New & Noteworthy
February 18, 2015
If you’re shipwrecked on a desert island, writing a message on a scrap of paper, sealing it in a bottle, and flinging it into the ocean could be your only chance at communication. As the song goes (see below), the message in a bottle is an S.O.S. to the world.
It turns out that cells may do something very similar. But instead of using a bottle, they enclose their messages in membrane-bound bubbles.
Many different mammalian cell types have been seen to form these extracellular vesicles (EVs). In mammalian cells, it’s known that EVs are used for cell-to-cell communication. They contain signals that allow cells to influence their neighbors, both for good (for example, regulating the immune response) and for bad (transmitting viruses or toxic peptides). In most cases these signals aren’t well-characterized, but the EVs may include DNAs and proteins, and they’re rich in RNAs.
Fungi have been found to produce EVs too, but they’ve been much less studied. In a new paper in Scientific Reports, da Silva and colleagues looked at the extracellular vesicles (EVs) produced by four different fungal species, including S. cerevisiae, and found that among other things, the vesicles actually include at least parts of many RNAs, both protein-coding and non-coding.
The scientists decided to look at S. cerevisiae and three species of fungal pathogens that infect humans: Cryptococcus neoformans, Paracoccidioides brasiliensis, and Candida albicans. (S. cerevisiae can be pathogenic too, but isn’t as virulent as any of those species.) They isolated EVs from each and treated the unbroken EVs with RNase to get rid of any RNA that might be contaminating their surfaces.
Then they broke open the vesicles to see what was inside. They found that the EVs contained many small RNAs, most less than 250 nucleotides in length. The scientists used RNA-seq analysis to determine the sequences of these small RNAs, and compared them to the genomic sequences that were already known for these organisms.
Many of the sequences corresponded to noncoding RNAs. For S. cerevisiae, the RNA sequences identified included the mitochondrial small and large ribosomal RNAs, RNA components of RNase enzyme complexes, a variety of small nuclear and small nucleolar RNAs, and tRNAs.
Sequences corresponding to several dozen S. cerevisiae mRNAs were also detected. There wasn’t much rhyme or reason to the kinds of proteins they encoded. But the set of mRNA fragments didn’t correspond simply to the set of most abundant mRNAs in the cell. So it seemed like the vesicles didn’t just contain random samples of the cytoplasm, but instead had been loaded selectively with particular mRNAs.
This study raises as many questions as it answers, and there is a lot of work to be done before fungal EVs will be understood. The intriguing discovery of RNA in EVs suggests the possibility that the RNAs could influence gene expression in cells that take up the EVs, either by regulating processes like splicing or translation, or even by encoding a protein that gets translated in the recipient cell.
Being able to influence neighboring fungal cells via EVs could be an advantage for fungi in the fierce competition for biological resources. Or perhaps EVs are used to subvert gene expression in host tissues during a fungal infection. Far from being an S.O.S., these messages could be threatening.
These speculations all need much more research. Fungi are an integral part of our world, and we need to pay careful attention to the messages that they send us!
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
“Message in a Bottle,” The Police, 1979
February 11, 2015
Finding a needle in a haystack would take a long time and would be very tedious (although it’s been done!) Finding a specific drug to fight malaria by testing the effect of each drug, one at a time, on a purified protein in vitro would be at least as tedious and maybe even more so.
Luckily, we don’t have to sift through a haystack. In a new study in ACS Chemical Biology, Frame and colleagues used our friend S. cerevisiae to find nine drugs out of a collection of more than 64,000 that are promising candidates for stopping the malaria parasite in its tracks. It is as if yeast allowed them to set fire to the haystack and see nine needles gleaming in the ashes.
Malaria is a huge problem for global health. Plasmodium falciparum, the organism that causes malaria, is fast developing resistance to the few effective drugs that we have left.
But P. falciparum has an Achilles heel—it can’t make its own purine nucleotides! Since these are the building blocks of DNA and, obviously, essential for life, if we can keep P. falciparum from being able to take them up, we can kill it.
P. falciparum imports purines via a major transporter protein, called PfENT1, located in the plasma membrane. So a drug that specifically inhibited this transporter could be a good way to attack the pathogen.
It’s possible to assay the activity of the transporter in vitro, adding different drugs one at a time and seeing which inhibits transport. But doing this for thousands of drugs might make you wish you were looking for a needle in a haystack. Frame and colleagues decided to harness the awesome power of yeast genetics to test a very large set of drugs more quickly.
The toxic nucleoside analog 5-fluorouridine (5-FUrd) is taken into yeast cells by the high-affinity uridine transporter Fui1. It kills normal yeast cells, but fui1 null mutant yeast can survive in the presence of 5-FUrd.
The researchers engineered a yeast codon-optimized version of pfENT1 and expressed it in the mutant, restoring 5-FUrd uptake. The nucleoside analog was again toxic to this strain, and the only way the yeast could survive was if the transporter activity of pfENT1 was inhibited.
This system allowed a simple and powerful screen for pfENT1 inhibitors. The yeast strain expressing pfENT1 would be able to grow in the presence of 5-FUrd only if pfENT1 transporter activity was blocked by the drug that was being tested.
Setting up the screen on a large scale, the scientists were able to test 64,560 compounds. They initially found 171 compounds that allowed the yeast to grow. They narrowed these down to 9 compounds that worked well and belonged to different structural classes of chemicals.
Because of the way the study was designed, it was likely that these compounds allowed yeast to grow because they prevented PfENT1 from pumping the toxic 5-FUrd into the cell. But what if the compounds were actually doing something different, and unexpected? To rule out this possibility, the researchers designed a secondary screen for the 9 top candidate drugs.
They used ade2 mutant yeast, which can’t make their own adenine and need to be fed it in order to survive. These mutants can make do with the related compound adenosine, but it can’t normally get inside the cell; yeast doesn’t have a transporter that will take it up. However, PfENT1 can transport adenosine, so ade2 mutants can grow on it if they are expressing PfENT1.
With this system, if the candidate drugs are working as expected, they should prevent yeast growth. And that is exactly what the researchers found. This confirmed that the drugs are working because they specifically inhibit PfENT1 and do not allow growth by some other, indirect mechanism.
To be completely sure of the mechanism, the scientists did a direct test. They found that the nine drugs prevented PfENT1-expressing cells from taking up radiolabeled adenosine.
This was all fine, but the ultimate goal of the study was to affect growth of the malaria parasite. So Frame and colleagues tested the drugs on P. falciparum.
In the presence of any of the nine drugs, the parasite couldn’t take up adenosine and also failed to grow. This even happened when the parasites were grown in medium containing much higher purine concentrations than found in human blood.
Even though PfENT1 was targeted by the drugs, all nine of the drugs also killed Pfent1 null mutants. This suggested that the drugs have a secondary target or targets in addition to PfENT1. This could be a real advantage, because it could help prevent the parasites from developing resistance to the drugs.
All nine of these diverse drugs are promising candidates for the treatment of malaria. And the same approach could be used to find chemicals that affect the function of other transporters from various organisms.
As usual, yeast is providing scientists with streamlined ways to find new treatments for serious human diseases. Instead of tediously rummaging about in a haystack, yeast lets us quickly and easily find the needles we need.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
February 4, 2015
tRNACUG may not just be for translation anymore:
Back before airplanes and cars, when times got tough people would often take trains to what they hoped were greener pastures. And to hitch a ride on a train, they’d usually need to have a ticket. Turns out the same is true for Gln3, a transcription factor in yeast.
Basically, Gln3 stays in the cytoplasm as long as there are good sources of nitrogen available to the cell. When these sources run out, Gln3 moves from the cytoplasm to the nucleus where it can turn on genes that can help the yeast cope with its new situation.
In a new study in GENETICS, Tate and coworkers have identified one of the tickets that lets Gln3 take the trip to the nucleus. And it was totally unexpected. To get to the nucleus, Gln3 needs a fully functional glutamine tRNACUG. No, really.
To get this evidence, Tate and coworkers used a reporter in which Gln3 was linked to GFP (green fluorescent protein). They tracked the location of Gln3 in the cell using fluorescence microscopy.
Using a temperature-sensitive mutant of tRNACUG, sup70-65, the authors showed that at the nonpermissive temperature of 30 degrees C, Gln3 could not translocate to the nucleus under a wide variety of conditions in which nitrogen was limiting. Gln3 had no problems translocating at the permissive temperature of 22 degrees C, and in wild-type cells Gln3 translocated at both temperatures. Clearly tRNACUG is doing something important in this process!
The next experiment showed that tRNACUG was more like a one-way ticket. Once Gln3 entered the nucleus under nitrogen starvation conditions at the permissive temperature, switching to the nonpermissive temperature had little effect. Gln3 stayed put.
A possible wrinkle in these experiments was that cells harboring sup70-65 formed chains reminiscent of pseudohyphae at the nonpermissive temperature no matter what the nitrogen conditions. One possible explanation for the results seen here was that many of these cells lacked nuclei. In this case, they might not see nuclear translocation because there was no nucleus to translocate to.
In the course of these studies, Tate and coworkers showed that adding rapamycin mimicked the effects of nitrogen starvation with one big difference—nuclear localization happened much more rapidly than with nitrogen starvation. This fast response allowed the authors to look at Gln3 localization while visualizing nuclei by staining DNA with DAPI (which gives a short-lived signal). They were able to use the DAPI to see that these cells did indeed have nuclei and that when they raised the temperature, Gln3 did not colocalize with the DAPI stained nuclei. Gln3 was being kept out of nuclei at the nonpermissive temperature.
So it really looks like Gln3 needs a working tRNACUG to get into the nucleus. There are a couple of possible ways that this tRNA could be needed for Gln3 to make the trip.
In the first model, the tRNA is part of a complex that allows Gln3 to make the trip to the nucleus. In this model, it is almost as if Gln3 (or one of its compatriots) is clutching its ticket, tRNACUG. In the second, less fun model, the tRNA is required to translate a protein involved in Gln3’s transit. Which model is the correct one is still up in the air, but it will be interesting to see which is the right one.
This was the most astonishing finding in the article, but it was by no means the only one. We don’t have the time to go into the other experiments, which, among other things, teased apart differences in the four or five distinguishable pathways that work to turn on the cell’s nitrogen response.
This work highlights a recurring theme in basic research: we may think we know everything that’s going on (tRNAs just help to translate proteins, right?) but just about every time we look more closely, there is much more to see than first meets the eye. Being in the right place at the right time is essential, whether you’re escaping the Dust Bowl in The Grapes of Wrath or a transcription factor responding to the lack of a nutrient. It’s not so surprising that the cell has drafted every possible player into this process, even a lowly tRNA.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Since the title has this song stuck in our heads, we thought you might want to hear it too. Enjoy!
February 1, 2015
We at SGD are happy to share this announcement from the organizers of ICYGMB 2015. We’ll keep you up to date on the latest news about the conference and important deadlines. You can also follow announcements on Twitter by searching for the hashtag #yeast2015.
It is our great pleasure to announce the 27th International Conference on Yeast Genetics and Molecular Biology (ICYGMB) to be held in Levico Terme, Trento, Italy, from 6th to 12th September 2015.
The conference, initially scheduled to take place in Lviv, was moved to the heart of the Italian Alps, following the assessment of the political situation in Ukraine, and is now being co-organized by the Italian and the Ukrainian Scientific Committees under the auspices of the International Committee on Finances and Policy, together with the help of some international scientific societies such FEMS and EMBO.
The goal of the conference is to bring together investigators from around the world to present and discuss research focused on yeasts as model for the understanding of molecular biology and genetic processes, and as a paramount biotechnological microorganism.
The Conference is historically an exciting forum on yeast biology, on our web site, you will have the opportunity to appreciate the scientific program and the beautiful scenery of this magnificent part of Italy where the Conference venue is located. In addition to the exceptional selection of speakers, we kept the opportunity, for brilliant researchers, to present their most recent work, since the fourth speaker of every plenary session will be selected from the abstracts submitted to the poster sessions. The science, the surroundings, and, of course, the food will be in the best tradition of Italian professional hospitality.
The conference is also part of EXPO2015, the world exposition that will be hosted in Italy in 2015 in Milano, comprising events in every Italian region. This year, the theme is to feed the planet, and we are committed to divulge to the public the importance of yeast in food and beverage productions. For this reason, the conference will be followed by a one-day EXPO2015 Symposium on Yeast Ecology and Food Production as well as on the fundamental role of the microscopic world in nutrition and human health (12th September 2015), together with a Roundtable on science as nurturer of peace. The registration to the 27th ICYGMB Conference will also include the participation to these extra events that will take place at the Museum of Modern Art of Rovereto, Italy.
Join us in this lovely part of the Trentino Region, in Italy, from 6th to 12th of September 2015 for this unique international event!
Help us spread the news by word of mouth and circulate the announcement you find here because for some colleagues we may not have had the updated e-mail address in our database.
Good science, food and location are waiting to make this a most memorable yeast Conference.
Duccio Cavalieri & Andriy Sibirniy (ICYGMB Organisers),
on behalf of the National and International Organizing Committees.