sty1 pathway. Based on our results, we propose that tschimganine and its derivatives extend CLS by activating the Sty1 pathway in fission yeast, and CR extends CLS via two distinct pathways, one Sty1-dependent and the other Sty1-independent. These findings provide the potential for creating an additive life span extension effect when combined with CR, as well as a better understanding of the mechanism of CLS., (© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

ecl1+, ecl2+ and ecl3+ genes encode highly homologous small proteins, and their over-expressions confer both H2O2 stress resistance and chronological lifespan extension on Schizosaccharomyces pombe. However, the mechanisms of how these Ecl1 family proteins function have not been elucidated. In this study, we conducted microarray analysis and identified that the expression of genes involved in sexual development and stress responses was affected by the over-expression of Ecl1 family proteins. In agreement with the mRNA expression profile, the cells over-expressing Ecl1 family proteins showed high mating efficiency and resistant phenotype to H2O2. We showed that the H2O2-resistant phenotype depends on catalase Ctt1, and over-expression of ctt1+ does not affect chronological lifespan. Furthermore, we showed that six genes, ste11+, spk1+, hsr1+, rsv2+, hsp9+ and lsd90+, whose expressions are increased in cells over-expressing Ecl1 family proteins are involved in chronological lifespan in fission yeast. Among these genes, the induction of ste11+ and hsr1+ was dependent on a transcription factor Prr1, and we showed that the extensions of chronological lifespan by Ecl1 family proteins are remarkably diminished in prr1 deletion mutant. From these results, we propose that Ecl1-family proteins conduct H2O2 stress resistance and chronological lifespan extension in ctt1+- and prr1+-dependent manner, respectively.

tmRNA, a product of ssrA gene, plays a crucial role in the quality control system that eliminates aberrant products of nonstop mRNAs in prokaryotes. Although tmRNA recycles ribosomes stalled at the 3' end of nonstop mRNAs, the fate of ribosomes that stall at the 3' end in the absence of tmRNA has not been extensively examined. Here we report our analysis of the translation status of nonstop mRNAs. Polysome analysis showed that nonstop mRNAs were translated efficiently, and peptidyl-tRNA was not found in any fraction in a DeltassrA strain. In vitro translation experiments using PURESYSTEM revealed that ribosomes translating nonstop mRNAs were dissociated from the 3' end of mRNA, and the peptidyl-tRNA was only weakly hydrolyzed in the monosome. These results suggest that the peptidyl-tRNA of a nonstop mRNA is hydrolyzed by an unknown factor(s) in vivo, thereby allowing a nonstop mRNA to be translated as efficiently as a normal mRNA. Possible factors involved in the hydrolysis of the peptidyl-tRNAs of nonstop mRNAs are discussed.

Background: We have shown recently that read-through of a normal stop codon by a suppressor tRNA in specific genes possessing a Rho-independent terminator leads to SsrA-mediated tagging of extended proteins in Escherichia coli cells. Miscoding antibiotics such as kanamycin and streptomycin reduce translational fidelity by binding to the 30S ribosomal subunit. The aim of the present study was to address how miscoding antibiotics affect the read-through of stop codons and SsrA-mediated protein tagging., Results: Miscoding antibiotics caused translational read-through of stop codons when added to the culture medium at sublethal concentrations. Under the same conditions, the drugs enhanced SsrA-mediated tagging of bulk cellular proteins, as observed in cells carrying an ochre suppressor tRNA. Translational read-through products generated from the crp gene in the presence of the antibiotics was efficiently tagged by the SsrA system, presumably because the ribosome reached the 3' end of the mRNA defined by the terminator hairpin. The SsrA-defective cells were more sensitive to the miscoding antibiotics compared to the wild-type cells., Conclusion: We conclude that the SsrA system contributes to the survival of cells by dealing with translational errors in the presence of low concentrations of miscoding antibiotics.

Background: Bacterial SsrA RNA (also known as tmRNA or 10Sa RNA) mediates the addition of a short peptide tag to the C-terminus of the nascent polypeptide when a ribosome is stalled at the 3' end of an mRNA lacking a stop codon. This process, called trans-translation, rescues the stalled ribosome and ensures degradation of tagged polypeptides by ATP-dependent proteases. To fully understand the physiological roles of SsrA RNA, it is essential to know how endogenous mRNA targets for the SsrA system are generated in cells. The aim of the present study is to examine how translational readthrough by suppressor tRNAs affects trans-translation in Escherichia coli., Results: We demonstrated that SsrA tagging of bulk cellular proteins was significantly enhanced by an ochre or an amber suppressor tRNA. Western blot analysis of proteins produced from specific genes possessing a Rho-independent terminator revealed that readthrough at the normal stop codon leads to an efficient tagging and proteolysis of the extended proteins. Size analyses of both protein and mRNA suggested that tagging of extended proteins occurs because ribosome passing through the normal stop codon presumably reach the 3' end of mRNA defined by the transcription terminator hairpin. The inhibitory effect of ssrA mutation on cell growth was markedly amplified in cells with an ochre suppressor tRNA., Conclusion: The present finding suggests that the SsrA system contributes to scavenge errors and/or problems caused by translational readthrough that occurs typically in the presence of a suppressor tRNA.

Background: The pts operon of Escherichia coli consists of three genes ptsH, ptsI and crr, each encoding for central components of the phosphoenolpyruvate: carbohydrate phosphotransferase system, HPr, enzyme I and IIAGlc, respectively. Transcription of the pts operon is stimulated when glucose is present in the culture medium. One of the two major promoters, P0, is responsible for this glucose induction. However, no regulatory protein responsible for the glucose induction of the pts operon has been identified yet and molecular mechanism by which glucose stimulates the pts transcription is not known., Results: We found by Northern blotting that the pts mRNA levels in cells lacking Mlc, a new global repressor of carbohydrate metabolism, were increased without external glucose and that the addition of glucose had no effect on the pts mRNA levels in the mutant cells. Western blotting revealed that the enzyme I level in the mlc- cells was also elevated without glucose and no further increase in the enzyme I level was observed in the presence of glucose. S1 analysis revealed that transcription of the glucose-sensitive promoter, P0, occurs constitutively in the mlc- cells independently from the external glucose. In vitro transcription studies indicated that Mlc strongly inhibited P0 transcription. DNase I footprinting experiment revealed that Mlc bound to P0 promoter region to prevent RNA polymerase binding at P0., Conclusion: We conclude that Mlc is a repressor for the pts transcription acting as a major regulatory protein involved in the glucose induction of pts operon. We propose that glucose induces the pts transcription by modulating the Mlc activity. The mechanism by which glucose modulates the Mlc action remains to be studied.

Background: Rho-independent terminators in Escherichia coli are DNA sequences of 30-50 bp consisting of a GC-rich dyad symmetry sequence followed by a run of T residues in the nontemplate strand. The transcription termination at the Rho-independent terminator occurs within the T-tract in vitro. It has been believed that the transcription termination at the Rho-independent terminator occurs within the T-tract in vivo, as established in vitro, and therefore the 3' ends of mRNAs are mostly generated as a direct result of transcription termination. However, how the transcription termination occurs and how the 3' ends of mRNAs are formed in living cells remains to be studied., Results: We developed a double terminator system in which a second Rho-independent terminator was placed downstream of the crp terminator. This system made it possible to detect transcripts that pass through the crp terminator by Northern blotting. We found that most of the crp transcripts extend beyond the crp terminator. The transcriptional read-through at the crp terminator was reduced when the translation of crp mRNA was interrupted. The level of the read-through transcript decreased with distance between the two terminators, suggesting that transcription termination occurs at multiple positions beyond the crp terminator., Conclusion: We conclude that most RNA polymerase reads through the crp terminator in the natural situation and terminates transcription over a wide region downstream of the crp terminator, resulting in heterogeneous primary transcripts that are subsequently processed back to the terminator hairpin. We propose that ribosome translation to the crp stop codon causes read-through of the terminator. The regulatory effect of translation on Rho-independent termination may be a general phenomenon at other operons.

Background: The inhibition of beta-galactosidase expression in glucose-lactose diauxie is a typical example of the glucose effect in Escherichia coli. It is generally believed that glucose exerts its effect at least partly by reducing the intracellular cAMP level. However, there is no direct evidence that the inhibitory effect of glucose on the expression of the lac operon is mediated by a reduction of the cAMP level in the glucose-lactose system., Results: To examine the roles of cAMP and the cAMP receptor protein (CRP) in the glucose effect, the intracellular levels of these factors were determined during diauxic growth in a glucose-lactose medium. We found that the levels of cAMP and CRP in a lactose-grown phase were not higher than those in a glucose-grown phase, although the cAMP levels increased transiently during the lag phase. The addition of exogenous cAMP eliminated diauxic growth but did not eliminate glucose repression. Glucose repression and diauxie were observed in cells which lack cAMP but produce a cAMP-independent CRP. In addition, inactivation of the lac repressor by the disruption of the lacI gene or the addition of IPTG, eliminated glucose repression., Conclusion: We conclude that the repression of beta-galactosidase expression by glucose is not due to the reduction of the cAMP-CRP level but due to an inducer exclusion mechanism which is mediated by the phosphoenolpyruvate-dependent sugar phosphotransferase system.