New & Noteworthy

New Yeast-Based Assay for Classifying BRCA1 Variants

May 13, 2022

Lifetime risk of developing ovarian or breast cancer is increased by germline mutations in the BRCA1 gene. While specific pathogenic variants have been well studied, new sequencing technologies continue to identify variants of uncertain significance (VUS). These variants are comparatively rare and cannot easily be studied in humans. Thus, a recent study in the International Journal of Molecular Sciences by Bellè et al. demonstrates a means to assess pathogenicity of a given variant in a cell-based assay in yeast. The new technique complements previous techniques (one in yeast, others computational) to improve the accuracy and sensitivity of assessing pathogenicity for the numerous variants of BRCA1.

From Bellè et al., 2022; red pathogenic, turquoise benign, black uncertain

Belle et al. approached their study by noting that BRCA1 mainly affects DNA repair and genome stability, and that yeast has a full toolbox for studying these processes. The team previously demonstrated that pathogenic BRCA1 variants increase the rates of intra- and interchromosomal homologous recombination (HR) and also gene reversion (GR) in yeast.

For the current study, they developed a diploid strain that allows simultaneous assessment of intra- and interchromosomal HR by use of two mutated markers, one that repairs by intrachromosomal exchange and the other by interchromosomal exchange. When they induced BRCA1 variants from a plasmid, they were able to compare rates of HR (compared to the WT BRCA1 gene) by the simple use of plate assays.  

Similarly, a haploid strain with a different mutated marker that repairs by gene reversion was used to assess rates of GR upon induction of a BRCA1 variant versus wild type.  

From Bellè et al., 2022

After multiple replications of the experiments, the authors plotted Waterfall distributions of the results to arrive at breakpoint values at which a variant could be called benign versus pathogenic for each assay. These results were then analyzed for rates of false positives and false negatives with respect to previous data.

From Bellè et al., 2022; red pathogenic, turquoise benign, gray undermined

The combination of the two yeast-based assays (HR/GR and SCP, together called yBRCA1) was evaluated for performance and shown to be highly accurate and reliable.

From Bellè et al., 2022

As full concordance of results was obtained for three out of ten VUS between the yeast-based combined method and other non-yeast methods, the authors conclude that no one technique is suitable for making a clinical assessment but that multiple techniques will reduce numbers of VUS and resolving conflicting interpretations. Measurement of DNA repair in yeast is a particularly potent example of how yeast can provide a whole-cell, functional assay that sheds light on cell-division disorders.

Categories: Research Spotlight

Tags: yeast model for human disease, cell-based assay, breast cancer, Saccharomyces cerevisiae

Role of Npl3p in regulating senescence rates

May 06, 2022

Telomeres, regions of repetitive DNA at the terminal ends of linear chromosomes, function as protective caps essential to maintain chromosomal structural integrity. Telomeres shorten with every cell cycle and eventually activate replicative senescence (a checkpoint-mediated cell cycle arrest) once they reach a critical length. Regulation of telomere length is essential as an uncontrolled shortening of telomeres can cause organismal aging, and the inability to trigger senescence can result in tumor development. In budding yeast, regulatory factors such as TERRA (telomere repeat containing RNA), a RNAPII-transcribed long non-coding RNA at all telomeres, and R-loops promote Homology-Directed Repair (HDR) at critically short telomeres. Whereas at long telomeres, RNase H2 and Rat1p are recruited and function to degrade TERRA and R loops during S phase.

An interesting new study by Perez-Martinez L et al. in EMBO identifies a telomere-binding protein, Npl3p, to stabilize R-loops at critically short telomeres to prevent premature senescence. The authors propose that at short telomeres, TERRA recruits Npl3p, and the bound protein stabilizes R-loops by limiting access to degrading enzymes like Rat1p and RNAse H2. As a result, the stabilized R-loops promote HDR, and telomeres are elongated to prevent early senescence. Conversely, the absence of Npl3p causes R-loop instability and a defective HDR mechanism, unable to constrain the senescence rates.

From Perez-Martinez L et al., 2022

The authors additionally show that like TERRA and R-loops, Npl3p levels are regulated in a cell-cycle-dependent manner, with increased accumulation during the early S phase followed by a decline in the late S phase. The study also points to the fact that several proteins or complexes, including Npl3p and Tlc1p, accumulate strongly at short telomeres and in the presence of RNAse A and RNAse H, this interaction is lost.

The study highlights Npl3p as an essential factor in studying the diseases associated with dysregulation of telomere length and senescence rates.

Categories: Research Spotlight

Tags: telomere, telomere length, senescence, Saccharomyces cerevisiae, Homology-Directed Repair

Degradation of Mmr1 protein vital for mitochondrial dynamics

April 29, 2022

Regulating mitochondrial dynamics during cell division is crucial for maintaining homeostasis in eukaryotic cells. In budding yeast, proteins such as Myo2p and Mmr1p are essential to transport mitochondria into the growing bud, which post cell division, exists as an independent daughter cell. Improper inheritance and/or distribution of mitochondria can generate reactive oxygen species (ROS) toxicity in daughter cells.

An interesting new study by Obara K et al. in Nature Communications shows that degradation of Mmr1p is crucial in maintaining mitochondrial homeostasis in budding yeast. During cell division, Mmr1p bridges mitochondria and Myo1p and assists in its transportation to the bud via actin cables. Once the transportation is complete, kinases such as Ste20p and Cla4p phosphorylate Mmr1, leading to its recognition and poly-ubiquitination by E3 ligases, Dma1p/Dma2p. The proteasome degrades the poly-ubiquitinated and phosphorylated Mmr1p, and the mitochondria are released from Myo2p and distributed in the bud. Once released, Myo2p translocates to the bud neck.  

From Obara K et al., 2022

The study shows that in the absence of Dma1p/Dma2p, the transported mitochondria are not released from the actin-myosin machinery but in fact, become expanded or deformed and accumulate at the bud tip and then at the bud neck (due to translocation of Myo2p). Double mutants with altered mitochondrial morphology exhibit elevated respiratory activity and increased generation of ROS, rendering cells hypersensitive to oxidative stress

From Obara K et al., 2022

Additionally, the authors show that bud-localized kinases, Ste20p, and Cla4p phosphorylate (most probably) the serine residue at amino acid 414 of Mmr1p and regulate its degradation only after the mitochondria are transported to the growing bud, a crucial step in proper mitochondrial distribution.

Thus, the study highlights the importance of Mmr1p, Dma1p, and Dma2p in maintaining mitochondrial morphology and distribution required to sustain cell homeostasis in yeast cells.

Categories: Research Spotlight

Tags: cell homeostatis, mitochondrial inheritance, cell division, Saccharomyces cerevisiae

Yeast Lifespan Impacted by rDNA Copy Number

April 22, 2022

Replicative lifespan (RLS), determined by the number of daughter cells a mother cell produces before death, has proved to be an effective model for studying aging in budding yeast. The chromatin-associated proteins Sir2p and Fob1p have been shown to modulate ribosomal DNA (rDNA) and impact the formation of extrachromosomal rDNA circles (ERCs), an accumulation of which is linked to a shorter lifespan.

A recent study by Hotz M et al. in PNAS has shown that chromosomal rDNA copy number (CN) positively correlates with RLS in budding yeast. The authors performed whole-genome sequencing (WGS) of 13 wild-type strains and analyzed the lifespan data, which showed an increased rDNA CN along with enhanced RLS. Additionally, the data showed that the rDNA CN explains the majority (~ 70%) of RLS variation observed in almost identical wild-type strains.

To understand this correlation, the authors analyzed ERC levels in aging cells and fob1-Δ strains, in which ERC levels are low. Together, the analysis concluded that ERCs are inversely correlated with rDNA CN. Exploring further, the authors found that cells with lower rDNA CN showed improved accessibility of the upstream activating factor (UAF) complex binding site at the SIR2 locus. This change in chromatin accessibility reduces the expression of SIR2, causing higher ERC levels and thus a shortened lifespan, implicating both Sir2p levels and ERCs as the underlying cause of the CN-RLS correlation.

 Additionally, the authors analyzed the CN-RLS relationship in a set of mutant strains (such as hda2-Δ, upb8-Δ, gpa2-Δ, etc.), all known to increase lifespan. The data showed that while some mutants appeared to impact the CN-RLS relationshiprDNA CN strongly influenced the RLS of these mutant strains (except for fob1-Δ). 

Thus, the study demonstrates how rDNA CN impacts yeast lifespan by regulating certain aging factors and highlights rDNA copy number as an essential parameter to examine in aging studies.

Categories: Research Spotlight

Tags: replicative life span, Saccharomyces cerevisiae, yeast model for aging

Yeast Reveals an Alzheimer’s Clue

April 15, 2022

In a recent issue of EMBO Molecular Medicine, an intriguing study with potential implications for Alzheimer’s disease (AD) by Ring et al. used yeast to look at why human amyloid beta 42 (Abeta42) kills cells. Upon overexpression in yeast, human Abeta42 protein oligomerizes into aggregates that translocate to mitochondria, where the aggregates cause oxidative stress and eventual necrotic-like cell death. Functional mitochondria are required for this Abeta42-mediated death, and a combined genetic and proteomic approach in yeast identified the HSP40-type chaperone Ydj1p as critical for stabilizing the oligomers and escorting them to mitochondria.

From Ring et al., 2022

The effect of ydj1Δ deletion was to lower toxicity, with the effect specific to Abeta42 and not to a different type of induced cell death in a yeast model for Parkinson’s disease. Further, Ydj1p protein directly interacted with Abeta42 in a co-immunoprecipitation experiment.

Yeast YDJ1 is homologous to human DnaJA1, which, when expressed in yeast, re-established the toxicity of Abeta42 to a ydj1Δ strain, indicating functional complementation. The human protein directly interacted with Abeta42 in a murine model for Alzheimer’s disease and also displayed dysregulation in post mortem brain samples of AD patients.

In a fly model for AD, deletion of Droj2 (the Drosophila melanogaster ortholog of YDJ1 and DnaJA1) not only reduced the toxicity of Abeta42 but significantly improved the short-term olfactory memory loss associated with Abeta42 expression. Together, the authors convincingly demonstrate the strong evolutionary conservation of this particular chaperone and its effects on exacerbating Abeta42-mediated toxicity, cell death, and memory loss in relevant model systems.  The use of yeast to identify this key factor indicates the power of a simplified and tractable system and will hopefully lead to progress in treating a terrible disease.

Categories: Research Spotlight

Tags: yeast model for human disease, Cell toxicity, alzheimer's, Saccharomyces cerevisiae

The Ties That Bind Mitochondria and Aging

April 08, 2022

Continuing our theme of highlighting lifespan in yeast, this week’s study by Liu et al. in eLife looks at a possible mechanism for age-related loss of mitochondrial quality. As loss of mitochondrial quality is linked to the rate of building new mitochondria, and as mitochondrial biogenesis depends on the import of new building blocks through the mitochondrial membranes, the abundance of transporters is an important determinant. The authors wondered whether cells were “smart” enough to coregulate the level of transporters with the level of new building blocks.

To get at this question, they overexpressed the translocases of the outer membrane (TOM) proteins to ask if the overabundance of a particular one caused the overabundance of representative mitochondrial proteins, i.e. displayed a regulatory role. Of these, only TOM70 overexpression (OE) increased the abundance of the four representative mitochondrial proteins. They further showed that TOM70 OE led to increased levels of mitochondrial DNA, likely via Mip1p, the mitochondrial DNA polymerase gamma. As further evidence of the role of TOM70 in regulation, the tom70 knockout led to the reverse effect from OE, i.e. reduction in levels of both mitochondrial proteins and mtDNA. Tom70p is a well-conserved receptor within the TOM complex, but this role in regulation of mitochondrial biogenesis is a novel finding.

From Liu et al., 2022

The authors then examined a number of possible intermediate signaling possibilities between Tom70p and the increased abundance of their target proteins. By disrupting various transcription factors and assessing reactive oxygen species, they saw only partial blockage by loss of any one signaling partner, and thus concluded that Tom70p works via multiple pathways.

As TOM70 appears to have a role in regulating new mitochondria, and mitochondrial defects are well established as an indicator of age, the authors asked about connections between TOM70 and aging. They first noted that Tom70p levels go down over time across many organisms. Is this a cause or an association? To counteract the wild-type reductions in Tom70p levels, they overexpressed TOM70 and then examined age-related phenotypes. Higher levels of Tom70p reversed age-associated loss of mitochondrial membrane potential, age-related reductions in other mitochondrial proteins, and, indeed, extended the lifespan of yeast. Further, the tom70∆ knockout once more showed the reverse effects, leading to accelerated aging and reduced lifespan.

From Liu et al., 2022

Using the remarkable power of yeast, the authors were then able to study the mechanics of the reduction in Tom70p over time. They found that mRNA levels reduce over time due to loss of transcriptional activity, which was rescuable by changes made to the TOM70 promoter. Further, they showed the degradation of Tom70p increased over time due to increased levels of the protein Dnm1p, involved in sorting proteins for degradation under age-related vacuole deacidification. Thus, TOM70 mRNA levels decrease and Tom70p protein becomes less stable, providing redundancy in reduction of a protein that activates mitochondrial biogenesis. With the relative ease possible in yeast, the authors made important headway in revealing these mechanistic links between mitochondrial quality and aging.

Categories: Research Spotlight

Tags: mitochondrial biogenesis, mitochondrial quality, yeast model for aging, mitochondrial regulation, Saccharomyces cerevisiae, mitochondrial import

Polarity Linked to Lifespan in Mother Yeast Cells

April 01, 2022

In last week’s post, we discussed asymmetry in pH between mother and daughter cells. This week we’re continuing the theme of mother/daughter inheritance patterns with a study of asymmetrical mitochondrial inheritance. The underlying hope is to garner clues about aging and lifespan, since mother cells have reduced lifespan relative to their daughters.

Yeast cells are normally symmetrical, i.e. round. One of the first steps during cell growth is the breaking of symmetry to create poles, where one pole will become the bud tip and the other pole will become the mother cell tip. In a recent paper in iScience, Yang et al. describe how the mother cell tip is normally distal to the bud tip and that higher-functioning mitochondria localize to both poles. The mitochondrial F box protein Mfb1p was shown to be key for tethering mitochondria at the mother cell tip, which prevents every higher-functioning mitochondria from going to the bud. Mfb1p remains associated with the mother cell tip throughout the entire cycle, and is the only protein known to do this.

From Yang et al., 2022

Interestingly, Mfb1p is itself asymmetrically localized, where it associates with mitochondria in the mother cell but remains excluded from the daughter cell until just before cytokinesis.

To assess the link between aging and polarity, the authors labeled bud scars with dyes that distinguished between new and old scars. In highly polarized cells, new scars will form directly next to one another. They found polarized bud site selection in >97% of young cells, which was quickly reduced to 89% after 6–10 divisions and plateaued at ~70% for the oldest cells. Thus, aging as a factor of polarity was detectable early in lifespan and continued to decline to a fixed level. Likewise, the polarized localization of Mfb1p to mother cell tips also declined with age, where young cells showed Mfb1p tethered to mitochondria almost exclusively at the mother cell tips, but over time the localization dispersed throughout the mother cell. Interestingly, deletion of RSR1/BUD1 (required for polarized bud site selection) disrupted this polarized localization of Mfb1p within the mother, yet didn’t cause Mfb1p to go to the bud (i.e. cause loss of asymmetry). Thus, polarization and asymmetry could be separated in the bud1Δ mutant cells—and these cells also showed reduced lifespan. The same was seen in bud2Δ and bud5Δ cells.

Given that both bud1Δ and mfb1Δ cells had reduced lifespan, the authors asked whether the two mutations affected lifespan in the same way by examining the double mutant for additive effects. Seeing no additive effects, they concluded that BUD1 and MFB1 operate in the same pathway for controlling mitochondrial distribution and the associated effects on aging.

Whereas the study we highlighted last week did not find a link between aging and pH in yeast, Yang et al. show that mitochondrial localization and inheritance patterns have a clear relationship with replicative lifespan. The secrets of aging continue to invite study, and future results should be equally intriguing.  

Categories: Research Spotlight

Tags: mitochondria, yeast inheritance, mitochondrial inheritance, polarity in yeast, Saccharomyces cerevisiae, yeast model for aging

Yeast Mother/Daughter Relationships

March 25, 2022

Yeast researchers have long observed asymmetry in cytosolic pH between mother and daughter cells. Likewise, researchers have detailed the accumulation of long-lived asymmetrically retained proteins (LARPs) in mother cells, one of which is the plasma membrane proton pump Pma1p. Pma1p transports protons out of the cytosol, thereby increasing pH, and mother cells show marked increases in vacuolar pH as they age. As mother cells have a shorter replicative life span (RLS) than daughter cells, and aging factors have been linked to pH, it is reasonable to ask whether accumulation of Pma1p itself reduces life span in mother cells.

To address this specific question, Yoon et al. in a recent issue of International Journal of Molecular Sciences described screening for suppressors of asymmetric inheritance of Pma1p. They identified three vacuolar protein sorting genes (VPS8, VPS9, and VPS21) for which mutation resulted in high percentages of abnormally symmetric distribution of Pma1p-GFP.  As all three of these genes are involved in endocytosis, they asked whether these mutations affected all proteins that are asymmetrically distributed between mother and daughter cells, or just those that reside in the plasma membrane. They found the latter, that the defect is restricted to PM proteins.

From Yoon et al.

Using these mutants with abnormal asymmetric distribution of the proton pump, the authors asked if defects in asymmetric distribution of Pma1p caused changes in aging. Did mother cells without accumulated Pma1p have a longer life span? The answer was a clear “No.” There was little difference in RLS between mutant and wild type, showing that asymmetric distribution of Pma1p does not correlate with aging.

From Yoon et al.

These results are in fact a bit surprising, because aging in mother cells had been previously correlated with a mutation in PMA1 (the pma1-105 allele, Henderson et al., 2014), which increased lifespan by about 30%. Thus, it appears that Pma1p plays a role in aging, but not via asymmetric distribution between mother and daughter cells.

This study adds to what is known about a complicated system. As usual, the awesome power of yeast genetics (#APOYG) provides an excellent forum in which to ask complicated questions in a simple system. Hopefully the links between pH, aging and asymmetry will be revealed in future experiments.

Categories: Research Spotlight

Tags: replicative life span, asymmetry in cell division, Saccharomyces cerevisiae, yeast model for aging

Guardians of (the) Chromosome Stability

March 18, 2022

Chromosome instability (CIN), recognized as a hallmark of several cancers, results from error in chromosome segregation leading to differences in both chromosome structure and numbers. Chromosome Segregation protein (CSE4) in budding yeast and CENP-A in humans are examples of an evolutionary conserved histone H3 variant in all eukaryotic centromeres which have a crucial role in efficient chromosome segregation. As such, overexpression of CSE4 (or CENP-A) are known to cause mislocalization of the protein to non-centromeric chromatin leading to CIN.

In an interesting new study in Nucleic Acids Research, Ohkuni et al. have shown how mislocalized Cse4p is removed and targeted for proteasomal degradation, thus preventing CIN. The authors show that Cdc48p-Npl4p-Ufd1p AAA ATPase complex recognizes the mislocalized and polyubiquinated Cse4p and facilitates its removal from the non-centromeric chromatin, targeting it for degradation. The authors demonstrate that the Cdc48p complex targets specifically the mislocalized chromatin-bound Cse4p and not the centromeric Cse4p. Another essential factor involved in this mechanism is an E3 ubiquitin ligase, Psh1p, which polyubiquinates Cse4p and promotes its recognition by Cdc48p-Npl4p-Ufd1p AAA ATPase complex via its cofactor Npl4p.

This paper demonstrates an important role for the Cdc48-Npl4-Ufd1 AAA ATPase complex in removing mislocalized Cse4p from non-centromeric chromatin. It infers that accumulation of mislocalized CENP-A may contribute to aneuploidy in human cancers, thus revealing another pathway to target for treating human diseases.

Categories: Research Spotlight

Tags: cancer, Protein Mislocalization, chromosome instability, Saccharomyces cerevisiae

Yeast Show No Fear of Commitment

March 11, 2022

Meiosis in budding yeast is typically induced by starvation, with a signal roughly translated as “make spores quick because things are terrible right now.” When the outside food supply dries up, meiosis gets initiated in the nucleus of the yeast cell. If food is resupplied early enough, meiosis can pause and mitosis—a normal cell division—can be undergone instead, a process called “return to growth” (RTG). There’s a sharp point-of-no-return in mid-prometaphase, though, after which no amount of food will make cells go back to mitosis—they’ve committed to meiosis.

From Gavade et al., 2022

The components involved in this commitment to meiosis have been poorly understood. It was shown by several studies that the transcription factor Ndt80p is required at high levels to establish meiotic commitment by inducing middle meiosis genes. When levels of Ndt80p are low, in contrast, cells show a commitment defect in which they go back to mitosis even after initiating meiosis, leading to defective polyploid germ cells with multiple nuclei.

From Gavade et al., 2022

Gavade et al., in a study that just came out in Current Biology, discover six novel regulators of meiotic commitment. The authors first identified proteins whose overexpression reversed the commitment defects of a low-Ndt80 strain. The pool of potential regulators was narrowed by looking at mutants of the genes identified in the overexpression screen to see if decreased levels would cause commitment defects. They found BCY1 (involved in nutrient sensing), IME1 (a meiosis-specific kinase), CDC5 (Polo kinase, with related roles in mitosis), BMH1 and BMH2 (14-3-3- proteins involved in numerous types of signaling), and PES4 (an RNA-binding protein with an uncharacterized role in cell cycle control) to all have roles in meiotic commitment. The authors further showed that Bmh1p and Bmh2p are direct regulators of both Ndt80p and Cdc5p, the former by protein stability and the latter by protein activation.  

Overview of meiotic commitment, from Gavade et al., 2022

This intriguing study is an excellent example of how complex processes—such as the effect of food on sexual reproduction—can be teased apart in yeast. A relatively simple screen was able to identify proteins not formerly known to have a role in meiotic commitment, and it will be exciting to see if these proteins have orthologs in mammals that play related roles.

Categories: Research Spotlight

Tags: response to stress, meiotic commitment, response to starvation, Saccharomyces cerevisiae, meiosis

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