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author:("Liu, beilong")
1.  Stress Granule-Defective Mutants Deregulate Stress Responsive Transcripts 
PLoS Genetics  2014;10(11):e1004763.
To reduce expression of gene products not required under stress conditions, eukaryotic cells form large and complex cytoplasmic aggregates of RNA and proteins (stress granules; SGs), where transcripts are kept translationally inert. The overall composition of SGs, as well as their assembly requirements and regulation through stress-activated signaling pathways remain largely unknown.
We have performed a genome-wide screen of S. cerevisiae gene deletion mutants for defects in SG formation upon glucose starvation stress. The screen revealed numerous genes not previously implicated in SG formation. Most mutants with strong phenotypes are equally SG defective when challenged with other stresses, but a considerable fraction is stress-specific. Proteins associated with SG defects are enriched in low-complexity regions, indicating that multiple weak macromolecule interactions are responsible for the structural integrity of SGs. Certain SG-defective mutants, but not all, display an enhanced heat-induced mutation rate. We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects. Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants.
Our findings are consistent with the view that, not only are SGs being regulated by stress signaling pathways, but SGs also modulate the extent of stress responses. We speculate that nucleocytoplasmic shuttling of RNA-binding proteins is required for gene expression regulation during stress, and that SGs modulate this traffic. The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress.
Author Summary
When cells encounter harsh conditions, they face an energy crisis since the stress will reduce their energy production, and at the same time cause extra demands on energy expenditure. To tackle this dilemma, cells under stress form giant agglomerates of RNA and protein, called stress granules. In these, mRNA molecules are kept silent, preventing waste of energy on producing proteins not needed under these conditions. A few mRNAs, encoding proteins required for the cell to survive, stay outside of stress granules and escape this silencing. This mechanism can protect plants and microbes against cold spells or heat shocks, and human cells exposed to oxidative damage or toxic drugs. We have investigated which genes are necessary to form stress granules, and their impact on the stress response. We discovered that mutant cells unable to form stress granules overreacted to stress, in that they produced much higher levels of the induced mRNAs. We think this means that gene regulatory proteins are sequestered inside stress granules, inhibiting their action. Stress granules may thus function as moderators that dampen the stress response, safeguarding the cell against excessive reactions.
doi:10.1371/journal.pgen.1004763
PMCID: PMC4222700  PMID: 25375155
2.  Essential Genetic Interactors of SIR2 Required for Spatial Sequestration and Asymmetrical Inheritance of Protein Aggregates 
PLoS Genetics  2014;10(7):e1004539.
Sir2 is a central regulator of yeast aging and its deficiency increases daughter cell inheritance of stress- and aging-induced misfolded proteins deposited in aggregates and inclusion bodies. Here, by quantifying traits predicted to affect aggregate inheritance in a passive manner, we found that a passive diffusion model cannot explain Sir2-dependent failures in mother-biased segregation of either the small aggregates formed by the misfolded Huntingtin, Htt103Q, disease protein or heat-induced Hsp104-associated aggregates. Instead, we found that the genetic interaction network of SIR2 comprises specific essential genes required for mother-biased segregation including those encoding components of the actin cytoskeleton, the actin-associated myosin V motor protein Myo2, and the actin organization protein calmodulin, Cmd1. Co-staining with Hsp104-GFP demonstrated that misfolded Htt103Q is sequestered into small aggregates, akin to stress foci formed upon heat stress, that fail to coalesce into inclusion bodies. Importantly, these Htt103Q foci, as well as the ATPase-defective Hsp104Y662A-associated structures previously shown to be stable stress foci, co-localized with Cmd1 and Myo2-enriched structures and super-resolution 3-D microscopy demonstrated that they are associated with actin cables. Moreover, we found that Hsp42 is required for formation of heat-induced Hsp104Y662A foci but not Htt103Q foci suggesting that the routes employed for foci formation are not identical. In addition to genes involved in actin-dependent processes, SIR2-interactors required for asymmetrical inheritance of Htt103Q and heat-induced aggregates encode essential sec genes involved in ER-to-Golgi trafficking/ER homeostasis.
Author Summary
Asymmetric cell division is key to cellular rejuvenation and budding yeast exploits this mode of cytokinesis to generate a young daughter cell from a mother cell that with each division grows progressively older. Thus, age physiognomies are reset in the progeny during division, a phenomenon that requires a mother-biased segregation of cytoplasmic ‘aging factors’, including damaged/aggregated proteins. There are two models for how aggregated proteins are segregating in a mother cell-biased fashion; one holds that asymmetric inheritance is a purely passive outcome of the aggregates' random but slow diffusion whereas the other model reasons that specific factors/organelles prevent free diffusion of aggregates into the daughter cell. In the present work, we tested whether the passive diffusion model or the factor-dependent model appear most relevant in explaining asymmetrical inheritance by quantifying traits predicted to affect inheritance by passive diffusion and identifying factors required for asymmetrical inheritance amongst essential genes interacting with SIR2; a gene shown previously to be required for mother-biased segregation. We show that passive diffusion of aggregates is not sufficient to establish mother-biased segregation and that ER to Golgi trafficking, in addition to the actin cytoskeleton, calmodulin, and the Myo2 motor protein, are key components restricting the inheritance of both heat stressed-induced aggregates and aggregates formed of the Huntington disease protein Htt103Q.
doi:10.1371/journal.pgen.1004539
PMCID: PMC4117435  PMID: 25079602
3.  Enhancing protein disaggregation restores proteasome activity in aged cells 
Aging (Albany NY)  2013;5(11):802-812.
The activity of the ubiquitin-proteasome system, UPS, declines during aging in several multicellular organisms. The reason behind this decline remains elusive. Here, using yeast as a model system, we show that while the level and potential capacity of the 26S proteasome is maintained in replicatively aged cells, the UPS is not functioning properly in vivo. As a consequence cytosolic UPS substrates, such as ΔssCPY* are stabilized, accumulate, and form inclusions. By integrating a pGPD-HSP104 recombinant gene into the genome, we were able to constitutively elevate protein disaggregase activity, which diminished the accumulation of protein inclusions during aging. Remarkably, this elevated disaggregation restored degradation of a 26S proteasome substrate in aged cells without elevating proteasome levels, demonstrating that age-associated aggregation obstructs UPS function. The data supports the existence of a negative feedback loop that accelerates aging by exacerbating proteostatic decline once misfolded and aggregation-prone proteins reach a critical level.
PMCID: PMC3868723  PMID: 24243762
Replicative aging; yeast; UPS; proteasome; disaggregation
5.  Mediator Promotes CENP-A Incorporation at Fission Yeast Centromeres 
Molecular and Cellular Biology  2012;32(19):4035-4043.
At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-ACnp1, which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-ACnp1 from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ cells appears to directly block CENP-ACnp1 incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.
doi:10.1128/MCB.00374-12
PMCID: PMC3457525  PMID: 22851695
6.  Mediator Influences Telomeric Silencing and Cellular Life Span ▿ 
Molecular and Cellular Biology  2011;31(12):2413-2421.
The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.
doi:10.1128/MCB.05242-11
PMCID: PMC3133415  PMID: 21482672
7.  Chemogenetic fingerprinting by analysis of cellular growth dynamics 
Background
A fundamental goal in chemical biology is the elucidation of on- and off-target effects of drugs and biocides. To this aim chemogenetic screens that quantify drug induced changes in cellular fitness, typically taken as changes in composite growth, is commonly applied.
Results
Using the model organism Saccharomyces cerevisiae we here report that resolving cellular growth dynamics into its individual components, growth lag, growth rate and growth efficiency, increases the predictive power of chemogenetic screens. Both in terms of drug-drug and gene-drug interactions did the individual growth variables capture distinct and only partially overlapping aspects of cell physiology. In fact, the impact on cellular growth dynamics represented functionally distinct chemical fingerprints.
Discussion
Our findings suggest that the resolution and quantification of all facets of growth increases the informational and interpretational output of chemogenetic screening. Hence, by facilitating a physiologically more complete analysis of gene-drug and drug-drug interactions the here reported results may simplify the assignment of mode-of-action to orphan bioactive compounds.
doi:10.1186/1472-6769-8-3
PMCID: PMC2532679  PMID: 18721464

Results 1-7 (7)