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Runaway Polymerases Can Wreak Havoc in Cells

October 16, 2014

A train without working brakes can cause a lot of destruction if it careens off the tracks. And it turns out that a runaway RNA polymerase II (pol II) can cause a lot of damage too.  But it doesn’t cause destruction, so much as disease.

Working brakes are important for both large and small machines, including RNA polymerase. Image from Wikimedia Commons

Unlike a train, which has its brakes built right in, pol II has to count on outside factors to stop it in its tracks. And one of these brakes in both humans and yeast is a helicase: Sen1 in yeast and Senataxin, the product of the SETX gene, in humans. 

Mutations in SETX are associated with two devastating neurological diseases: amyotrophic lateral sclerosis type 4 (ALS4) and ataxia oculomotor apraxia type 2 (AOA2), both of which strike children and adolescents.  One idea is that these mutations may short circuit the brakes on pol II, causing it to keep on transcribing after it shouldn’t. And this is just what Chen and colleagues found in a new paper in GENETICS.

The researchers used the simple yet informative yeast model system to look at some of these mutations, and found that they disrupted the helicase function of Sen1 and caused abnormal read-through of some transcriptional terminators.  Looks like bad brakes may indeed have a role in causing these devastating diseases.

Some human proteins can function perfectly well in yeast. Unfortunately, Senataxin isn’t one of those; it could not rescue a sen1 null mutant yeast, so Chen and coworkers couldn’t study Senataxin function directly in yeast. But because Senataxin and Sen1 share significant homology,  they could instead study the yeast protein and make inferences about Senataxin from it.

First, they sliced and diced the SEN1 gene to see which regions were essential to its function. They found that the most important part, needed to keep yeast cells alive, was the helicase domain. But this wasn’t the only key region.

Some flanking residues on either side were also important, but either the N-terminal flanking region or the C-terminal flanking region was sufficient. Looking into those flanking regions more closely, the researchers found that each contained a nuclear localization sequence (NLS) that directed Sen1 into the nucleus. This makes perfect sense of course…the brakes need to go where the train is!  If we don’t put the brakes on the train, it won’t matter how well they work, the train still won’t stop.

These flanking sequences appeared to do more than direct the protein to the nuclear pol II, though.  When the authors tried to use an NLS derived from the SV40 virus instead, they found that it couldn’t completely replace the function of these flanking regions even though it did efficiently direct Sen1 to the nucleus.

Next the researchers set out to study the disease mutations found in patients affected with the neurological disease AOA2.  They re-created the equivalents of 13 AOA2-associated SETX mutations, all within the helicase domain, at the homologous codons of yeast SEN1.

Six of the 13 mutations completely destroyed the function of Sen1; yeast cells could not survive when carrying only the mutant gene. When these mutant proteins were expressed from a plasmid in otherwise wild-type cells, five of them had a dominant negative effect, interfering with transcription termination at a reporter gene. This lends support to the idea that Sen1 is important for transcription termination and that the disease mutations affected this function.

The remaining 7 of the 13 mutant genes could support life as the only copy of SEN1 in yeast. However, 5 of the mutant strains displayed heat-sensitive growth, and 4 of these showed increased transcriptional readthrough.

Taken together, these results show that the helicase domains of Senataxin and Sen1 are extremely important for their function. They also show that Sen1 can be used as a model to discover the effects of individual disease mutations in SETX, as long as those mutations are within regions that are homologous between the two proteins.

It still isn’t clear exactly how helicase activity can put the brakes on that RNA polymerase train, nor why runaway RNA polymerase can have such specific effects on the human nervous system. These questions need more investigation, and the yeast model system is now in place to help with that.

So, although it might not be obvious to the lay person (or politician) that brainless yeast cells could tell us anything about neurological diseases, in fact they can. Yeast may not have brains, but they definitely have RNA polymerase. And once we learn how the brakes work for pol II in yeast cells, we may have a clue how to repair them in humans.

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

SGD Fall 2014 Newsletter

October 15, 2014

SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This Fall 2014 newsletter is also available on the community wiki. If you would like to receive the SGD newsletter in the future please use the Colleague Submission/Update form to let us know.

You Can Thank Fruit Flies for Those Yummy Beers

October 14, 2014

A wonderful side effect of the close relationship between yeast and fruit flies is great tasting beer. Image from Wikimedia Commons

It is as simple as this, beer tastes good.  And if a new study in Cell Reports by Christiaens and coworkers pans out, you can thank fruit flies for some of those delicious flavors.

No, fruit flies aren’t in your beer. Instead, they have forced the evolution of our favorite beast, Saccharomyces cerevisiae, down a path towards making the aromatic compounds that make beer so darned tasty.

See, yeast can’t get around on their own and so they often rely on insects to move to new pastures. In order to have this happen, they need to attract insects. Plants have worked this out by evolving colorful flowers and sweet nectar. And one way that yeast may do this is by generating aromas that fruit flies find irresistible.

The researchers in this study first stumbled onto this possibility around fifteen years ago. Back then the P.I. was a graduate student who left his yeast flasks out on the bench over the weekend.  Over that same weekend fruit flies escaped from a neighboring Drosophila lab and invaded the yeast lab.

In a “you got peanut butter on my chocolate” moment, the yeast researchers found the fruit flies swarming around one set of flasks and ignoring some of the others. A quick look at the flasks showed that fruit flies were ignoring the yeast strains in which the ATF1 gene was knocked out.

The ATF1 gene encodes the alcohol acetyltransferase responsible for making most of a yeast’s fruity acetate esters.  So it makes perfect sense that fruit flies ignored strains deleted for ATF1 because they didn’t smell as good anymore. To confirm this hypothesis, the authors did a fun, controlled experiment.

In this experiment, the authors set up a chamber where they could use cameras to track fruit fly movement.  One corner of the chamber had the smells from a wild type yeast strain and another corner had smells from that same strain deleted for ATF1.  As you can see in the video here, the fruit flies cluster in the corner with the wild type strains.  Fruit flies definitely prefer yeast that can make flowery sorts of acetate esters.

Christiaens and coworkers took this one step further by actually looking at the effect these chemicals had on Drosophila neurons.  They used a strain of fruit fly containing a marker for neuronal response, so that the researchers could “see” how the flies were reacting to wild type and atf1 mutant yeast smells.  As expected from the previous experiments, the olfactory sensory neurons responded differently to each smell.

To confirm that the esters were responsible for this difference, the authors observed the effect of adding esters back to media in which the atf1 mutant yeast were growing. They found that as more esters were added, the activity pattern of the Drosophila neurons shifted towards that seen with the wild type yeast.

OK, so fruit flies like good smelling yeast.  The next question the researchers asked was whether this had any effect on the dispersal of the yeast – and it definitely did.

To test this, they labeled wild type and atf1 mutant yeast with two different fluorescent markers, so the strains could be distinguished from each other. They then spotted each strain opposite from one another on a specially designed yeast plate and let a fruit fly roam the plate. They then removed the fly and the original spots of the yeast cells.

After letting the plate incubate for 48 hours, so that any yeast cells that had been moved around on the plate could grow up into colonies, they washed the plate to remove the cells that had been dispersed by the fly and used flow cytometry to determine the amount of each strain. They found that wild type yeast were transported about four times more often than the aft1 mutant yeast.

These results show that fruit flies are more likely to disperse yeast if the yeast are producing fruity smells.  Given the close relationship between fruit flies and yeast, and the fact that insect vectors are very important for yeast out in the wild, it is reasonable to think that yeast may smell good in order to attract fruit flies to carry them to new places.

This research also again points to the importance of expanding studies to include more than one organism (see our last blog here). By increasing the diversity of organisms in an experiment, we can learn much more about how things work in the real world. And maybe even learn why yeast evolved to give us such delicious beer.

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

New and improved Locus Summary pages

October 13, 2014

We are pleased to announce that the redesign of our gene-specific pages, which has been ongoing over the past year, is now complete with the release of the reworked Locus Summary page. The page contains all of the information on the previous Locus Summary page, and has a more modern look and feel. Note that the order and organization of the sections has changed, and the order of the tabs across the top of the page has changed as well. New elements on the page include a navigation bar on the left to take you to the different sections of the page, a redesigned map showing genomic context in the sequence section, and a new interactive histogram summarizing expression data. Biochemical pathway information now appears in its own section (see an example), and we have added a History section to replace the previous Locus History tab. If there are no data of a particular type (for example, Pathways), then that section is absent from the page.

Please explore this new page and send us your feedback.

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