December 19, 2022
Stanford University will be closed for two weeks starting Wednesday, December 21, and will reopen on Wednesday, January 4, 2023. SGD staff members will be taking time off, but the website will be up and running throughout the winter break, and we will resume responding to user requests and questions in the new year.
December 15, 2022
We are proud that SGD has been included in the first list of Global Core Biodata Resources (GCBRs) announced by the Global Biodata Coalition (GBC)! This collection of 37 resources comprises deposition databases which archive and preserve primary research data, and knowledgebases, such as SGD, that add value to research data through expert curation and annotation. The list is meant to highlight those data resources whose long term funding and sustainability is critical to life science and biomedical research worldwide.
GCBRs represent the most crucial resources within the global life science data community. SGD’s selection as a key global data resource recognizes that SGD is essential to the global research endeavor.
November 15, 2022
Gene transcription — the elaborate process that our cells use to read genetic information stored in DNA – was long thought to be turned on only when certain regulatory factors traveled to specific DNA sequences. In a new study published in Genes & Development, Mittal et al., 2022, discovered that a subset of genes has their transcription regulatory factors and cofactors already in place, but in a latent state. With the appropriate signals, these “poised” genes become highly active.
The authors calculated the DNA-bound preinitiation complex/TBP-associated factor (PIC/TAF) ratio at all yeast genes and identified two major classes. The first, and largest, group provides basic “housekeeping” functions and are usually “on” at very low levels all the time (i.e., “constitutive”). The second class, the “inducible” genes, has a whole entourage of “poised” proteins assembled nearby which provides a guiding hand to transcription machinery when triggered by environmental signals, resulting in high levels of “induced” transcription.
Using CRISPR-Cas9 mediated protein depletion/degradation and gene knockout techniques, Mittal et al. removed parts of the SAGA and TFIID cofactors to systematically examine the role they play in regulating the above gene classes. They discovered that the constitutive class largely depended on TFIID, whereas the inducible genes required both SAGA and TFIID, suggesting an integrated pathway of PIC assembly at inducible genes. If true, such a possibility would be different from previous models where the two cofactors were thought to engage in somewhat distinct mechanisms of PIC assembly.
The authors further examined the above results and were surprised to find that at the inducible promoters, SAGA stabilized Taf1p (and thus TFIID) which helped in a rapid and robust transcription initiation of these genes upon acute environmental changes.
In addition, Mittal et al. also removed gene-specific transcription factors (TFs) to address how these factors recruited TBP upon sensing changes in environmental signals. They showed that TFs such as Hsf1p and associated cofactors were already bound to gene promoters prior to induction, instead of traveling to cognate sites upon induction. These cofactors, thus, lend a helping hand in recruiting TBP and the associated machinery at the induced genes.
Lastly, Mittal et al. removed Gcn5p and DUB subunits of SAGA to examine the effect on histone acetylation and ubiquitylation, respectively. They showed that global histone acetylation and ubiquitylation levels require active Gcn5 and the deubiquitination (DUB) activities, suggesting a SAGA independent moiety contributing towards maintaining total cellular acetylated and ubiquitylated histone pools.
Contributed by Chitvan Mittal.
Categories: Research Spotlight
October 20, 2022
SGD has updated our RNA pages to add secondary structures provided by RNAcentral and generated by R2DT. Thumbnails and linkouts to RNAcentral via RNAcentral IDs are shown on the Summary and Sequence pages.
Interactive secondary structure viewers are available on the Sequence pages.
Take the pages for a spin! For more information about the structures, please see the Help page at RNAcentral.
Tags: RNA structure
September 09, 2022
There are ~400 origins of replication in yeast, each of which can be “licensed” by the binding of the conserved origin recognition complex (ORC) and then the MCM replicative helicase complex, all of which happens in G1 phase. During the subsequent S phase, origins are then “activated” by binding of several other replication factors, leading to unwinding and then nascent strand synthesis.
The regulation of origin licensing and activation is a complex, multi-level process, for which numerous aspects of the picture remain unclear. As yeast has the most tools for studying this process, including a full map of origins and numerous options for genetic and biochemical modulations, it presents the ideal model in which to ask probing questions. A recent study by Regan-Mochrie et al. in Genes & Development has revealed the key role of sumoylation in regulation of genome replication.
The origin recognition complex comprises six subunits, encoded by ORC1 to ORC6. The authors built on previous results showing sumoylation of these proteins during DNA damage to ask about their modification status under normal growth. They were able to assess sumoylation status for four of the six subunits, observing various degrees of sumoylation of Orc1p, Orc2p, Orc4p, and Orc5p. They then created a construct to hypersumoylate Orc2p to ask how this affects cells, and found it led to cell lethality. This lethal effect was specific to the Orc protein, as other non-Orc hypersumoylated proteins were tolerated.
The lethality could be rescued by reducing the levels of a SUMO-conjugating enzyme, further indicating the specificity of the effect. The authors identified a subset of early origins that were preferentially inhibited upon hypersumoylation and, upon study, determined that the extra sumoylation interfered with loading of the MCM complex.
After identifying the residues becoming sumoylated on Orc2p, the authors were able to generate mutants to ask about the converse, i.e. hyposumoylation. Indeed, as might be hypothesized, lack of sumoylation caused DNA replication defects via abnormal increased firing of early origins.
Thus, by close study in yeast, the role of sumoylation in genomic stability becomes more clear, where sumoylation of ORC subunits affects loading of the MCM complex, which is itself the substrate for loading of activation factors. Modulated sumoylation status appears to provide a key level of regulatory control.
Categories: Research Spotlight
September 02, 2022
While glycolysis is highly conserved between unicellular yeast and multicellular humans, it appears that not only the glycolytic functions but also the secondary (aka “moonlighting”) functions of the relevant proteins remain largely consistent. This consistency presents unique opportunities for better understanding human glycolysis in muscle tissue and elsewhere.
The technical challenges of studying glycolytic enzymes are not trivial—largely due to genetic redundancy for such critical components—but a recent report by Boonekamp et al. in Cell Reports explains how the authors built on previous work to overcome the obstacles in yeast. Employing a minimized set of glycolytic genes that were also relocated to a single chromosome (strain SwYG) made it possible to swap in human orthologs with relative ease.
The authors first looked at direct complementation of yeast genes by 25 human glycolytic enzymes. Remarkably, 22 of 25 human genes readily complemented their yeast counterpart. The three exceptions were hexokinases 1, 2, and 3 (HsHK1, HsHK2, and HsHK3), which are roughly twice the size of their yeast orthologs and have lower sequence conservation.
As glycolysis cannot take place without the hexokinases, the authors first looked more closely at HsHK1 and HsHK2 to ask why they fail to function in yeast. Upon exposure to glucose, for which the native human proteins could not support growth, they created conditions to select for systematic mutations that conferred improved glycolysis. The mutant proteins proved to be less sensitive to inhibition by glucose-6-phosphate (G6P), a potent allosteric inhibitor of hexokinase activity.
With this understanding, the authors set out to complement the entire pathway in yeast with human proteins. They created two different strains, one with HsHK2 because it is considered the main isoenzyme in human muscle (strain HsGly-HK2), and one with HsHK4 because it shows less inhibition by G6P (strain HsGly-HK4). The HsGly-HK2 strain could not grow well on glucose until after a long lag phase in which mutations were selected near the G6P-binding site of HsHK2.
Comparing the uptake and output of the two humanized yeast strains to the native yeast strain revealed a number of intriguing differences, especially around the different enzyme activities between human and yeast. These differences led to different behaviors on different carbon sources and overall slower growth and glycolytic flux for the humanized strains versus native yeast.
Three glycolytic yeast enzymes have secondary “moonlighting” roles beyond their function in glycolysis. Hexokinases are involved in glucose repression of genes such as invertase (SUC2), and, indeed, it appears that human hexokinases can also at least partially complement this secondary function. Yeast aldolase (FBA1) plays a secondary role in vacuolar function that is required for growth at alkaline pH. This function is likewise complemented by the human orthologs, where the humanized strains can grow at pH 7.5. The third moonlighter is enolase, where yeast ENO2 is required for mitochondrial import of tRNALys, thereby allowing growth at higher temperatures and on non-fermentable carbon sources. All three human enolases can at least partially complement this growth defect, and thus appear to have the same secondary function.
Despite the high conservation of functions, the humanized yeast strains have a slow growth phenotype. The authors used this phenotype to employ adaptive laboratory evolution to see which genomic changes restore growth. Interestingly, the mutations that restored growth were mostly not in the glycolytic enzymes themselves, but in associated factors that regulate enzyme abundance and activity. The identity of these regulators and the potential for targeting them in human muscle have already rewarded the successful transfer of skeletal muscle glycolysis into yeast.
Categories: Research Spotlight
August 25, 2022
Perturbations in iron homeostasis affect aging, but how this happens has remained a bit of a black box. A new study by Patnaik et al. in Cell Reports illuminates this box by looking more closely at the transcription factors that are first to respond when iron becomes limiting.
Key among these are Atf1p and Atf2p, which activate the full suite of iron-mobilization genes, among which is TIS11/CTH2, which encodes an RNA-binding protein that targets specific messages for decay.
The targeted messages flagged for decay encode mitochondrial proteins, as these use iron but are not the most essential in the set. The most essential Fe-requiring enzymes are those involved in DNA synthesis and repair, such that slowing/shutting mitochondrial function is a response to iron deficiency. Intriguingly, mitochondrial function also happens to decline with age.
To find the specific mechanisms linking iron with aging, the authors used an unbiased analysis of genes involved in iron homeostasis to see which showed connection with aging. The strain with a tis11Δ mutation lived longer than any others, with a lifespan extended by 51.1%. In a broader sense, they found that genes involved in different aspects of response to iron deficiency also had different effects on fitness and aging.
Delving more deeply into the role of Tis11p/Cth2p in aging, the authors used RNA-seq and Ribo-seq to look at temporal changes in transcription versus translation in aging cells. They showed how, overall, aging leads to inhibition of translation—except for certain genes which are upregulated instead. Interestingly, most of the upregulated genes are in the Fe regulon that gets activated by the first responder Atf1p.
While the expression of TIS11/CTH2 increases both with aging and with iron deficiency, the deletion of the gene extends lifespan. Thus, multiple lines of evidence suggest Tis11p/Cth2p is a negative regulator of longevity. The key connection appears to be mitochondrial translation, where the function of Tis11p/Cth2p to inhibit translation of mitochondrial transcripts for repressing non-essential Fe-requiring enzymes serves to simultaneously repress overall mitochondrial respiration, which speeds aging.
As not all genes translationally upregulated in the tis11Δ mutant contained appropriate binding sites in the 3’ UTR, the authors looked further and found binding sequences for Puf3p, a protein known to bind and inhibit translation of mRNAs coding for mitochondrial ribosome proteins. Thus, Puf3p appears to be a critical partner for Tis11p/Cth2p in mediating downregulation of mitochondrial function. Further, they questioned the relationship with the Hap4p transcription factor, which regulates numerous components of the electron transport chain and whose overexpression extends lifespan. As the combination of a tis11Δ deletion with HAP4 overexpression had no additive effect in an epistasis experiment, they concluded that Tis11p-dependent repression acts through Hap4p.
The role of phosphorylation of Tis11p/Cth2p was examined by mutating N-terminal serine residues, which impairs degradation of the protein. Consistent with the converse result of extended lifespan in null mutants, the nondegradable version of the protein shortens lifespan.
Thus, the ease of the yeast model once more illuminates intricate connections between critical proteins, facilitating potential drug discovery around several new aging factors.
Categories: Research Spotlight
August 12, 2022
One way to imagine DNA is as a busy road with a lot of competing traffic. Say, a small village in southern Italy…where someone must mediate conflicts between competing vehicles to avoid disaster.
It turns out that the “someone” in yeast cells is Sen1p. Two recent papers from associated groups describe the intriguing detail of how the Sen1p helicase plays this role for RNA polymerase III transcription. The paper by Aiello et al. in Molecular Cell shows how Sen1p regulates transcription-driven conflicts between the several machineries all engaged with DNA. In the related paper by Xie et al. in Science Advances, the authors show how the Sen1p helicase mediates “fail-safe” methods of transcription termination for RNA Pol III, thereby promoting efficiency and avoiding conflict with other pieces of machinery.
The key conflict preventing RNA Pol III from transcribing noncoding genes is with RNA Pol II, which is busy transcribing coding genes. Aiello et al. show how Sen1p has two strategies for mediating these conflicts, both of which involve interactions between Sen1p and the replisome. One involves temporary release of RNA Pol II from DNA while the other resolves genotoxic R-loops in nascent RNA. Both are critical for preventing genome instability.
In the related paper by Xie et al., the authors focus on how termination of transcription of noncoding genes by RNA Pol III is achieved, and the role that Sen1p plays in termination. They show how Sen1p can interact with all three polymerases and also with the other two subunits (Nrd1p and Nab3p) of the NRD1 snoRNA termination (NNS) complex. More specifically, they show by mutation and co-immunoprecipitation that it is the N-terminal domain (NTD) of Sen1p that interacts with RNA Pol III and the replisome.
The authors use metagene analysis of RNA Pol II distribution at mRNA-coding genes to show how Sen1p can promote the release of RNA Pol II to resolve transcription-replication conflicts (TRCs). They further show how the association of Sen1p with the replisome is required for limiting TRCs at the ribosomal replication fork barrier, and how this action appears redundant with that of RNases H. The cooperation and redundancy in this role are key means to protect genome stability.
Not only is Sen1p required for termination of RNA Pol III transcription, but the authors show how this function is independent of the NNS complex. Unlike resolution of conflicts between RNA Pol II and RNA Pol III, the termination function of Sen1p does not require the replisome.
They asked the question of whether Sen1p acts via the primary termination site for RNA Pol III or, rather, a backup secondary termination that catches errors (i.e., when RNA Pol III reads through a weak termination site). Termination for RNA Pol III employs a tract of T nucleotides (T-tract) in the nontemplate strand and these T-tracts can be relatively weak or strong. When T-tracts prove insufficient to stop the polymerase, Sen1p plays a role by means of secondary structures in nascent RNAs, which act as auxiliary cis-acting elements. This backup method is termed the “fail-safe transcription termination pathway.” The RNA secondary structures are not absolutely required for RNAPIII termination, but can function as auxiliary elements that bypass weak or defective termination signals.
Once more, it is the power of the yeast model that has allowed investigation to such exquisite molecular detail. That cells preserve genomic stability and avoid pile-ups amid so much traffic along DNA remains truly remarkable–even when we know more of how it works.
Categories: Research Spotlight
August 05, 2022
Cells are efficient machines, honed by selection. Recent studies are showing us how cells are more efficient than we realized, with proteins discovered to have multiple functions that can be surprisingly different under changes in environment. A new paper in Molecular Cell describes Get3p as one such protein. Under non-stress conditions, Get3p acts as an ATP-dependent guide for post-translational insertion of tail-anchored proteins into membrane. Under stress, however, Get3p morphs into an antioxidant “guard” protein that prevents toxic protein aggregation by functioning as a chaperone. What are the mechanics of such a switch?
The finding came about in something of a backward way, where Ulrich et al. set out to find a eukaryotic chaperone protein specifically activated during oxidative stress. They looked for similarity to Hsp33, a bacterial protein that protects against oxidative damage by activation of a zinc-coordinating, four-cysteine motif. Their search led them to Get3p, which shares little overall homology but has the same conserved CXC and CXXC motifs.
Whereas the bacterial protein acts only in stress, yeast Get3p also has a non-stress role that differs from its role in stress response. Under non-stress conditions, it is the CXXC motif that plays the major role, where the two cysteines of the motif bind zinc, which aids the protein in dimerization. In the switch to the chaperone-active form, however, it is the nearby CXC motif that plays the critical role.
During oxidative stress, the switch to the chaperone form of the Get3p protein requires two things: (1) loss of bound ATP (with accompanying loss of ATPase activity, see below); and (2) oxidation of the free thiol groups (-SH) on residues C240 and C242 of the CXC motif. Once oxidized from thiol groups to sulfur groups, the two sulfurs of the CXC can form an intramolecular disulfide bond. Thus, in the presence of an ROS like H2O2, the new bond reconfigures the protein to become a non-ATP-requiring active chaperone. The CXC motif, then, acts as a “redox” switch.
The role of ATP with respect to the redox switch is illustrated in the above schematic, where the presence of ATP makes the two thiol groups inaccessible to oxidation. When ATP is lost (due to the oxidative stress that reduces cellular ATP levels), the -SH groups can be oxidized to -S groups that bind to one another and partially unfold the protein, making it chaperone active.
The authors further show how the CXC motif is dispensable for the normal protein-targeting function of Get3p but absolutely required for the chaperone function. Thus, Get3p has two cysteine motifs that act in fully different ways, where CXXC is required for normal guide function while CXC is required for protection from oxidative stress.
Whereas the toxicity of reactive oxygen species is well established, the elucidation of a redox switch-triggered stress response in yeast provides an important clue to how eukaryotes detoxify these oxidants. It would not be surprising to find other cysteine-motif proteins acting as antioxidants in related processes that save cells from damage. As usual, yeast is an excellent model for understanding these intricate relationships.
Categories: Research Spotlight
July 29, 2022
Sequencing of Saccharomyces cerevisiae in the 1990s revealed blocks of duplicated genes, suggesting an ancient whole-genome duplication. The original duplication has been postulated as a means to restore fertility to an interspecies hybrid of ancestral yeast parents; however, the question remains as to why so many paralog doublets have been retained through eons of evolution. Are these paralogs the residual clutter that has yet to be selected away, or has it represented an opportunity that created positive selection? Said another way, if every gene once had a twin, why have some been retained and others lost?
To dig further into understanding doublet evolution, a study by Purkanti and Thattai in a recent issue of Scientific Reports looks at the functional significance of these yeast paralog doublets from an evolutionary perspective. To ask whether retention of doublets across sub-clades of the yeast phylogenic tree might suggest selective advantage, they looked at which Gene Ontology biological processes showed the greatest enrichment of observed over expected for the 887 genes with conserved doublets. The greatest enrichment for over-represented doublets was in the GO process of endocytosis, with its parent term of vesicle-mediated transport also showing significant enrichment.
The authors then assigned vesicle-traffic genes to function-specific and pathway-specific modules to ask about doublet retention. Among the modules, the greatest enrichment of doublets was observed for coat/adaptor genes and lipid control genes. Other modules, such as the endosomal sorting complex required for transport (ESCRT), had no doublets at all, i.e., the genes in this module have completely reverted to singletons.
Intriguingly, the classes with singletons and the classes with doublets can be broadly grouped, where doublets tend to be retained in secretory and early endocytic pathways, whereas singletons largely act in retrograde Golgi traffic and late endocytic steps.
The authors separated all 360 ancestral vesicle traffic doublets into two groups: those that are retained as doublets in present-day species and those that are not. They looked at the nucleotide sequence identity as a proxy for evolutionary rate and found that retention of doublets is strongly associated with lower evolutionary rates. From this they conclude that doublet retention is under significant selection, as the purifying selection that lowers evolution rates is well correlated with functional significance.
While it may be mere coincidence that budding yeast happened to undergo ancestral genome duplication, it appears that this event has proven fortuitous for making S. cerevisiae even more useful as a model. As revealed in this study, the ease of manipulation and the vast knowledge base in yeast makes it possible to ask these deeply important questions about evolutionary impact on paralog doublets.
Categories: Research Spotlight