New & Noteworthy
April 15, 2015
In the science fiction novel Dune, the most precious thing in the universe is spice. It is harvested from the sands of the planet Dune under very dangerous conditions—every time people start to mine it, a gigantic worm comes to kill them. The spice is so valuable, though, that the miners harvest it until the very last second. As time runs out, a carryall whisks them away as the worm rises out of the desert.
While nothing quite so exciting happens in the nucleus of a yeast cell, one of the closest situations may be at the rDNA locus. Here the precious commodity is ribosomes and yeast cells need them to be made almost constantly. The only pauses in production are when this part of the genome needs to be replicated and when it needs to be segregated into a daughter cell. In both cases, as something akin to giant sandworms comes crashing onto the scene, harvesting stops and the machinery is removed with a cellular carryall.
Of course there aren’t harvesters extracting whole ribosomes from the yeast nucleus! Instead, it is RNA polymerase I (Pol I) and RNA polymerase III (Pol III) making the raw stuff of ribosomes, the 35S and 5S rRNAs, respectively. These polymerases need to stop transcribing and clear the way for the rDNA replicating replisome during S phase and for condensins at the end of anaphase. If they don’t, the rDNA locus becomes unstable, resulting in the mother yeast cell living a shorter life and in its daughter not getting the rDNA locus (and the rest of the chromosome it is on) at all.
In a new study in Nature Communications, Iacovella and coworkers have identified the carryall that helps to remove Pol I from the rDNA locus. Surprisingly, it is a kinase, named Rio1, that was previously known to be involved in rRNA processing and building ribosomes in the cytoplasm.
Rio1 does not physically remove Pol I from the 35S gene. What this study suggests is that it phosphorylates one of the 14 subunits of Pol I, Rpa43, so that Pol I no longer interacts as strongly with the transcription factor Rrn3. The end result is the untethering of the polymerase and its release from the DNA. Now condensins can glom onto the rDNA locus at the end of anaphase and DNA polymerase can barrel through the region in S phase without wreaking genomic havoc.
The first key finding in this study was that Rio1 isn’t just active in the cytoplasm, but also in the nucleus. In fact, it was most active in the nucleolus, a small moon shaped section of the nucleus that is formed around the rDNA locus.
The researchers went on to show that Rio1 is present in the nucleolus only at certain times in the cell cycle (S phase and anaphase). Using chromatin immunoprecipitation (ChIP) assays, they were able to show that Rio1 was enriched specifically at the rDNA’s 35S promoter and coding sequence.
They next created a conditional Rio1 mutant that could not enter the nucleus in the presence of galactose. When Rio1 was kept out of the nucleus, nucleoli became fragmented, there were no condensins on the rDNA locus at anaphase, and the mother yeast did not pass the replicated nucleolus to her daughter. Obviously Rio1 is a critical housekeeper for the rDNA locus!
They next used ChIP assays to show that when Rio1 was kept out of the nucleus, there was around 3-fold more Pol I at the 35S promoter and gene during anaphase than when Rio1 was allowed to go nuclear. This resulted in a 5-8 fold increase in 35S rRNA levels—implying that Pol I was still there cranking out rRNA.
The most likely explanation is that all the hyperactive Pol I transcribing the rDNA locus prevents condensins from binding the DNA and that removal of Pol I by Rio1 allows the condensins to bind. The condensins then shrink the rDNA region such that it can move though the tiny bud opening into the daughter.
They got similar results in S phase, where lack of nuclear Rio1 caused an increase in Pol I occupancy at the rDNA and an increase in 35S transcription as well. Here the lack of Rio1 has more devastating consequences to the genome. Its absence most likely causes the replisome to collide head-on with the transcribing Pol I, resulting in double strand DNA breaks. Because the rDNA locus is such a repetitive region, the cell makes mistakes when it repairs the break using homologous recombination. The end result is nucleolar fragmentation and a shorter life span.
The final set of experiments showed that Rio1 most likely affects Pol I occupancy by phosphorylating one of its subunits, Rpa43. First the authors used Western blot analysis to show that Rpa43 is less phosphorylated when Rio1 is kept out of the nucleus and when a mutant version of Rio1 lacking its kinase function is used. They also showed that Rio1 could phosphorylate Rpa43 in vitro.
They postulate that this phosphorylation causes the interaction between Pol I and the transcription factor Rrn3 to weaken, allowing Pol I release. Alternatively, Rpa43 phosphorylation could lead to the disintegration of the Pol I enzyme itself. Confirmation of one or the other will require more research.
Taken together, these studies paint a fascinating picture of the rDNA locus. Here Pol I is frenetically transcribing as much 35S rRNA as it possibly can to keep up with the yeast cell’s unquenchable thirst for ribosomes. The only time it stops is when continuing could harm the cell, during DNA replication in S phase and DNA transmission in anaphase. And even then, like spice hunters on Dune, they stay until the very last minute. A spicy tale, indeed.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Rise, replisome, rise!
April 9, 2015
Have you ever heard of the Baader-Meinhof phenomenon? (Don’t worry, we hadn’t either!) But you’ve surely experienced it.
The phenomenon describes the experience of suddenly seeing something everywhere, after you’ve noticed it for the first time. For those of us in Northern climates, it’s like like robins coming back in the springtime: one day, you see a single robin hopping on the grass; the next day, you look around and realize they’re all over.
Something similar happened to Godin and colleagues as they investigated the S. cerevisiae Shu2 protein. People knew about the protein and its SWIM domain but no one had looked to see just how conserved it was.
When the researchers looked for homologous proteins that contained the characteristic SWIM domain, they found homologs in everything from Archaea through primitive eukaryotes, fungi, plants, and animals. They were practically everywhere (in the evolutionary sense).
In a new paper in GENETICS, Godin and coworkers described their studies on this relatively little-studied, unsung protein. They found that it is an important player in the essential process of DNA double-strand break repair via homologous recombination, and its SWIM domain is critical to this function. Not only that, but being able to compare the SWIM domains from so many different homologs allowed them to refine its consensus sequence, identifying a previously unrecognized alanine within the domain was both highly conserved and very important.
Double-strand DNA breaks (DSBs) can happen because of exposure to DNA damaging agents, but they are also formed normally during meiotic recombination, in which a nuclease actually cuts chromosomes to start the process. Homologous recombination to repair DSBs is a key part of both mitosis and meiosis.
During homologous recombination, one strand of each broken DNA end is nibbled back to form a single-stranded region. This region is then coated with a DNA-binding protein or proteins, forming filaments that are necessary for those ends to find homologous regions and for the DSB to be repaired.
Shu2 is one of the proteins that participates in the formation of these filaments in S. cerevisiae. It was known that it was part of a complex called the Shu complex, and a human homolog, SWS1, had been identified. But the exact role of Shu2 and the significance of the SWIM (SWI2/SNF2 and MuDR) zinc finger-like domain that it contains were open questions.
One of the first questions the authors asked was whether Shu2 was widely conserved across the tree of life. Genes with similar sequences had been seen in fission yeast (Schizosaccharomyces pombe) and humans, but no one had searched systematically for orthologs. They used PSI-BLAST, a variation of the Basic Local Alignment Search Tool (BLAST) algorithm that that is very good at finding distantly related proteins, to search all available sequences.
Querying with both yeast Shu2 and human SWS1, the researchers found hits all across the tree of life—both in “lower” organisms such as Archaea, protozoa, algae, oomycetes, slime molds, and fungi, and in more complex organisms like fruit flies, nematode worms, and plants. The homologous proteins that they found across all these species also had the SWIM domain, suggesting that it might be important.
The sequence similarity was all well and good, but did these putative Shu2 orthologs actually do the same job in other organisms that Shu2 does in yeast? One way to test this is to do co-evolutionary analysis. Proteins that work together are subject to the same evolutionary pressures, so they tend to evolve at similar rates. Godin and colleagues found that evolutionary rates of the members of the Shu complex in fungi and fruit fly did generally correlate with those of other proteins involved in mitosis and meiosis.
The awesome power of yeast genetics offered Godin and coworkers a way to look at the function of Shu2. They first tested the phenotype of the shu2 null mutation, and found that it decreased the efficiency of forming filaments of the Rad51 DNA-binding protein on the single-stranded DNA ends that are created at DSBs. Formation of these filaments is a necessary step in repairing the DSBs by homologous recombination.
The comparison of SWIM domains from so many different proteins highlighted one particular alanine residue. This alanine hadn’t previously been considered part of the domain’s consensus sequence, but it was conserved in all the domains.
When the researchers changed the invariant alanine residue in yeast Shu2, the mutant protein bound less strongly to its interaction partner in the Shu complex, Psy3. When they mutated the analogous residue in the human Shu2 ortholog SWS1, this also decreased its binding to its partner, SWSAP1.
Other mutations within the SWIM domain of Shu2 also affected its interactions with other members of the Shu complex, and made the mutant cells especially sensitive to the DNA-damaging agent MMS. Diploid cells with a homozygous mutation in the Shu2 SWIM domain had very poor spore viability, suggesting that the SWIM domain is important for normal meiosis.
As one more indication of the SWIM domain’s importance, Godin and colleagues took a look in the COSMIC database, which collects the sequences of mutations found in cancer cells. Sure enough, a human cancer patient carried a mutation in that invariant alanine residue of the SWIM domain in the Shu2 ortholog, SWS1.
There’s still much more to be done to figure out exactly what Shu2 and the Shu complex are doing during homologous recombination. Yeast obviously provides a wonderful experimental system, and the discovery of Shu2 orthologs in two other model organisms that also have awesomely powerful genetics and happen to be multicellular, Drosophila melanogaster and Caenorhabditis elegans, expands the experimental possibilities even further.
There’s also a lot to be learned about the SWIM domain in particular. The discoveries that it affects the binding behavior of these proteins and that it is mutated in a cancer patient show that it’s very important, but just what does it do in Shu2? It will be fascinating to find out exactly how this domain works to help cells recover from the lethal danger of broken chromosomes. And it is amazing what you can see, once you start looking.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
April 2, 2015
Editing is an essential part of producing good writing. You might cringe when your masterpiece comes back covered in red ink, but in the end your paper is better for it.
These days, computers have made editing much, much faster and easier than using ink on paper. Some of us remember when cutting and pasting literally mean cutting the piece of paper on which your manuscript was typed and pasting the sections in a different order!
The same kind of revolution has happened when it comes to editing genomes. A seemingly obscure system used by bacteria to defend against invading phages, discovered in the 1950s, led to the development of restriction enzymes as the reagents that have enabled virtually all modern molecular biology. And now, an equally “obscure” system of bacterial immunity has opened the door to genome editing that is as precise and nearly as fast as your thesis advisor using Microsoft Word to edit your dissertation (maybe faster!).
This bacterial system is called CRISPR/Cas. Bacteria use it to defend against the foreign DNA—from infecting phages, bacteria that transfer plasmids via conjugation, or other sources—that constantly assaults them. And now scientists are using it to edit the genomes of most any beast they want, including our favorite yeast.
What makes this technique so powerful is that it is easily programmable. You can make virtually any sequence change that you wish, and even more impressively, make lots of changes all at once. CRISPR/Cas may be one of those technological leaps that changes everything.
The incredible potential of this approach, and the ethics of its use in humans, are hot topics right now. But while researchers and philosophers are hammering out guidelines for using the CRISPR/Cas system in larger organisms, yeast researchers are free to forge ahead and edit the S. cerevisiae genome to their hearts’ content. This has now been made much easier with the availability of a whole toolkit, created at the Delft University of Technology and described in a new paper by Mans et al.
The yeast system has three essential components. The first is a plasmid that will express a specific guide RNA (gRNA). The gRNA leads a nuclease to the right place in the genome. Mans and colleagues made a whole set of plasmids, with different nutritional and antibiotic resistance markers, that can express one or more gRNAs.
The second component of the system is the nuclease that binds to the gRNA and makes a double-stranded cut in the target DNA. The researchers used the Cas9 nuclease from Streptococcus pyogenes, and engineered a set of yeast strains that had the cas9 gene stably integrated into a chromosome and expressed from a strong yeast promoter.
The third component is one or more repair fragments: pieces of DNA that specify the modified sequence that the researcher wants to engineer into the genome.
So, for example, if a scientist wants to delete a gene precisely, she can create a gRNA that targets the gene, and then co-transform a Cas9-expressing yeast strain with both the plasmid expressing the gRNA and a repair fragment that corresponds to the gene’s upstream and downstream flanking sequences, fused together. When the gRNA is expressed in the transformant, it leads Cas9 to the gene, where it makes a double-stranded cut at a precise position in the DNA.
Now the researcher can let yeast do the rest of the work. S. cerevisiae has a powerful homologous recombination system, and it’s greatly stimulated by double-stranded breaks in DNA.
After Cas9 cuts the gene, yeast will repair the break, using as a template the repair fragment that the researcher designed. In this case, the upstream and downstream sequences will recombine with the homologous sequences in the chromosome, but since the coding sequence is missing from the repair fragment, the resulting strain will have a precise deletion of the gene of interest.
This example illustrates one of the simplest uses of the technique. Mans and colleagues tried successively more complicated tasks and were able to accomplish some amazing feats.
They were able to precisely delete six genes in one step by transforming with repair fragments for all six, along with three plasmids that each expressed two gRNAs. Also in one step, they replaced one yeast gene with six Enterococcus faecalis genes encoding subunits of the pyruvate dehydrogenase complex and other enzymes in the pathway. The E. faecalis genes were specified on six overlapping repair fragments that were cotransformed into the strain.
The CRISPR/Cas9 system designed by Mans and colleagues can do much more than gene deletions and replacements. By designing repair fragments specifying particular mutations, the method can also be used to create point mutations or other modifications.
For this technique to work, it’s important that there are no mismatches between the gRNA sequence and the chromosomal target sequence, and other sequence characteristics can influence the efficiency of the method. So the authors created an online tool that helps researchers select optimal Cas9 targets in regions of interest and design gRNA sequences. Since they incorporated the sequences of 33 different S. cerevisiae strains into the tool, researchers can specify a strain and retrieve information on the best sequences for targets and gRNAs for their gene(s) of interest based on sequences found in that particular strain.
Importantly, the CRISPR/Cas9 technique allows researchers to make multiple changes in a single step. This is a big advantage, since transformation itself can be mutagenic. For example, a strain that has been commonly used to investigate the function of hexose transporters was engineered to carry multiple deletions in the conventional manner, using successive rounds of transformation and selection. Its genomic sequence, which was recently determined, reveals that its genome is a complete mess, with many rearrangements and deletions.
With the development of this toolkit, editing the S. cerevisiae genome is beginning to be almost as easy as editing a text document. And since Mans and colleagues have made all of the strains, plasmids, and online tool freely available to the world, everyone will be able to take advantage of them. Just think of the stories that yeast researchers will be able to write!
CRISPR/Cas in Bacteria
Way before this became a powerful tool for researchers, it was a very cool immune system for bacteria, allowing them to defend against assaults from foreign invaders.
Bacteria collect foreign DNA sequences from invaders that threaten them, just like collecting mug shots of notorious criminals. They store these mug shots in a special place in their genome, integrated between blocks of a repeated sequence. (The CRISPR acronym refers to these repeats.) This region is transcribed, and the RNA is chopped into pieces containing individual mug shots. Because these mug shots, called crRNAs, are complementary to the DNA sequences of invaders, they can recognize and hybridize with those invading sequences.
Bacteria have an additional small RNA, the tacrRNA, that binds to both the crRNA and to a CRISPR-associated (Cas) nuclease. This forms a RNA-DNA-protein complex on the foreign DNA and allows the nuclease to do its work, cutting both strands of the DNA and neutralizing the invader.
To use this system for genome engineering, scientists have fused the two RNAs of the bacterial system into a single RNA, the guide RNA (gRNA). It contains both the mug shot (with sequences complementary to the target) and the RNA sequence that recruits the nuclease. The gRNA, a Cas nuclease, and a repair fragment are the essential components of the system.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
March 25, 2015
In a classic Apple ad, the world is a gray, dreary place where everyone is the same. In charges a woman dressed in brightly colored clothing who hurls a hammer into a big black and white screen, smashing the old conformist world order. Individuality can now blossom.
This is a powerful story for people, but it turns out that if Mother Nature had her druthers, she would like that woman to either stay at home or dress and act like everyone else. At least this is true if we are talking about individuals that are genetically identical. In this case, alleles that cut down on cell to cell variation tend to be the ones that prosper.
This is confirmed in a new study in Nature in which Metzger and colleagues in the Wittkopp lab showed that there is a selection against mutations that cause increased variation between individuals in the S. cerevisiae TDH3 gene. In other words, mutations that cause more “noise” are selected against. The squeaky wheel is eliminated.
The TDH3 gene encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important metabolic enzyme. Yeast cells can survive a deletion of this gene, but their fitness is greatly reduced. Overexpression of the gene also has noticeable effects. This suggested to the researchers that the level of TDH3 expression would be under selection pressure during evolution.
Metzger and coworkers compared the promoters of the TDH3 gene from 85 different strains of S. cerevisiae and found that the promoter had undergone selection out in the wild. The authors were interested in why certain polymorphisms were selected for and why others were selected against. To try to tease this out, they compared the activities of evolved changes to randomly selected ones.
The authors first used the sequences of the 27 haplotypes they saw in the 85 strains to predict what the original, ancestral promoter probably looked like. They then re-created this sequence and also sequences that represented the most likely intermediates on the way to the current promoters. They linked each of these promoter sequences to a yellow fluorescent protein (YFP) reporter and looked at mean activity and expression noise. In other words, they looked at how much promoter activity there was in aggregate and how much it varied between individual cells for the 10,000 cells in each culture.
They next set out to generate a pool of polymorphisms that didn’t make the cut during evolution, so they could compare these to the successful ones. To do this, they individually mutated 236 G:C to A:T transitions throughout the promoter region. They chose this transition because these are the most common spontaneous mutations seen in yeast and the most common SNP seen in this promoter out in the wild.
Now they were ready to do their experiment! Comparing the randomly created mutations to the evolved changes, they looked at both the overall level of expression and how much variation there was between each of the 10,000 individual cells in the tested culture.
What they found was that the effects on the mean level of activity were pretty comparable between the “selected” mutations and the random ones. But the same was not true for individual variation. The random mutations were much more likely to increase expression noise compared to the “selected” mutations.
From these results the authors conclude that there is a selection against mutations that increase the level of noise. In fact, they go a step further and conclude that at least for the TDH3 promoter, there was more of a selection against noise than there was a selection for a particular level of activity. It was more important that individuals had consistent activity than it was to have some mean level of activity.
This makes some sense, as a cell is a finely tuned machine where all the parts need to work in harmony together to succeed. If one part is erratic and shows different levels of activity in different individuals, then some of those individuals won’t do as well and so won’t survive.
This also means that certain paths to a more fit organism will be selected over others. And it could be that organisms miss out on some potential fitter states because they can’t survive the dangerous evolutionary journey that would be needed to get there.
So the cell prefers that all the parts work together in a predictable way. When you’re a population of individuals that are more or less genetically identical, nonconformists are dangerous. The gray sameness of the Nineteen Eighty-Four world is preferable to a more bohemian atmosphere where diversity is celebrated.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics