New & Noteworthy

A Redox Switch that Remakes a Guide Protein into an Antioxidant

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.

From Ulrich et al., 2022

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.

From Ulrich et al., 2022

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.  

From Ulrich et al., 2022

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

Tags: chaperones, Saccharomyces cerevisiae, oxidative stress, redox switch

Insight into Evolution of Paralog Doublets

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.   

Enriched biological processes; those related to vesicle traffic are in bold, from Purkanti and Thattai, 2022

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.

Doublets per module, from Purkanti and Thattai, 2022

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

Tags: paralog doublets, whole genome duplication, vesicle traffic, Saccharomyces cerevisiae, paralogous genes, evolution

SGD Newsletter, Summer 2022

July 28, 2022

About this newsletter:
This is the Summer 2022 issue of the SGD newsletter. The goal of this newsletter is to inform our users about new features in SGD and to foster communication within the yeast community. You can view this newsletter as well as previous newsletters on our Community Wiki.

Performance updates at SGD

sgd maintenanceguy.jpeg

Thousands of SGD users run their data against SGD data every day and this can put a heavy load on servers. To improve performance, we have invested in dockerizing our full database so that loads can be better distributed in real time. We are currently at the stage of testing the frontend of our software to look for bugs, which we do for every update. The next phase will entail dockerizing and testing the backend, so we can improve our data uploads.

We hope to see improved performance for you, our users. If you happen to notice faster speeds or fewer hiccups…we’d be quite glad to hear about it! (A message to the SGD Helpdesk reaches us all.)

Research Spotlights are back

ResearchSpotlight 2022-07-26 at 9.32.28 AM.png

SGD triages all the papers that come out each week to find those that add value to our database. In the process, we are continually impressed by the quality of the research done in yeast and we decided it was time to bring back the “Research Spotlight” as a post that appears on the SGD home page in the “New and Noteworthy” section.

The goal is to highlight interesting new work that either continues an ongoing story, makes a twist in the story we all thought we knew (such as an unexpected side gig for a protein), or offers a new technique or perspective for mining the most value from the yeast model. There are numerous intriguing papers in yeast, including those shedding light on human disease by dissecting the cellular biology in yeast, using our remarkable tools, so as to identify new targets or drugs for humans.

As the model organism databases move toward forming the Alliance of Genome Resources, which will streamline and integrate our combined data, we expect this power to make useful connections will only grow.

Upcoming Yeast Genetics Meeting

YGM2022.png

We may have forgotten how to talk to other people in person, but the Yeast Genetics Meeting is going to give us a chance to practice. The meeting will be held in person for the first time in four years at UCLA, from August 17-21. We hope to see you there!

Of course, it being the COVID era, you can also register to attend virtually, and the cost will not be higher for late registration.


A big congratulations to the award recipients who are the invited speakers for 2022. Tom Petes is receiving the YGM Lifetime Achievement Award, Trisha Davis is giving the Winge-Lindegren Address, Maya Schuldiner is receiving the Ira Herskowitz Award, and Michael Desai is giving the Lee Hartwell Lecture. Learn more about these researchers and their work at the YGM website.

Congratulations to Jeremy Thorner

JeremyThorner.png

While on the subject of congratulations, a past recipient of the YGM Lifetime Achievement Award and a longtime friend and advisor of SGD has been further honored with a full issue of the journal Biomolecules dedicated to his work, Transmembrane and Intracellular Signal Transduction Mechanisms: A Themed Issue in Honor of Professor Jeremy Thorner


We are grateful to have Dr. Thorner’s work integrated into the SGD database and into our wider community’s ongoing mission to understand yeast.

SGD collaborates to add new data links

AlphaFoldPrediction.png

On the topic of integration, SGD is happy to integrate new data sets that add value to the database. We have the ability to incorporate these datasets directly from research groups, rather than from publications. Most recently we integrated the AlphaFold predicted 3D structures for complexes as links on the SGD Interaction and Protein pages. It is now possible to look for your own proteins or complexes of interest and go straight to the predicted structure.

Another recent example is AnalogYeast, a dataset of analogs to yeast proteins in non-fungal organisms predicted by sequence similarity, which was created by the Schuldiner lab. Links have been added to the Resources sections of SGD Protein and Homology pages.

We are open to more of this collaboration and would be glad to hear from community members who think they have data useful to other researchers. Get in touch!

microPublications enter log phase

MicroPub.png

microPublication is part of the emerging genre of rapidly-published research communications. We are seeing a strong set of microPublications come through the database and are glad for this venue to publish brief, novel findings, negative and/or reproduced results, and results which may lack a broader scientific narrative. Each article is peer-reviewed, assigned a DOI, and indexed through PubMed and PubMedCentral.


Consider microPubublications when you have a result that doesn’t necessarily fit into a larger story, but will be of value to others.


To see recent micropublications in yeast, visit the list of micropubs in SGD.

Alliance of Genome Resources – Release 5.2

alliance logo.png

The Alliance of Genome Resources, a collaborative effort from SGD and other model organism databases (MOD), released version 5.2 this past May.

  • The Alliance is now using the latest version of the DRSC Integrative Ortholog Prediction Tool (DIOPT): Version 9. This version includes a data refresh from all orthology sources, the addition of the SonicParanoid algorithm, and the removal of both TreeFam and RoundUp data sources.
  • Much of this release focused on backend enhancements, particularly the continued efforts to develop software to provide a single interface for Alliance curators from all the Model Organism Databases (MODs). This single interface will reduce software development redundancies and free up resources for new features. Future releases will continue to add more modules for entering different data types.

Upcoming Conferences and Courses

  • Yeast Genetics & Genomics – modern and intensive laboratory course that teaches students the full repertoire of genetic and genomic approaches needed to dissect complex problems using the yeast Saccharomyces cerevisiae
    • Cold Spring Harbor Laboratory, NY
    • July 26 – August 15, 2022
  • Yeast Genetics Meeting – the premier meeting for students, postdoctoral scholars, research staff, and principal investigators studying various aspects of eukaryotic biology in yeast
    • University of California, Los Angeles
    • August 17 – 21, 2022
  • Gene Ontology Consortium Fall Meeting
    • Virtual plus in person
    • California Institute of Technology, Pasadena
    • October 11 – 13, 2022
  • Fungal Pathogen Genomics – Hands-on training in web-based data-mining resources for fungal genomes
    • Wellcome Genome Campus, Hinxton, Cambridge, UK
    • May 07 to May 12, 2023

Categories: Newsletter

Cln2p Moonlights in Cohesion

July 22, 2022

In an interesting new development, two different teams using two different sets of genetic interactions came to the same conclusion that Cln2p has a role in sister chromatid cohesion separate from its well-known role as a cyclin.

In a recent issue of the journal G3 (Bethesda), Buskirk and Skibbens report how deletion of the G1 cyclin CLN2 can rescue the temperature-sensitive growth defects of a strain lacking both ECO1 and RAD61, which are involved in regulation of sister chromatid cohesion. The authors show that neither CLN1 nor CLN3 deletion has the same effect, so the function is unique to CLN2. This result is especially interesting considering that CLN1 and CLN2 are paralogs resulting from whole genome duplication.

The authors show by genetic interaction how the role of Cln2p in sister chromatid cohesion is independent of its role in the G1/S cell cycle transition. Given that Cln2p activates Cdc28p within the CLN2-CDC28 kinase complex (Cln2-CDK) to trigger post-Start processes, the authors asked whether the whole complex has a role in cohesion. By looking closely at phenotypes of condensation, they conclude that this particular cyclin kinase complex plays a role in DNA hypercondensation, but that other CDKs interact with Eco1p at other points in the cell cycle.

Multifaceted and opposing roles for CDK regulation of Eco1 activity, from Buskirk and Skibbens, 2022

Using a similar but different approach, Choudhary et al. in a recent issue of mBio likewise looked for suppressors of a chromatid cohesion defect causing temperature-sensitive growth. In this case, they use a double mutant lacking Pds5p, a protein associated with the cohesin complex, and Elg1p, a protein involved in DNA replication. The elg1Δ mutation can suppress the pds5-1 temperature-sensitive allele but not the full deletion, such that the double mutant is inviable.

In a screen for mutants that rescued this lethality, 23 of 40 isolates carried mutations in the CLN2 gene. Deletion of the paralog CLN1 could not rescue the lethality of the pds5Δ elg1Δ double mutant, again suggesting these paralogs are not functionally redundant. Further investigation showed how cln2Δ deletion caused overexpression of MCD1, encoding the alpha-kleisin subunit of the cohesin complex. MCD1 has two promoter elements bound by the MBF transcription complex, and removal of these elements abrogated the overexpression. Thus, consistent with previous studies, the absence of CLN2 somehow caused overexpression of genes regulated by the MBF complex, in this case a critical cohesin subunit.

Model for the bypass of Pds5 function by elg1Δ cln2Δ, from Choudhary et al., 2022

We expect to hear more about the role of Cln2p in cohesion, for this secondary role is intriguing. It may be the case that other MBF-regulated genes are overexpressed when CLN2 is absent, and this might give rise to other roles. Given the complexity of these multifunction proteins, the yeast model provides a strong platform for answering the next questions.

Categories: Research Spotlight

Tags: cell cycle regulation, checkpoint proteins, sister chromatid cohesion, mitotic cell cycle, Saccharomyces cerevisiae

Acetylation Regulates the Nuclear Pore Complex

July 15, 2022

Several recent studies have done an excellent job characterizing the architecture of the yeast nuclear pore complex (NPC). With so much new information, researchers are now able to ask probing questions about how NPCs mediate communication between the nucleus and the rest of the cell. Considering that signals perceived from the environment need to reach the transcriptional machinery in the nucleus, and that mRNA transcripts made in response to these signals need to get back out to get translated, the NPC has a lot of communicating to do. A study in a recent issue of the EMBO Journal by Gomar-Alba et al. makes strong strides toward understanding how this communication is accomplished.

On the nuclear side of the NPC resides a substructure called the nuclear basket that has previously been shown to play roles in regulating gene expression and mRNA export. The nuclear basket also interacts with lysine acetyltransferases (KATs) and deacetylases (KDACs) that are best known for modulating transcription via reversible acetylation of histones in chromatin. These enzymes, however, can also act on non-histone proteins and have been linked to numerous cell processes, including DNA damage repair, cell division, and signal transduction.

From Gomar-Alba et al, 2022

Promotion of mRNA export is another function linked to acetylation, specifically by the NuA4 histone acetyltransferase complex, for which the catalytic subunit is Esa1p. Gomar-Alba et al. show in this recent study that Esa1p is the primary lysine acetyltransferase that promotes cell cycle entry—and also that it acetylates the nuclear pore protein Nup60p.

Acetylation of Nup60p promotes mRNA export, which in turn triggers fast entry into the Start phase of the cell cycle, thereby promoting cell division. Nup60p accomplishes this increased export by recruiting the TREX-2 transcription-export complex to the nuclear basket once Nup60p becomes acetylated. The deacetylated form of Nup60p has lower affinity for TREX-2 and thus mRNA export decreases. Deacetylation of Nup60p is performed by Hos3p, which acts in opposition to Esa1p in removing Esa1p-transferred acetyl residues.

Perhaps the most intriguing finding in this study is that Hos3p localizes primarily to daughter cells after cell division, causing displacement of the mRNA export complex and thus slowing G1/S phase transition. This action prevents premature division in the smaller daughter cells, as they require additional growth to meet the size control threshold for entry into a new cell cycle. Accomplishing this level of control with a single enzyme acting on a single nuclear pore protein is a simple, elegant solution.

As usual, studies in yeast make enormous impact on understanding cell division in other organisms.

Categories: Research Spotlight

Tags: mRNA export, cell cycle control, acetylation, Saccharomyces cerevisiae, nuclear pore, nuclear export

Pathway Between Copper and Disease Revealed in Yeast

July 08, 2022

Several lethal genetic disorders in humans are caused by mutations that cause symptoms of copper (Cu) deficiency, even in the presence of copper. These copper-deficiency disorders are fatal and include Menkes disease, Friedreich’s ataxia, and neurological and cardiac defects in infants due to lack of copper supply to cytochrome c oxidase in mitochondria. Given that no treatments are currently available for these terrible disorders, researchers have been interested in drugs that might improve copper bioavailability. The copper-binding oncological drug elesclomol (ES) has been identified as a candidate.

In a recent report by Garza et al. in the Journal of Biological Chemistry, the authors use the facility of yeast genetics to ask detailed questions about how ES affects metal homeostasis in yeast cells. It was previously established that perturbation in the levels of one metal tends to cause perturbations in supply of other metals, and thus they asked directed questions about both copper and iron (Fe). Deficiency in copper can cause linked deficiencies in bioavailable iron, and both are critically important for metal-dependent enzymes in mitochondria.

The crux of the team’s findings was that supplementing copper-deficient yeast cells with Cu-bound ES (ES-Cu) not only increased Cu levels, but nearly doubled Fe levels in mitochondria. They were able to show that ES transports copper by an alternate route that bypasses the major yeast copper importer (Ctr1p). While this is an intriguing and perhaps encouraging result, perturbations in metals have so much potential to be toxic that it remains critically important to understand the relationships between the components.

From Garza et al., 2022

The authors found that application of preformed ES-Cu complex is more efficient than ES at transporting Cu, and that transport of Cu across the plasma membrane by the drug occurs by passive transfusion, not active transport. Further, they made the critical discovery that copper delivered by ES goes first to the Golgi lumen, not directly to mitochondria as the authors had expected based on studies in other models. At the Golgi, the Cu is made available to the copper-transporting ATPase Ccc2p, which in turn assists in metalating Fet3p, a multicopper oxidase that oxidizes ferrous (Fe2+) to ferric iron (Fe3+). Once activated with copper in the Golgi, Fet3p-Cu is transported to the plasma membrane, where it oxidizes iron to Fe3+, the form that can be taken up by the iron transporter Ftr1p. This increased transport of iron leads to increased bioavailability in mitochondria. Thus, the link between copper and iron by means of an alternative copper transporter becomes more clear.

Interestingly, copper and iron metabolism are linked in both humans and yeast. The yeast protein Fet3p has two homologs in humans (ceruloplasmin and hephaestin), both of which are multicopper oxidase proteins critical for normal iron metabolism. From these powerful studies in yeast, it appears that disorders of Cu metabolism cause defects in Fe metabolism due to disrupted metalation of these cuproenzymes within the Golgi. Indeed, this is an intriguing finding that opens avenues for possible therapies, and it would be hard to imagine making this connection without the use of the yeast model.

Categories: Research Spotlight

Tags: yeast model for human disease, mitochondria, copper transport, copper, Saccharomyces cerevisiae, iron, iron transport, cytochrome c oxidase

Snf5p Senses pH to Combat Starvation

July 01, 2022

Yeast are keenly sensitive to internal pH. Several membrane proteins pump H+ ions out of the cell to keep the internal pH near neutral.  When carbon becomes scarce, however, it is essential for survival that these pumps get inactivated so the internal space is rapidly acidified. This acidification is postulated to conserve energy and trigger a number of subsequent pathways to combat starvation. Key among these adaptive responses is the derepression of the glucose-repressed genes. The well-studied SWI/SNF complex has been established as a key mediator for this, but the details of how the transcriptional boost is effected have not been known.

A recent study in eLife by Gutierrez et al. has shown the pivotal subunit of the SWI/SNF complex to be Snf5p, which performs a regulatory role by sensing pH. But how would a protein sense pH?

from Gutierrez et al., 2022

In studying the sequence and structure of the eleven subunits of the SWI/SNF complex, the authors noted that ten of the eleven subunits had large intrinsically disordered regions and that four of the eleven contained glutamine-rich low-complexity sequences (QLCs) that contain multiple histidine residues. QLCs were previously identified as important for binding transcription factors.

In looking for a link between pH and activation, the authors postulated that the histidine residues might be important because the histidine sidechain has an intrinsic pKa of 6.9, and thus might change conformation when pH drops.

Detailed comparative analysis of QLCs from yeast and other organisms led the authors to conclude that, in yeast, the histidines are salient features of QLCs that have been evolutionarily conserved. Given this, they noted that the N terminus of Snf5p has one of the largest QLCs in the whole yeast proteome and is in the top three for number of histidines.

from Gutierrez et al., 2022

Naturally, given the tools of the yeast model, the next step was to mutate the protein, for which they compared a full deletion against an N-terminal deletion of the QLC and a targeted allele with four histidines within the QLC mutated to alanine.

from Gutierrez et al., 2022

They found that total loss of the gene was phenotypically distinct from either of the QLC-targeted mutants. Total absence of the protein caused disruption of the SWI/SNF architecture, while QLC-mutants maintained an intact complex but showed disruptions in transcriptional reprogramming in response to starvation, as specifically measured by derepression of the ADH2 gene.

from Gutierrez et al., 2022

By a subsequent series of elegant biochemical experiments—conducted both in vivo and in vitro—the authors show with great precision how the Snf5p QLC specifically senses pH to trigger widespread reprogramming of genes that will help yeast metabolize non-preferred carbon sources. Even more specifically, they show how acidification leads to protonation of the histidines in the QLC, causing that region of the protein to expand and change conformation, thereby affecting the binding properties of the whole SWI/SNF complex.

from Gutierrez et al., 2022

The ability to do these experiments and develop a model of how the cell accomplishes delicate regulation once more astounds us with the awesome power of yeast genetics.

Categories: Research Spotlight

Tags: glucose starvation, glucose-repressed genes, glucose repression, signal transduction, Saccharomyces cerevisiae, glucose metabolism, pH sensing

The Remarkable Nuclear Pore Complex

June 24, 2022

The nuclear pore complex (NPC) is a complicated assembly embedded in the nuclear envelope that has the ability not only to assemble and disassemble quickly, but to adapt to changing needs for transport of macromolecules. The critical function of this elaborate complex has led researchers to invest intensive study, which has recently yielded remarkable new understanding.

In a January study in Cell, Akey et al. describe resolving the yeast nuclear pore complex to astounding detail. Using both in situ and isolated complexes, they dissect the layered organization of the pore to characterize the flexible inner ring, the adaptin-like central layer, and then the membrane-interacting layer that anchors the complex.

from Akey et al., 2022

Each of these layers employs complex protein connections that together form “spokes” in the pore. The authors speculate that the multiple layers and flexible connectors provide the means for NPCs to assemble and disassemble as quickly as they do, giving the ability to react to cell cycle stages and environmental conditions.

from Akey et al., 2022

Interestingly, upon close examination of crystalized structures, the authors observed that yeast has both single and double outer rings. The double outer ring has been observed in two other fungi to date and appears to represent a functional variant.

The double outer ring in a subset of yeast NPCs, from Akey et al., 2022

Upon further examination, the authors identified a third variant in yeast and were able to show that the variants co-exist in cells. One variant has two single outer rings that frame the inner ring, a second form has a single ring on the cytoplasmic surface and a double ring on the nuclear surface (both with nuclear baskets); while a third variant has two single rings, no baskets, and is specifically enriched over the nucleolus.

NPC variants in the same cell, from Akey et al., 2022

These variants provide further clues as to how the NPC might assemble as modular structures with multiple forms that adapt to different conditions. Further, the inner ring appears to have the ability to dilate and contract to allow smaller versus larger macromolecules to pass through, thereby adding another means of adaptability.

Understanding the yeast nuclear pore complex provides a foundation for understanding the eukaryotic NPC in general. A paper released this past week by Petrovic et al. in Science looked closely at just this set of relationships, comparing the human NPC to both the S. cerevisiae and Chaetomium thermophilum fungal NPCs. They show how, despite low conservation of sequence among nucleoporins and the other components of the pore complex, there is strong evolutionary conservation of the linker-scaffold architecture between humans and fungi. Once more, studies on model organisms throw bright light on the inner workings of our own cells.

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, nucleocytoplasmic transport, yeast model for human cells, nuclear pore

Mpe1p essential for mRNA processing

June 17, 2022

Messenger RNA (mRNA) 3′ end processing is an evolutionarily conserved and highly controlled process which requires several components from translation/transcription machinery. This processing involves monitoring nascent mRNAs for specific sequences, endonucleolytic cleavage, adding poly(A) tails, and triggering transcription termination. In budding yeast, the 3′ end processing machinery involves the cleavage and polyadenylation factor (CPF) complex and RNA-binding cleavage factors CF IA and CF IB. Based on the different enzymatic roles, the CPF complex has three distinct modules: polymerase, phosphatase, and nuclease.

CPF complex from Casañal A et al., 2017

A new study by Rodriguez-Molina et al. in Molecular Cell provides exciting insights into the role of the CPF complex in the 3′ end processing. The study provides strong evidence that Mpe1p, a nuclease module subunit of CPF, is involved in polyadenylation, cleavage, and transcription termination. The data show that in the presence of RNA, Mpe1p directly interacts with the polymerase module subunit Pfs2p through residues 207-268. The authors designate this region as a pre-mRNA-sensing region (PSR) of Mpe1p. However, another nuclease module subunit, Cft2p, hinders this Mpe1-polymerase module interaction. 

From Rodriguez-Molina JB et al., 2022

It is known that the polymerase module subunit Yth1p, interacts with RNA via a polyadenylation signal (PAS), a conserved sequence of A1A2U3A4A5A6. The authors show that in addition to Yth1p, Mpe1 interacts with PAS through the P215 residue. The authors hypothesize that Mpe1-PSR interacts with the polymerase module only after the Yth1p recognizes the PAS RNA, suggesting that Mpe1p may be able to ‘sense’ the RNA-polymerase module binding. 

Further, to understand the importance of the PSR region in Mpe1p, the authors analyzed mutants where its interaction with Pfs2p (mpe1-W257A,Y260A) or PAS (mpe1-P215G) is disrupted. Both mutants show reduced endonuclease and polyadenylation activities. Thus, suggesting that the same residues in Mpe1p involved in PSR-RNA/Pfs2 binding are also responsible in activating cleavage and regulating polyadenylation. A similar effect on endonuclease and polyadenylation activities is observed in a mutant where the CPF complex lacks the Mpe1p subunit, further corroborating the role of Mpe1p in mRNA 3′ end processing.

In the CPF complex, Mpe1p interacts with Ysh1p, another nuclease module subunit, via its N-terminal ubiquitin-like domain (UBL). This interaction stabilizes Mpe1p with the nuclease module (and CPF complex) even in the absence of RNA. To evaluate the importance of this interaction, a variant where the UBL region of Mpe1p is disrupted (mpe1-F9A,D45K,R76E,P78G) was generated. This variant is unable to form a stable CPF complex and show deficiencies in activating cleavage and polyadenylation activities. Thus the authors conclude that Mpe1p-Ysh1p interaction is essential for proper processing of 3′ end mRNA.

From Rodriguez-Molina JB et al., 2022

Another important aspect of mRNA 3′ end processing is the timely termination of transcription. The data show that the CPF complex lacking Mpe1p is unable to successfully terminate the transcription in time. Furthermore, the authors show that the PSR region specifically influences the role of Mpe1p in timely transcription termination.

Thus, the study highlights the role of Mpe1p as an essential subunit of the CPF complex in mRNA 3′ end processing, specifically in cleavage, polyadenylation, and transcription termination. 

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, Mpe1p, CPF complex, mRNA 3' end processing, transcription

Chromatin Modulators Act in Fungal Drug Resistance

June 10, 2022

Candida glabrata and Saccharomyces cerevisiae are closely related yeasts of which the former is pathogenic to mammals and the latter is used to make bread, wine, and beer. At present, the ~400,000 annual cases of life-threatening Candida infection are at risk of increase due to multidrug-resistant strains.  Fungal drug resistance relies on the fungal-specific pleiotropic drug resistance (PDR) pathway, in which ATP is consumed to pump drugs out of cells. The PDR pathway is similar among related fungi and a recent study by Nikolov et al. in Nature Communications leveraged this similarity to delve further into genetic regulation of PDR drug resistance in the tractable and benign S. cerevisiae. The team then applied these findings to the virulent C. glabrata.

The case study of the pleiotropic drug response in Saccharomyces cerevisiae, from Lelandais and Devaux, 2010.
 

In S. cerevisiae, the genes involved in PDR share a common promoter element called the PDR responsive element (PDRE). Nikolov et al. discovered that the histone chaperone Rtt106p has an unexpectedly strong binding affinity for PDREs, suggesting a role in transcriptional activation separate from histone modulation. They further show that Rtt106p works through the PDR gene PDR3, a transcriptional activator, to regulate basal expression of PDR-related genes. They found that basal expression of PDR5, the main multidrug transporter of the PDR pathway, requires Rtt106p binding the PDR5 promoter in concert with Pdr3p.

In response to drugs, however, a different set of regulators come into play. The authors show that neither RTT106 nor PDR3 is responsible for drug induction of resistance genes, but rather that PDR1 is critical for drug-induced upregulation of PDR5. By means of the minichromosome isolation technique, they further show how a different histone-modulating mechanism—the SWI/SNF ATP-dependent chromatin-remodeling complex—both binds to and activates the PDR5 promoter in response to an azole-type antifungal drug.

From Nikolov et al., 2022

As the goal of this work was to better understand PDR in pathogenic yeasts, the team next looked at the orthologs of the histone modulation genes in C. glabrata. The summary of their findings is that neither CgRtt106 nor CgSWI/SNF acts in maintenance of basal expression of PDR network genes, but each is clearly required for drug-induced expression. From this work, the authors conclude that these chromatin modulators studied in budding yeast constitute potential therapeutic targets in the pathogenic yeast. The use of a tractable model yeast has once more proven its value in combat of fungal multidrug resistance.

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, fungal drug resistance, fungal multidrug resistance, pleiotropic drug resistance, yeast model for pathogenic yeasts

Next