New & Noteworthy
January 12, 2017
Not too long ago, it was common to see people pouring used motor oil into street drains. Or to have people dumping old prescription drugs down their sinks.
Practices like these were (and are) terrible for the environment. Nature simply can’t deal with a buildup of this stuff (click here for some examples of the effects of pharmaceuticals on the environment).
Which is why it is so great that there are now ways to deal with waste like this. We can recycle it or at the very least dispose of it more carefully.
Turns out that things are similar in a cell. When its trash isn’t disposed of properly and/or recycled, the cell can suffer. And if cells suffer, so can the person made up of those cells.
One case where something like this is probably happening is in patients with the neurological disorder Pontocerebellar hypoplasia type 1B (PCH1B). These people have a mutated EXOSC3, an important gene for a cell’s RNA exosome. Presumably, this terrible disease is the result of certain cells not being able to properly clear some of their old RNAs.
In a new study out in GENETICS, Fasken and coworkers use good old Saccharomyces cerevisiae to begin to figure out what might be going on in the cells of these patients. They found that the most severe mutation seems to make it harder for the mutated protein to be part of the RNA exosome. As a result of being left out, the mutant protein is degraded more quickly leading to a buildup of some RNAs.
These sets of experiments were made a bit more complicated by the fact that human EXOSC3 cannot substitute for RRP40, the equivalent gene in yeast. This meant the researchers needed to focus on only those disease-causing mutations that hit the most highly conserved residues: EXOSC3-G31A, D132A and W238R.
Of these three, only the W238R yeast equivalent, rrp40-195R had much of an effect on the yeast. Fasken and coworkers propose that this is because this is the most deleterious of the three mutants.
Yeast harboring rrp40-195R grew more slowly at both 30 and 37 degrees C with the more pronounced effect at the higher temperature. At 37 degrees C, this mutant had higher levels of certain RNAs but not others. The RNA exosome was compromised for some but not all yeast RNAs.
And it wasn’t compromised everywhere. Although the RNA exosome works both in the nucleus and the cytoplasm, this mutant appeared to only be compromised in the nucleus. (Check the paper out for the cool way they figured this out.)
Next, the authors wanted to work out what this mutation did to the protein and the exosome. They were able to show that the mutant protein was more unstable than the wild type version and, interestingly, was even less stable when co-expressed with the wild type protein. They also showed that the mutant protein associated less well with the exosome complex and, again, this was exacerbated if the wild type protein was also present.
A reasonable model here is that RRP40 is more prone to degradation when it is not part of the RNA exosome. If true, then the mutant version of the protein is less stable because it is less often a part of the RNA exosome. And wild type RRP40 outcompetes the mutant protein making the mutant stay out of the complex for even more time.
OK, so they have done some good work showing why this mutant of RRP40 affects the growth of a yeast cell. But what we really want to know is if these results explain what is going on in the cells of people with the disease.
Fasken and coworkers tackled this by looking at the effect of expressing the equivalent mouse Exosc3 mutant in the presence of wild type endogenous Exosc3 in mouse neuronal cells (the types of cells affected in PCH1B patients). They found that just like in yeast cells, the mutant was less stable in mouse neuronal cells.
So it looks like the recycling machinery for RNA is broken in these cells because of an unstable component and that this leads to a buildup of toxic RNAs. But if the yeast experiments hold up, not all RNAs are affected.
It is more like people still being able to recycle their cans and bottles but not their motor oil. Certain parts of the environment like waterways take a hit but other parts are left relatively unscathed.
This makes sense when you think about PCH1B. Only a few cell types are affected by the mutation in the EXOSC3 gene. In other words, most cells can deal with a slightly wonky RNA exosome.
Yeast has again helped researchers better understand a genetic disease. Awesome indeed. #APOYG
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
December 20, 2016
We want to take this opportunity to wish you and your family, friends and lab mates the best during the upcoming holidays.
Stanford University will be closed for two weeks from Wednesday, December 21, 2016 through Tuesday, January 3, 2017. Regular operations will resume on Wednesday, January 4, 2017.
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Happy Holidays and best wishes for all good things in the coming New Year!
December 20, 2016
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December 16, 2016
Some people get the jitters from a single espresso while others need a triple shot just to get started in the morning. Some of this is due to caffeine tolerance—a buildup of resistance to the marvelous effects of that wonderfully addictive substance, caffeine. But the rest has to do with genetic differences that affect how well each of us processes caffeine—our caffeine sensitivity.
Our best buddy Saccharomyces cerevisiae is a real wimp when it comes to caffeine. In fact, like a lot of other microorganisms, caffeine actually kills this yeast. S. cerevisiae is indeed a sensitive soul when it comes to caffeine.
In a new study in the Journal of Agricultural and Food Chemistry, Wang and coworkers were able to toughen up budding yeast against caffeine by adding bfr1, a gene from Schizosaccharomyces pombe that encodes the ABC transporter that shunts caffeine out of the cell. And then, using random mutagenesis, they were able to make bfr1 even better at its caffeine-exporting job. Although the yeast don’t get any of the pleasurable effects of caffeine, at least they can now happily grow in cultures that have more caffeine than a strong cup of coffee.
This new attribute could prove to be incredibly useful if caffeine producers ever want to start making caffeine biologically instead of synthetically. You can imagine adding the caffeine pathway from coffee to yeast and having the yeast merrily exporting caffeine to the culture medium where it can be harvested. And who knows, maybe they can have the yeast make caffeine and alcohol at the same time creating the equivalent of a vodka and Red Bull in a single step!
Previous research had shown that bfr1 was an important player in helping S. pombe deal with caffeine. When Wang and coworkers added the gene to S. cerevisiae, this newly engineered yeast could now better tolerate caffeine. For example, whereas wild type yeast barely grew with 8 mg/ml caffeine, the engineered yeast did OK.
These authors next turned to random mutagenesis of the bfr1 gene to screen for mutants that could tolerate even more caffeine. And boy did they win the lottery on this one! A mutant that they named bfr1-B did great even at concentrations of 25 mg/ml caffeine. Now they were getting somewhere.
Bfr1 doesn’t just export caffeine; it actually exports many different compounds. The authors found that bfr1-B was fairly specific for increased resistance to caffeine. For example, when they tested the bfr1-B mutant with theophylline, a structurally similar compound, and atropine, a structurally distinct compound, they found that S. cerevisiae expressing the mutant were, if anything, more sensitive to these compounds. They found what looked like a caffeine-specific mutant.
When they looked at the mutant, Wang and coworkers found that there were 11 amino acid substitutions scattered across the protein. The next step was to figure out which ones mattered and which ones didn’t.
Using a bit of modeling with the 3-D structure of other ABC transporters, they settled on testing three mutations individually. Two of the mutations, S36 and D340 were in the nucleotide binding domain (NBD) and the third, Y497, was in the transmembrane domain (TMD). The NBD is where ATP binds to the transporter to supply the energy to move caffeine across the membrane.
Of the three, only D340 in the nucleotide binding domain conferred caffeine resistance. While not as robust as bfr1-B, this mutant allowed yeast expressing it to tolerate caffeine concentrations up to 15 mg/ml, conditions under which cells with wild type bfr1 failed to grow.
So while this mutation explains a lot of why bfr1-B is so good at dealing with caffeine, it is not the whole story. At least some of those other 10 mutations contribute to how well bfr1-B does with caffeine.
In the end we have a bullet-proof yeast when it comes to caffeine that should prove useful for anyone who wants yeast to synthesize caffeine for them. Of course unlike even the most grizzled 30 year coffee drinker with ideal genetics, the yeast almost certainly gets no joy from its morning Joe. But at least that cup of coffee won’t kill it!
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics