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Heme is an essential prosthetic group that serves as a co-factor and a signaling molecule. Heme levels decline with age, and its deficiency is associated with multiple hallmarks of aging, including anemia, mitochondrial dysfunction, and oxidative stress. Dysregulation of heme homeostasis has been also implicated in aging in model organisms suggesting that heme may play an evolutionarily conserved role in controlling lifespan. However, the underlying mechanisms and whether heme homeostasis can be targeted to promote healthy aging remain unclear. Here, we used Saccharomyces cerevisiae as a model to investigate the role of heme in aging. For this, we have engineered a heme auxotrophic yeast strain expressing a plasma membrane-bound heme permease from Caenorhabditis elegans (ceHRG-4). This system can be used to control intracellular heme levels independently of the biosynthetic enzymes by manipulating heme concentration in the media. We observed that heme supplementation leads to a significant extension of yeast replicative lifespan. Our findings revealed that the effect of heme on lifespan is independent of the Hap4 transcription factor. Surprisingly, heme-supplemented cells had impaired growth on YPG medium, which requires mitochondrial respiration to be used, suggesting that these cells are respiratory deficient. Together, our results demonstrate that heme homeostasis is fundamentally important for aging biology, and manipulating heme levels can be used as a promising therapeutic target for promoting longevity., (© 2024. The Author(s).)

Here, we present a protocol for modulating the function of the Cth2 mRNA-binding protein (RBP) in Saccharomyces cerevisiae. We describe steps to amplify and integrate mutations in Cth2 that affect its stability and function. Next, we detail the functional assay to verify the activity of the wild-type and mutant versions of Cth2 in yeast cells. This protocol can be adopted to modify the function of other RBPs with their respective functional mutations. For complete details on the use and execution of this protocol, please refer to Patnaik et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Iron dyshomeostasis contributes to aging, but little information is available about the molecular mechanisms. Here, we provide evidence that in Saccharomyces cerevisiae, aging is associated with altered expression of genes involved in iron homeostasis. We further demonstrate that defects in the conserved mRNA-binding protein Cth2, which controls stability and translation of mRNAs encoding iron-containing proteins, increase lifespan by alleviating its repressive effects on mitochondrial function. Mutation of the conserved cysteine residue in Cth2 that inhibits its RNA-binding activity is sufficient to confer longevity, whereas Cth2 gain of function shortens replicative lifespan. Consistent with its function in RNA degradation, Cth2 deficiency relieves Cth2-mediated post-transcriptional repression of nuclear-encoded components of the electron transport chain. Our findings uncover a major role of the RNA-binding protein Cth2 in the regulation of lifespan and suggest that modulation of iron starvation signaling can serve as a target for potential aging interventions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein synthesis is an essential process that affects major cellular functions including growth, energy production, cell signaling, and enzymatic reactions. However, how it is impacted by aging and how the translation of specific proteins is changed during the aging process remain understudied. Although yeast is a widely used model for studying eukaryotic aging, analysis of age-related translational changes using ribosome profiling in this organism has been challenging due to the need for isolating large quantities of old cells. Here, we provide a detailed protocol for genome-wide analysis of protein synthesis using ribosome profiling in replicatively aged yeast. By combining genetic enrichment of old cells with the biotin affinity purification step, this method allows large-scale isolation of aged cells sufficient for generating ribosome profiling libraries. We also describe a strategy for normalization of samples using a spike-in with worm lysates that permits quantitative comparison of absolute translation levels between young and old cells.

The yeast genome becomes unstable during stress, which often results in adaptive aneuploidy, allowing rapid activation of protective mechanisms that restore cellular homeostasis. In this study, we performed a genetic screen in Saccharomyces cerevisiae to identify genome adaptations that confer resistance to tunicamycin-induced endoplasmic reticulum (ER) stress. Whole-genome sequencing of tunicamycin-resistant mutants revealed that ER stress resistance correlated significantly with gains of chromosomes II and XIII. We found that chromosome duplications allow adaptation of yeast cells to ER stress independently of the unfolded protein response, and that the gain of an extra copy of chromosome II alone is sufficient to induce protection from tunicamycin. Moreover, the protective effect of disomic chromosomes can be recapitulated by overexpression of several genes located on chromosome II. Among these genes, overexpression of UDP- N -acetylglucosamine-1-P transferase ( ALG7 ), a subunit of the 20S proteasome ( PRE7 ), and YBR085C-A induced tunicamycin resistance in wild-type cells, whereas deletion of all three genes completely reversed the tunicamycin-resistance phenotype. Together, our data demonstrate that aneuploidy plays a critical role in adaptation to ER stress by increasing the copy number of ER stress protective genes. While aneuploidy itself leads to proteotoxic stress, the gene-specific effects of chromosome II aneuploidy counteract the negative effect resulting in improved protein folding., Competing Interests: The authors declare no conflict of interest.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Transcriptional regulation plays an important role in the control of gene expression during aging. However, translation efficiency likely plays an equally important role in determining protein abundance, but it has been relatively understudied in this context. Here, we used RNA sequencing (RNA-seq) and ribosome profiling to investigate the role of translational regulation in lifespan extension by CAN1 gene deletion in yeast. Through comparison of the transcriptional and translational changes in cells lacking CAN1 with other long-lived mutants, we were able to identify critical regulatory factors, including transcription factors and mRNA-binding proteins, that coordinate transcriptional and translational responses. Together, our data support a model in which deletion of CAN1 extends replicative lifespan through increased translation of proteins that facilitate cellular response to stress. This study extends our understanding of the importance of translational control in regulating stress resistance and longevity., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Being a simple eukaryotic organism, Saccharomyces cerevisiae provides numerous advantages for expression and functional characterization of proteins from higher eukaryotes, including humans. However, studies of complex exogenous pathways using yeast as a host have been hampered by the lack of tools to engineer strains expressing a large number of genetic components. In addition to inserting multiple genes, it is often desirable to knock out or replace multiple endogenous genes that might interfere with the processes studied. Here, we describe the "insertion Green Monster" (iGM) set of expression vectors that enable precise insertion of many heterologous genes into the yeast genome in a rapid and reproducible manner and permit simultaneous replacement of selected yeast genes. As a proof of principle, we have used the iGM method to replace components of the yeast pathway for methionine sulfoxide reduction with genes encoding the human selenoprotein biosynthesis machinery and generated a single yeast strain carrying 11 exogenous components of the selenoprotein biosynthetic pathway in precisely engineered loci., (Copyright © 2014 Labunskyy et al.)