New & Noteworthy

Shmoos Lost in Translation

August 07, 2014

To mate, the yeast Saccharomyces cerevisiae needs to shmoo — to generate a projection that reaches out to a nearby yeast of the opposite sex, until the yeast cell is shaped like the Al Capp cartoon character.  And to shmoo yeast needs, among other things, polyamines like spermidine.

Spermidine is important for one of the most interesting proteins in the world, the translation initiation and elongation factor eIF5A.  Not only is this protein pretty much conserved in just about every living thing, but it is also the only protein to have the unique amino acid hypusine.  And to make things even more fascinating, there are two other conserved proteins whose only job is to convert a single lysine residue of eIF5A into hypusine, using polyamines like spermidine.  Simply mind boggling.

In a new study in GENETICS, Li and coworkers provide compelling evidence that spermidine is important in yeast shmooing because of its involvement in the hypusinylation of eIF5A.   They also found that one reason eIF5A is so important in this process is that it is necessary for translating Bni1p, a formin needed to organize the actin cables of the shmoo.  Without these actin cables, the shmoo can’t form.

It looks like yeast needs eIF5A to translate Bni1p because of the long stretches of prolines found in this protein.  This suggests that like its bacterial ortholog EF-P, a key job for eIF5A is to help the cell deal with polyproline stretches in proteins.

To show this the researchers made a set of targeted mutations to check whether hypusinylation of eIF5A is necessary for shmooing.  When they knocked out LIA1, one of the enzymes that uses spermidine to convert lysine to hypusine, the resulting yeast failed to shmoo.  Since the only known target of the Lia1 protein is eIF5A, this suggests that hypusinylation of eIF5A is critical to its function in shmooing.

They also used temperature sensitive mutants of eIF5A to show that this gene (HYP2, also known as TIF51A) is involved in shmooing.  At the nonpermissive temperature, only 7.7% of yeast with the less severe mutant allele, tif51A-1, shmooed, while none of the yeast with the more severe mutation, tif51A-3, were able to shmoo.  These two results taken together establish the importance of eIF5A in shmooing.

Because eIF5A was known to be important for translating polyproline regions, the researchers looked for yeast proteins with such stretches, with the idea that their failure to be translated may be behind the need for eIF5A in shmooing.  They found 549 such proteins, and a comparison of their Gene Ontology (GO) annotations showed four overrepresented categories including “mating projection” (shmoo).  They focused on a protein from this group, Bni1p, because it was known to be involved in shmoo formation and it was one of only two proteins with ten or more prolines in a row. 

Bni1p is important for organizing the actin cables that are needed to make a shmoo.  Li and coworkers showed that the temperature sensitive mutants of eIF5A and bni1 mutants had similar phenotypes in terms of actin organization in the shmoo.

So the idea here is that yeast need eIF5A to shmoo because they need eIF5A to translate Bni1p, and Bni1p is needed to set up the actin framework of the shmoo.  In this hypothesis, it is the indirect action of eIF5A that prevents the shmooing. To test this hypothesis, the authors generated a bni1 mutant that lacked the polyproline regions. 

They compared the transcript levels of wild type BNI1 and the mutant lacking the polyproline stretches using RT-qPCR and found that the presence of eIF5A didn’t matter much.  The transcript levels of the mutant and wild type BNI1 were pretty much the same.

It was a different story for the protein levels.  Using Western blots Li and coworkers saw very little wild type Bni1p, but lots of the mutant protein.  The yeast cells struggled to translate wild type Bni1p but had no trouble with the mutant.  The easiest explanation is that eIF5A is needed to help the yeast translate polyproline regions of proteins, including Bni1p. 

Finally, to confirm the eIF5A and Bni1p connection, they showed that additional Bni1p could partially overcome the shmoo defect of the temperature sensitive mutants of eIF5A.  Since this suppression was only partial, and since the mutant phenotype of the eIF5A mutant is more severe than that of the bni1 mutant, there are probably other proteins involved in shmooing that require eIF5A for translation. Some likely candidates are those proteins containing polyproline stretches that are annotated to the GO term “mating projection”.

Although a connection between oddly-shaped yeast cells and human fertility and/or disease may not seem obvious, there might indeed be one. It turns out that eIF5A is so highly conserved  that human eIF5A works just fine when expressed in yeast, and mammalian formins, like Bni1p, are also proline-rich. Formins are necessary for polarized growth, which is a feature of both reproductive cell and cancer cell growth, and spermidine is required for fertilization.

Hard to believe that yeast channeling a cartoon character can teach us so much about the most fascinating of proteins, eIF5A.  And maybe even shed light on our own fertility. 

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: eIF5A, hypusine, Saccharomyces cerevisiae, translation

Time Flies Like an Arrow, Fruit Flies Like a Grande Yeast

July 31, 2014

Here at SGD we tend to have a totally positive opinion of yeast.  As we have said before, they give us bread, booze, a great model organism, and even our livelihoods.  But in truth, Saccharomyces cerevisiae has a few minor faults.

For example, you can thank yeast for all those irritating fruit flies buzzing around your brown bananas.  Fruit flies aren’t attracted to the rotting fruit itself.  They are instead attracted to chemicals the yeast cells are pumping out as they nosh on that old banana.

In a new study, Schiabor and coworkers set out to identify the genetic differences that make some yeast strains more attractive to fruit flies as compared to other strains.  They found that the flies can actually tell the difference between “petite” yeast, with defective mitochondria, and “grande” yeast whose mitochondria are normal.  The mitochondria play a huge role in determining which volatile chemicals a yeast will release, and so determine which yeast are the most attractive to a fruit fly.  But the mitochondria are probably not involved in the way that you might be thinking…

In the first experiment, the authors tested a bunch of different yeast strains to find the ones that fruit flies prefer. As expected, they found a wide range of yeast attractiveness.  They decided to focus on BY4741 as the more appealing strain and BY4742 as the less appealing one.

Schiabor and coworkers chose these two strains both because they are isogenic and because they are the strains from which the systematic yeast deletion collection was made.  These two attributes mean that it should be relatively easy to track down the genetic difference in each strain’s attractiveness to fruit flies.

The first obvious candidate was the different auxotrophies in each strain. Although the strains are isogenic overall, they have a few small differences: BY4741 is a met17 mutant and is mating type a, while BY4742 is a leu2 mutant and is mating type α. Since amino acids are very important in creating various volatile chemicals, the mutations in the amino acid biosynthetic genes seemed a likely cause of the difference in the way the two strains smelled to fruit flies. However, the authors found that none of the auxotrophic mutations mattered.  When they mated the two strains and did tetrad analysis to obtain every possible genetic combination, they found that each of the eight new strains was preferred over BY4742.

Given the non-autosomal inheritance of attractiveness, an obvious candidate was the mitochondria. This hunch was confirmed in a couple of ways.  First, Schiabor and coworkers showed that every strain except BY4742 grew well on glycerol, and second, they found that an isolate of BY4742 with functional mitochondria, BY4742g, was as attractive to fruit flies as BY4741.  Apparently their stock of BY4742 had lost mitochondrial function (which can happen fairly easily for some strains), and clearly the mitochondria matter here!

Through a series of experiments we don’t have the space to describe here, the authors found that the lack of attractiveness was not due to an inability to respire.  Instead, by growing each strain on different nitrogen sources, they were able to provide evidence that mitochondrial functions like proline catabolism and/or branched amino acid anabolism were more likely to be involved.  It can sometimes be hard to remember that the mitochondrion is more than the powerhouse of the cell we all learned about in high school: a lot of very important metabolic reactions other than respiration happen within the mitochondrial compartment.

The authors think that yeast with good working mitochondria are the most useful to fruit flies, which is why fruit flies have evolved to be attracted to those yeast.  This all makes sense, as yeast and fruit flies have a mutually beneficial relationship.  Yeast serve as food for fruit fly larvae, and the ethanol they produce also protects those same fruit fly larvae from predators.  Fruit flies can open up parts of the fruit the yeast can’t get to and help move the yeast to different places.   

The bottom line is that you can blame yeast mitochondria for that swarm of fruit flies hovering over your fruit bowl.  One day maybe we can come up with a way that our fruit will only allow petite yeast to grow.  Then we’ll have a bit of time to enjoy fruit that isn’t attractive to fruit flies.  Until, of course, the flies evolve to prefer petite yeast…

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: Drosophila, mitochondria, Saccharomyces cerevisiae

Esa1p, the Balancing Artist

July 15, 2014

In the art of rock balancing, the artist positions large rocks with exquisite precision. If he or she succeeds, the rocks counterbalance each other and stay in seemingly impossible positions to make a surprising and beautiful sculpture. But a little uneven pressure is enough to make the whole thing collapse.

It turns out that the cellular acetylation state is just as precisely balanced. In a new GENETICS paper, Torres-Machorro and Pillus identify Esa1p, an acetyltransferase, as the balancing artist in Saccharomyces cerevisiae cells.

Acetylation is an important type of protein modification. Histones, the proteins that interact with DNA to provide structure to chromosomes, are acetylated by histone acetyltransferases (HATs) and deacetylated by histone deacetylases (HDACs). Some HATs and HDACs also act on non-histone proteins.

The acetylation state in a cell is a dynamic process.  All those HATs are adding acetyl groups at the same time that HDACs are removing them.  The final level of acetylation depends on the activities of each of these classes of proteins.

Acetylation of histones has been associated with increases in gene expression and deacetylation with decreases.  So to keep gene expression levels in balance, it is very important to keep acetylation balanced as well.  Throwing acetylation patterns just a bit out of whack can have profound consequences on global gene expression that can ultimately lead to cell death. 

The authors focused on one particular HAT, Esa1p, that acetylates histones H4 and H2A and also has non-histone targets. They were intrigued by the fact that yeast cells cannot survive without Esa1p, since no other HAT or HDAC subunit is essential in yeast.

An obvious explanation for lethality is that losing this protein leads to too low a level of acetylation.  They reasoned that if they also knocked out an HDAC, then the overall acetylation levels might increase and so rescue the esa1 null mutant.  And they were right.

Using a plasmid-shuffling method, they created various double mutant strains of esa1 and HDAC genes, and found that a strain that was mutant in esa1 and also in either the SDS3 or DEP1 genes was viable. SDS3 and DEP1 both encode subunits of the Rpd3L HDAC complex.

Torres-Machorro and Pillus next characterized the esa1 sds3 double mutant further.  They found that although the sds3 mutation suppressed the inviability of the esa1 mutant, it did not suppress other phenotypes such as sensitivity to high temperature and DNA damaging agents.

The authors found that the sds3 mutation subtly increased histone H4 acetylation, which was low in the absence of Esa1p.  However, acetylation levels of a different histone, H3, remained high even in the absence of Esa1p. This suggested that the fundamental problem in the esa1 null mutant was an imbalance in the global state of histone acetylation.

To test this hypothesis, the researchers used a variety of different genetic methods to tweak the balance of cellular acetylation in the esa1 sds3 mutant. They created mutations in histones H3 and H4 that made it seem as if acetylation was low or high, and they also mutated other genes for HDAC subunits. It is as if they were passers-by who decided to poke at a balanced rock sculpture to see what it took to bring the whole thing down.

Although the details are too numerous to report here, the results showed that by using these genetic methods to tweak the overall acetylation state of the cell, the fitness of the esa1 sds3 strain could be improved: phenotypes such as slow growth, sensitivity to high temperature or DNA damaging agents, or cell cycle defects were suppressed to some extent by the various manipulations.  This lends support to the hypothesis that Esa1p is the master balancer of acetylation levels in the cell and that this is its essential function.

This balancing act may happen in human cells too. Esa1p has a human ortholog, TIP60, that has been implicated in cancer and other diseases. Like Esa1p, TIP60 is essential and is involved in the DNA damage response.

So yeast teaches us that the acetylation of proteins is balanced on a knife’s edge.  Even the slightest changes can lead to a collapse in global gene regulation, which can have catastrophic effects like cancer. All that we learn about Esa1p, the acetylation balancing artist, may have much broader implications for human health.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: histone acetylation, Saccharomyces cerevisiae, yeast model for human disease

Adding Introns to Synthetic Biology’s Toolbox

July 03, 2014

As any good handyman knows, the more tools you have in your tool chest, the better the chance that you can find what you need to solve a problem.  The same goes for synthetic biologists.  The more parts they can mix and match, the more likely they are to engineer the exact level of gene expression they need.

In the last few years synthetic biologists have amassed a wide variety of transcription and translation elements that can be combined in different ways to exquisitely tune the level of expression of their gene of interest.  And now, in a new study out in PLOS Genetics, Yofe and coworkers have added introns to the list of parts available for our favorite yeast Saccharomyces cerevisiae.

Yeast isn’t loaded with introns, but it does have a reasonable number that can be co-opted for synthetic biology.  The authors inserted 240 of these introns individually into the same position near the 5’ end of the yellow fluorescent protein (YFP) gene and monitored the level of fluorescence of each individual strain over a 24 hour period.  They chose the 5’ end of the gene because yeast has a bias for introns being located there.

The authors found that these reporters spanned a 100-fold range of gene expression, that every intron caused a decrease in the level of gene expression, and that even though many of these introns respond to environmental stimuli in their natural context, their effect on gene expression here was immune to the environmental changes the authors tested.  Taken together, these results suggest that introns could be used in yeast systems for dampening over-exuberant gene expression in ways that are independent of growth conditions.  If all of this holds up, introns will prove to be very useful tools indeed.

Yofe and coworkers next wanted to use this library to figure out some of the rules for why some introns cause lowered activity compared to others.  The simplest possibility, that longer introns cause a larger decrease in gene expression, turned out not to be true.  There was no correlation between the size of the intron and its effect on the level of fluorescence. 

Next they scanned the sequences of their constructs to look for elements that might increase or decrease splicing efficiency.  These splicing regulatory elements (SREs) are better understood in larger eukaryotes, but there is evidence that they are important in yeast as well.  The authors identified a number of intron splicing enhancers (ISEs) and intron splicing silencers (ISSs) that were highly enriched near the splice sites. 

To confirm that these sequences did in fact affect splicing efficiency (and hence gene expression), they showed that mutating the enhancer motif TTTATGCT to the silencer motif TTTGTGTA in two reporters resulted in a 22% and a 13% decrease in gene expression.  This proof of principle experiment suggests that future synthetic biologists may be able to further tweak the expression of their genes by manipulating these SREs.

In a final set of experiments the authors used the library to identify rules that can be used to predict how inserting various introns into different positions will affect a gene’s activity.  They found that the most important features were the presence of SREs and the RNA structures at the intron-exon junction.  Synthetic biologists should be able to use these rules to intelligently design their reporter systems.

These experiments are the first step towards adding introns to the ever growing set of tools available to synthetic biologists for modulating gene expression.  We are getting closer to figuring out how genes are controlled and being able to use that knowledge to our advantage.  Or to put it another way, we have taken another baby step towards being able to control a gene as well as a yeast cell does.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, splicing, synthetic biology

Polygamous DNA Replication

June 26, 2014

Once someone is married, there are lots of things that keep them from starting a second marriage at the same time.  Laws, fear of losing the first spouse, social mores and so on all create a situation where the vast majority of people have only a single spouse at any one time. 

As each of these inhibitions is lifted, people will be more or less inclined towards polygamy, depending on who they are and the culture they live in.  For example, if having multiple spouses becomes acceptable socially, then some people might dive right in while others might hold off.

It turns out that origins of replication are similar.  There are many layers of control that keep an origin from firing more than once during any cell cycle.  But just like people and polygamy, when a few inhibitory layers are removed, some origins are more likely to fire more than once in a cell cycle than are others.

In a new study out in PLOS Genetics, Richardson and Li have identified a DNA sequence that makes nearby origins of replication fire more than once during a cell cycle when certain regulatory mechanisms have been disabled.  The authors hypothesize that these reinitiation promoters (RIPs) may be important for promoting genetic diversity by causing genomic duplication of specific regions under certain circumstances. 

This lab had previously shown that the origin ARS317 reinitiates more frequently when global regulation is removed from some key players in initiation: Cdc6p, the Mcm2-7 complex, and the origin recognition complex (ORC).  They disabled the regulation of all three of these by mutating each to prevent their recognition by the master regulator cyclin-dependent kinase (CDK, whose catalytic subunit is Cdc28p). In this study, they identified a second origin, ARS1238, that also reinitiated more often under these conditions.  The authors next set out to identify why these origins reinitiated under these conditions.

The first thing they found was that chromosomal context didn’t matter a whole lot.  Both origins reinitiated at around the same rate when they were in their natural context or when moved to other chromosomes.  The ability to reinitiate must be contained in the sequence of the DNA that was moved.

They next showed through deletion and linker scanning analysis that the two origins both required an AT-rich, ~60 base pair sequence to reinitiate.  This sequence needed to be within around 35-75 base pairs of the origin to promote reinitiation. Not any old stretch of AT-rich DNA would do; a specific DNA sequence was necessary, suggesting that this DNA is not required for reinitiation just because it is more easily unwound. 

These authors have shed light on a key process in the life of a cell—the firing of an origin of replication once and only once during any cell cycle.  It is critical for a cell that origins do not routinely reinitiate to prevent widespread genomic duplications that left unchecked would be very dangerous to the cell.

Richardson and Li have shown that not all origins are created equally, in that some are more likely to reinitiate under certain conditions than are others. If similar regions in mammalian cells turn out to be hotspots for genetic changes in cancers, then scientists may be able to target them to prevent the cancer’s genetic progression.  We may be able to reintroduce laws to keep polygamy at bay.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: DNA replication, replication origin, Saccharomyces cerevisiae

Like People, Prions Need Intimate Contact to Spread

June 19, 2014

In the Matrix Trilogy, the delicate balance of a virtual world is upset by a rogue computer program that goes by the name of Agent Smith.  This program finds and touches other agent programs, converting them into copies of itself.  Eventually, all the agent programs are copies of Agent Smith and only the hero Neo can save humanity in an epic battle within the virtual world of the Matrix.

A new study out in GENETICS by Li and Du provides additional evidence that prions in the yeast Saccharomyces cerevisiae work similarly to Agent Smith, in that they spread through a direct contact model.  These prions are proteins that have entered a rogue conformation, and they end up converting all copies of the same protein into a similar rogue conformation.  The proteins change from a hardworking Agent Smith trying to do its job into something that mucks up the working of a cell.  And the results, at least in humans, can be as catastrophic for the cell as Agent Smith was for the Matrix. 

Mad cow disease, for example, is caused by prions converting the prion protein (PrP) in the brain cells of people from a useful conformation to a dangerous one that spreads.  As the conformation spreads throughout the cell, these prions form amyloid fibrils that eventually kill the cell.  When enough brain cells are killed, the person dies.

The authors chose to work in yeast because unlike in people, there are multiple examples of proteins in yeast that can go prion.  The list includes Sup35p, Ure2p, Rnq1p, Swi1p, Cyc8p, Mot3p, Sfp1p, Mod5p and Nup100p.  As you might guess from the sheer number of these prion-ready proteins, prions actually do more than kill a cell in yeast; they can serve useful functions. Scientists have yet to identify any useful functions for the prion form of PrP in people. 

Having multiple prions in a cell allowed Li and Du to perform some experiments to try to distinguish between two models of prion conformation spreading.  In the first, called the cross-seeding model, the prion acts very much like Agent Smith in that it needs to contact a “healthy” protein to convert it into a prion.  In the second model, the titration model, factors in the cell that prevent prion formation are titrated out when prions form.  As the factors are taken out of commission, prions are free to form.

The main evidence in this study that supports the cross-seeding model has to do with the localization of pre-existing prions during the de novo formation of a new prion.  Li and Du found that the prion [SWI+] localized to newly forming [PSI+] prions but not to already formed [PSI+] prions.  This is not the result we would expect if prion formation were due to titrating out of inhibitors of prion formation.  If that were the mechanism, then there would be no reason for [SWI+] to colocalize with newly forming [PSI+]. These experiments are like having a google map of the Matrix where we could see Smiths converting other agents by touch and then moving on and touching other agents.

Work like this is important for helping to find treatments for prion associated diseases and, perhaps, other amyloid fibril forming diseases like Huntington’s or Alzheimer’s.  Scientists need to focus on the amyloid fiber forming proteins themselves instead of trying, for example, to ramp up the activity of factors that inhibit formation.  Scientists probably need to eliminate Agent Smith to prevent the destruction of the Matrix and all of mankind.

---

This is how prions turn other proteins into copies of themselves:

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight, Yeast and Human Disease

Tags: prions, Saccharomyces cerevisiae

Holding Back the Translation Torrent

June 12, 2014

We all know the story of the little Dutch boy who stuck his finger into a hole in a dike to keep his village from being flooded.  Now, a new study out in Molecular Cell by Pircher and coworkers has identified a novel regulatory mechanism involving a small 18-nucleotide RNA that behaves similarly to this boy.

A big difference in our story is that unlike the broken dike, the “water flow” in yeast cells is usually a good thing.  It is the continuous stream of protein translation that goes through the ribosome. 

When it’s under stress, though, yeast needs to slow down translation in order to make sure that it is making and folding each protein correctly. This gives it a better shot at surviving the stress.  Once the stress is gone, translation can ramp up again. This is where that 18-nt RNA comes in.

This group identified this 18-nt RNA as a ribosome binding RNA in a previous study. Because there are only a couple of known cases where a noncoding RNA (ncRNA) regulates the ribosome directly, Zywicki and colleagues had wanted to see whether this happens in yeast.  They found about 20 ncRNAs that bound to the ribosome, with the most abundant being an 18-nt fragment that corresponded to part of the coding sequence of the TRM10 gene that encodes a tRNA methyltransferase.

In the current study Pircher and coworkers reconfirmed that in yeast cells about 80% of this 18-nt RNA is associated with ribosomes. To verify whether it really bound to the ribosome rather than to the mRNA being translated, they broke apart polysomes with the chelating agent EDTA. This separated the large and small ribosomal subunits from each other and from mRNA.

All of the 18-mer stayed with the large subunit, showing that it really does interact with the ribosome. The researchers also found that under normal conditions it is bound to nontranslating ribosomes, while in stressed cells it shifts to actively translating polysomes.

The mutant phenotype of the trm10 null mutant suggested that the 18-mer might have a role in adapting to stress conditions. This mutant looks normal under standard conditions, but grows slower than wild type when under osmotic stress.

Pircher and colleagues used a clever strategy to find out whether this phenotype was due to the absence of Trm10p or to the absence of the 18-mer. First, they added a stop codon into the TRM10 gene, outside the region encoding the 18-mer. This mutation blocked production of Trm10p, but didn’t affect the 18-mer. The mutant looked just like wild type under osmotic stress conditions, showing that Trm10p isn’t involved in the stress response.

Second, to see directly whether the 18-mer is important, they mutated its sequence by changing some of the codons within it to other, synonymous codons encoding the same amino acid. So the Trm10p derived from this gene was wild-type, although the 18-mer sequence was different.

A couple of mutants of this type both showed the same phenotype of slow growth under osmotic stress. So production of the 18-mer is in fact important for maintaining growth rate under stress conditions. These mutant 18-mers also failed to bind to ribosomes. 

To find out what this little RNA actually does, they used electroporation to load up each cell with about 200,000 molecules of the 18-mer. This was about the same as the number of ribosomes per cell. Translation was almost completely inhibited. When they did the same experiment with an 18-mer with a scrambled sequence, it had no effect.

Further in vitro experiments confirmed the inhibitory effect of the 18-mer on translation, and showed that the inhibited step is translation initiation. It’s not completely clear why slowing down translation promotes cell growth during stress, but the authors speculate that it leads to more accurate translation and protein folding, which improves protein homeostasis and adaptation to stress. It also remains to be determined whether the 18-mer is created by processing of the TRM10 mRNA or is transcribed independently.

This regulatory mechanism is surprising and relatively novel: there are just a couple of known cases of ncRNAs regulating the ribosome directly. But it makes sense that regulating translation in this way allows the cell to react very quickly to changing environmental conditions, without needing to synthesize any new molecules.

Small ncRNAs like microRNAs or small interfering RNAs are emerging as big players in regulation in many organisms. However, miRNAs and siRNAs are not found in S. cerevisiae. But as this study shows, this does not mean that yeast doesn’t use small RNAs for regulation.  And one of the most surprising things about this story is that such a tiny scrap of RNA can regulate the ribosome, with its 5.5 kb of rRNA and 80 proteins. The little Dutch boy’s finger is immense by comparison!

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: ncRNA, osmotic stress, Saccharomyces cerevisiae, translation

Saddling Mom with the Burden of Old Age

June 05, 2014

In A World Out of Time by Larry Niven, people live forever by teleporting the unfolded protein aggregates associated with aging out of their cells.  Turns out that our very clever yeast Saccharomyces cerevisiae can do the same thing and it doesn’t need a machine.  

Instead of teleporting the aggregates away, yeast saddles the mother cell with them when it buds.  The daughter has now regained her youth and the mother is left to struggle with old age.

In a new study in Science, Hill and coworkers show that the yeast metacaspase gene MCA1 is critical in this process.  But it doesn’t look like it is involved in segregating these bundles to the mother cell.  Instead, it appears to help clear away many of the bundles left in the daughter.   If it were in Niven’s original story, Mca1p might be a little nanobot that chewed up any aggregates the teleporter missed.  

This all makes sense given caspases’ role in multicellular beasts.  There, these executioner proteases chew up cellular proteins during apoptosis, the process of programmed cell death that is a critically important part of development and growth. 

Although apoptosis has been observed in yeast and Mca1p is involved in the process, it has always been a bit of a mystery why a single-celled organism needs a mechanism for suicide.  This study now suggests that yeast’s only caspase, Mca1p, has a role as a healer as well as an executioner. It saves the daughter by degrading and proteolytically clearing away the aggregated bundles clogging up her cell. 

Scientists already knew that HSP104 was a key player in making sure that aggregates stayed with mom.  Hill and coworkers used this fact and performed a genetic interaction screen using HSP104 to identify MCA1 as required to keep protein aggregates out of the daughter in response to a heat shock.  Follow up work confirmed this result by showing that overexpressing MCA1 led to more efficient segregation of aggregates and that deleting it led to poor segregation of aggregates.

Digging deeper, these authors found that this poor segregation was because Mca1p was not eliminating aggregates in the daughter, as opposed to affecting the segregation itself.  They also showed that the protease activity of Mca1p was needed for this effect.

In the final set of experiments we’ll discuss, the authors looked to see what effect MCA1 has on the life span of a yeast cell.  They saw little effect of deleting MCA1 unless a second gene was also deleted: YDJ1, which encodes an HSP40 co-chaperone.  The double deletion mutant yeast were able to divide fewer times before petering out.  Consistent with this, overexpressing MCA1 led to increased life span and this effect was enhanced in the absence of YDJ1.

Finally, cells lived for a shorter time if just the active site of the Mca1p protease was compromised in a ydj1 deletion background.  This again confirms that proteolysis is key to MCA1’s effects on aging. 

So yeast attains eternal youth by both dumping its age-related aggregates on its mother and by using Mca1p to destroy any aggregates that managed to get into the daughter.  The daughter gets a reset until she builds up too many aggregates, in which case she gets saddled with them.

Yeast may be showing us another way to live a longer life.  If we can specifically degrade our aggregates without causing our cells to commit mass suicide, maybe we can extend our lives.  And we don’t even need fancy teleporting machinery; we just need to adapt the molecular machinery yeast is born with.  Feel free to use this idea for a new science fiction story!

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: ageing, caspase, protein aggregates, Saccharomyces cerevisiae

Slipping Through Haldane’s Sieve

May 22, 2014

Imagine you are panning for gold in a river and there are two kinds of nuggets.  One type is naked gold while the other is gold hidden inside of normal rock.  Pretty easy to figure out which nuggets you’ll gather first!

Now imagine instead that the process of panning is a rough one that knocks the shell off of the second type of nugget revealing the gold inside.  Now there won’t be any difference between the two.  You will be just as likely to keep both types of nuggets.

The same sort of situation applies to new beneficial mutations in a changing environment.  Back in 1927, J. B. S. Haldane predicted that the more dominant a mutation, the more likely it was to help a diploid beast adapt to a new environment.  The naked gold was more likely to be taken over the covered gold.

Gerstein and coworkers show in a new study that at least in the yeast Saccharomyces cerevisiae, Haldane’s sieve (as it is called) may not always apply.  The process of adapting to a new environment can strip away the dominant older allele, revealing the recessive one.  Loss of heterozygosity (LOH) uncovers the hidden gold of the recessive phenotype.

The authors had previously identified haploid mutants that were able to survive in the presence of the fungicide nystatin.  They mated these mutants to create either heterozygotes or homozygous recessive mutants and compared these to wild-type diploids growing either in the presence or absence of nystatin. 

Gerstein and coworkers found a wide range of effects of these mutations in the absence of nystatin.  Sometimes heterozygotes grew better than either homozygote, sometimes homozygous recessive strains did best, and sometimes wild type grew best.   Phenotypes were all over the map.

The story was very different in the presence of nystatin where only the homozygous recessives managed to grow.  This appears to contradict Haldane’s sieve.  Here there were no dominant mutations that allowed for survival.

Gerstein and coworkers found that some heterozygote replicates started to grow after a prolonged lag period.  A closer look at the heterozygotes that grew showed that they had lost the dominant allele so that they could now show the recessive phenotype and survive.  LOH had broken Haldane’s sieve. 

The authors found that the lower the nystatin levels, the more likely a population was to break through Haldane’s sieve.  They postulate that the populations survive longer at lower levels of nystatin, which increases the chances that a LOH will happen.  It is a race between survival and eliminating the dominant allele that keeps them from growing. 

The next step was to determine if LOH was common enough that populations with a small percentage of heterozygotes could survive.  They found that even in populations where only 2% were heterozygotes, around 5% of the 96 replicate populations managed to lose an allele and grow.  So even at low levels, a recessive mutation can give a population the advantage it needs to adapt and survive.

Combining the awesome power of yeast genetics with cheap sequencing is allowing scientists to test fundamental models of genetics that will unearth how populations adapt and survive in new environments.  We are finding those nuggets of scientific knowledge that have remained hidden.

Now of course, not every diploid is as numerous or as genetically flexible as yeast.  Cows, chickens, lizards, and people may all still be slaves to Haldane’s sieve.  We will need more studies to see if our recessive treasures can be uncovered in time to save us. 

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: antifungal resistance, evolution, Saccharomyces cerevisiae

Yeast: A One Man Band for Finding New Drug Leads

May 15, 2014

Imagine you are in a band and the only instruments you have are guitars.  Yes, you can play some beautiful music, but there will be a whole lot of music that your band won’t be able to play. 

In some ways, finding chemical leads to develop into drugs is similar to an all guitar band.  The compounds in available libraries all tend to have a lot in common.  They are like a vast array of subtly different guitars.

In a new study, Klein and coworkers use synthetic biology to have the yeast Saccharomyces cerevisiae make more varied libraries on its own.  As an added bonus, the authors also use the yeast to assay the new leads.  Not only have they expanded the range of instruments available to your band, but they’ve also made it so you can play all the instruments.  You are now a one man band!

The first step in all of this is to have an assay that can easily pick out the important leads.  Klein and coworkers use a galactose inducible Brome Mosaic Virus (BMV) system they had previously developed.

In this system, if one of the viral genes is on, then it produces a fusion protein that includes the Ura3 protein.  When the URA3 gene is expressed, yeast die in the presence of 5-fluoroorotic acid (5-FOA).  So any yeast that can make a compound that can inhibit viral expression will survive in 5-FOA.

The next step in creating these in vivo libraries was to randomly assemble various biochemical pathways into yeast artificial chromosomes (YACs) and to transform them into yeast.  These pathways were chosen because they have yielded important compounds before or because they come from medically important beasts.  This work was described in detail in a previous paper.

Specifically, Klein and coworkers randomly combined cDNA genes from eight biochemical pathways into YACs and transformed them into the BMV replication yeast strain.  They found 74 compounds that allowed the yeast to survive in the presence of 5-FOA.  Of these, 28 had activity in a secondary BMV assay. 

A close look at the 74 compounds showed that by and large, most had characteristics that put them in the right ballpark to be useful leads.  They had low molecular weight and the right hydrophobicity, and were chemically complex. In addition, many could easily be improved chemically (this last point is called optimizability).  Most importantly, they were pretty unique from a drug lead point of view. 

Over 75% of the compounds resembled nothing in known libraries.  And the compounds were not similar to one another.  Klein and coworkers had created a wide range of instruments other than guitars.

Of course, keeping a yeast strain alive is hardly reason to look for a new drug.  But that isn’t all these compounds can do.  At least some of these leads show excellent activity against two viruses related to BMV, Dengue and hepatitis C, and one looks particularly promising. 

With a random combination of genes from a variety of biochemical pathways, yeast has been coaxed into synthesizing chemical leads that can target two medically relevant viruses.  Scientists should be able to use a similar approach to tackle other diseases.  All they need is a yeast strain with the right assay.

Yeast can make our bread rise, get us drunk, and now maybe cure us of disease.  Is there anything yeast can’t do?  Well, they still can’t play a guitar. 

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight, Yeast and Human Disease

Tags: drug discovery, Saccharomyces cerevisiae

Next