Budding Yeast Research Articles

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

In vitro observation of histone-hexamer association with and dissociation from the amino-terminal region of budding yeast Mcm2, a subunit of the replicative helicase.

During DNA replication, core histones that form nucleosomes on template strands are evicted and associate with newly synthesized strands to reform nucleosomes. Mcm2, a subunit of the Mcm2-7 complex, which is a core component of the replicative helicase, interacts with histones in the amino-terminal region (Mcm2N) and is involved in the parental histone recycling to lagging strands. Herein, the interaction of Mcm2N with histones was biochemically analyzed to reveal the molecular mechanisms underlying histone recycling by Mcm2N. With the addition of Mcm2N, a histone hexamer, comprising an H3-H4 tetramer and an H2A-H2B dimer, was excised from the histone octamer to form a complex with Mcm2N. The histone hexamer, but not H3-H4 tetramer was released from Mcm2N in the presence of Nap1, a histone chaperone. FACT, another histone chaperone, stabilized Mcm2N-histone hexamer complex to protect from Nap1-dependent dissociation. This study indicates cooperative histone transfer via Mcm2N and histone chaperones.

Optogenetic control of phosphate-responsive genes using single component fusion proteins in Saccharomyces cerevisiae.

Blue light illumination can be detected by Light-Oxygen-Voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling (PHO) pathway in the budding yeast Saccharomyces cerevisiae, exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration, and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (Pi) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionality they can deliver and biological questions that can be probed.

Modeling the START transition in the budding yeast cell cycle.

Budding yeast, Saccharomyces cerevisiae, is widely used as a model organism to study the genetics underlying eukaryotic cellular processes and growth critical to cancer development, such as cell division and cell cycle progression. The budding yeast cell cycle is also one of the best-studied dynamical systems owing to its thoroughly resolved genetics. However, the dynamics underlying the crucial cell cycle decision point called the START transition, at which the cell commits to a new round of DNA replication and cell division, are under-studied. The START machinery involves a central cyclin-dependent kinase; cyclins responsible for starting the transition, bud formation, and initiating DNA synthesis; and their transcriptional regulators. However, evidence has shown that the mechanism is more complicated than a simple irreversible transition switch. Activating a key transcription regulator SBF requires the phosphorylation of its inhibitor, Whi5, or an SBF/MBF monomeric component, Swi6, but not necessarily both. Also, the timing and mechanism of the inhibitor Whi5's nuclear export, while important, are not critical for the timing and execution of START. Therefore, there is a need for a consolidated model for the budding yeast START transition, reconciling regulatory and spatial dynamics. We built a detailed mathematical model (START-BYCC) for the START transition in the budding yeast cell cycle based on established molecular interactions and experimental phenotypes. START-BYCC recapitulates the underlying dynamics and correctly emulates key phenotypic traits of ~150 known START mutants, including regulation of size control, localization of inhibitor/transcription factor complexes, and the nutritional effects on size control. Such a detailed mechanistic understanding of the underlying dynamics gets us closer towards deconvoluting the aberrant cellular development in cancer.

Evidence of 14-3-3 proteins contributing to kinetochore integrity and chromosome congression during mitosis.

The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions in the cell. Homo- and hetero-dimers of these proteins mainly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or cells survive with severe growth defects, including gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work, we investigated their centromere- and kinetochore-related functions of Bmh1 and Bmh2. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and non-shared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, Bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.

Distinct roles of spindle checkpoint proteins in meiosis

Gametes are produced via meiosis, a specialized cell division associated with frequent errors that cause birth defects and infertility. Uniquely in meiosis I, homologous chromosomes segregate to opposite poles, usually requiring their linkage by chiasmata, the products of crossover recombination.1 The spindle checkpoint delays cell-cycle progression until all chromosomes are properly attached to microtubules,2 but the steps leading to the capture and alignment of chromosomes on the meiosis I spindle remain poorly understood. In budding yeast meiosis I, Mad2 and Mad3BUBR1 are equally important for spindle checkpoint delay, but biorientation of homologs on the meiosis I spindle requires Mad2, but not Mad3BUBR1.3,4 Here we reveal the distinct functions of Mad2 and Mad3BUBR1 in meiosis I chromosome segregation. Mad2 promotes the prophase to metaphase I transition, while Mad3BUBR1 associates with the TOGL1 domain of Stu1CLASP, a conserved plus-end microtubule protein that is important for chromosome capture onto the spindle. Homologous chromosome pairs that are proficient in crossover formation but fail to biorient rely on Mad3BUBR1-Stu1CLASP to ensure their efficient attachment to microtubules and segregation during meiosis I. Furthermore, we show that Mad3BUBR1-Stu1CLASP are essential to rescue the segregation of mini-chromosomes lacking crossovers. Our findings define a new pathway ensuring microtubule-dependent chromosome capture and demonstrate that spindle checkpoint proteins safeguard the fidelity of chromosome segregation both by actively promoting chromosome alignment and by delaying cell-cycle progression until this has occurred.

Strategic targeting of Cas9 nickase induces large segmental duplications

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.

Temperature regulates negative supercoils to modulate meiotic crossovers and chromosome organization.

Crossover recombination is a hallmark of meiosis that holds the paternal and maternal chromosomes (homologs) together for their faithful segregation, while promoting genetic diversity of the progeny. The pattern of crossover is mainly controlled by the architecture of the meiotic chromosomes. Environmental factors, especially temperature, also play an important role in modulating crossovers. However, it is unclear how temperature affects crossovers. Here, we examined the distribution of budding yeast axis components (Red1, Hop1, and Rec8) and the crossover-associated Zip3 foci in detail at different temperatures, and found that both increased and decreased temperatures result in shorter meiotic chromosome axes and more crossovers. Further investigations showed that temperature changes coordinately enhanced the hyperabundant accumulation of Hop1 and Red1 on chromosomes and the number of Zip3 foci. Most importantly, temperature-induced changes in the distribution of axis proteins and Zip3 foci depend on changes in DNA negative supercoils. These results suggest that yeast meiosis senses temperature changes by increasing the level of negative supercoils to increase crossovers and modulate chromosome organization. These findings provide a new perspective on understanding the effect and mechanism of temperature on meiotic recombination and chromosome organization, with important implications for evolution and breeding.

CMG helicase disassembly is essential and driven by two pathways in budding yeast

The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.

A common mechanism for recruiting the Rrm3 and RTEL1 accessory helicases to the eukaryotic replisome

The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these ‘accessory’ helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown. Here, by combining AlphaFold structural modelling with experimental validation, we show that the budding yeast Rrm3 accessory helicase contains two Short Linear Interaction Motifs (SLIMs) in its disordered N-terminus, which interact with CMG and the leading-strand DNA polymerase Polε on one side of the replisome. This flexible tether positions Rrm3 adjacent to the lagging strand template on which it translocates, and is critical for replication termination in vitro and Rrm3 function in vivo. The primary accessory helicase in metazoa, RTEL1, is evolutionarily unrelated to Rrm3, but binds to CMG and Polε in an analogous manner, revealing a conserved docking mechanism for accessory helicases in the eukaryotic replisome.

Plasticity of the mitotic spindle in response to karyotype variation

Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two "mega chromosomes," to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.

CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator

In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription.

Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae.

Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

Introducing carbon assimilation in yeasts using photosynthetic directed endosymbiosis

Conversion of heterotrophic organisms into partially or completely autotrophic organisms is primarily accomplished by extensive metabolic engineering and laboratory evolution efforts that channel CO2 into central carbon metabolism. Here, we develop a directed endosymbiosis approach to introduce carbon assimilation in budding yeasts. Particularly, we engineer carbon assimilating and sugar-secreting photosynthetic cyanobacterial endosymbionts within the yeast cells, which results in the generation of yeast/cyanobacteria chimeras that propagate under photosynthetic conditions in the presence of CO2 and in the absence of feedstock carbon sources like glucose or glycerol. We demonstrate that the yeast/cyanobacteria chimera can be engineered to biosynthesize natural products under the photosynthetic conditions. Additionally, we expand our directed endosymbiosis approach to standard laboratory strains of yeasts, which transforms them into photosynthetic yeast/cyanobacteria chimeras. We anticipate that our studies will have significant implications for sustainable biotechnology, synthetic biology, and experimentally studying the evolutionary adaptation of an additional organelle in yeast.

High-resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii.

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

Chk2 homolog Mek1 limits exonuclease 1-dependent DNA end resection during meiotic recombination in Saccharomyces cerevisiae.

The conserved Rad2/XPG family 5'-3' exonuclease, exonuclease 1 (Exo1), plays many roles in DNA metabolism including during resolution of DNA double-strand breaks via homologous recombination. Prior studies provided evidence that the end resection activity of Exo1 is downregulated in yeast and mammals by Cdk1/2 family cyclin-dependent and checkpoint kinases, including budding yeast kinase Rad53 which functions in mitotic cells. Here, we provide evidence that the master meiotic kinase Mek1, a paralog of Rad53, limits 5'-3' single-strand resection at the sites of programmed meiotic DNA breaks. Mutational analysis suggests that the mechanism of Exo1 suppression by Mek1 differs from that of Rad53.

Ancestral Sequence Reconstruction as a Tool to Detect and Study De Novo Gene Emergence.

New protein-coding genes can evolve from previously noncoding genomic regions through a process known as de novo gene emergence. Evidence suggests that this process has likely occurred throughout evolution and across the tree of life. Yet, confidently identifying de novo emerged genes remains challenging. Ancestral sequence reconstruction is a promising approach for inferring whether a gene has emerged de novo or not, as it allows us to inspect whether a given genomic locus ancestrally harbored protein-coding capacity. However, the use of ancestral sequence reconstruction in the context of de novo emergence is still in its infancy and its capabilities, limitations, and overall potential are largely unknown. Notably, it is difficult to formally evaluate the protein-coding capacity of ancestral sequences, particularly when new gene candidates are short. How well-suited is ancestral sequence reconstruction as a tool for the detection and study of de novo genes? Here, we address this question by designing an ancestral sequence reconstruction workflow incorporating different tools and sets of parameters and by introducing a formal criterion that allows to estimate, within a desired level of confidence, when protein-coding capacity originated at a particular locus. Applying this workflow on ∼2,600 short, annotated budding yeast genes (<1,000 nucleotides), we found that ancestral sequence reconstruction robustly predicts an ancient origin for the most widely conserved genes, which constitute "easy" cases. For less robust cases, we calculated a randomization-based empirical P-value estimating whether the observed conservation between the extant and ancestral reading frame could be attributed to chance. This formal criterion allowed us to pinpoint a branch of origin for most of the less robust cases, identifying 49 genes that can unequivocally be considered de novo originated since the split of the Saccharomyces genus, including 37 Saccharomyces cerevisiae-specific genes. We find that for the remaining equivocal cases we cannot rule out different evolutionary scenarios including rapid evolution, multiple gene losses, or a recent de novo origin. Overall, our findings suggest that ancestral sequence reconstruction is a valuable tool to study de novo gene emergence but should be applied with caution and awareness of its limitations.

A biallelic variant of the RNA exosome gene, EXOSC4, associated with neurodevelopmental defects impairs RNA exosome function and translation

The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Pathogenic variants in EXOSC genes, which encode structural subunits of this complex, are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene that causes a collection of clinical features in two affected siblings. This missense variant (NM_019037.3: exon3:c.560T>C) changes a leucine residue within a conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals show prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified by exome sequencing with Sanger sequencing to confirm segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show growth defects. Steady-state levels of both Rrp41-L187P and EXOSC4-L187P are decreased compared to controls, and EXOSC4-L187P shows decreased copurification with other RNA exosome subunits. RNA exosome target transcripts accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a decrease in actively translating ribosomes in rrp41-L187P cells as compared to control cells with the incorporation of 7S pre-rRNA into polysomes. This work adds EXOSC4 to the structural subunits of the RNA exosome that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse phenotypes caused by EXOSC pathogenic variants.

Involvement of lipid-translocating exporter family proteins in determination of myriocin sensitivity in budding yeast

Myriocin is an inhibitor of serine palmitoyltransferase involved in the initial biosynthetic step for sphingolipids, and causes potent growth inhibition in eukaryotic cells. In budding yeast, Rsb1, Rta1, Pug1, and Ylr046c are known as the Lipid-Translocating Exporter (LTE) family and believed to contribute to export of various cytotoxic lipophilic compounds. It was reported that Rsb1 is a transporter responsible for export of intracellularly accumulated long-chain bases, which alleviate the cytotoxicity. In this study, it was found that LTE family genes are involved in determination of myriocin sensitivity in yeast. Analyses of effects of deletion and overexpression of LTE family genes suggested that all LTEs contribute to suppression of cytotoxicity of myriocin. It was confirmed that RSB1 overexpression suppressed reduction in complex sphingolipid levels caused by myriocin treatment, possibly exporting myriocin to outside of the cell. These results suggested that LTE family genes function as a defense mechanism against myriocin.