New & Noteworthy

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.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: ALS, helicase, RNA polymerase II, Saccharomyces cerevisiae, transcription

You Can Thank Fruit Flies for Those Yummy Beers

October 14, 2014

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

Categories: Research Spotlight

Tags: beer, Drosophila, Saccharomyces cerevisiae

Yeast Gets By With a Little Help From its Friends

October 09, 2014

If you spend any time looking at social media, you’ve seen the viral videos about interspecies “friendships” – heartwarming scenes of elephants playing with dogs, or lions cuddling with antelopes. These animal relationships strike a chord with most people. Maybe they make us feel there’s hope for harmony within the human species, if such different creatures can get along with each other.

It may not give you quite as warm and fuzzy a feeling, but in a recent Cell paper, Jarosz and colleagues have shown that yeast and bacteria enjoy a friendship too.  However, these microbes have taken it a step further than the larger animals.

Not only do the yeast and bacteria get something good out of the relationship, but the yeast also get a permanent change that they can pass down to their daughters. It is as if being friends with an elephant could give a dog (and her puppies!) the ability to survive on grasses and fruit.

Like koalas with their eucalyptus leaves and pandas with their bamboo, yeast is a nutritional specialist. It is very good at consuming glucose, and will eat nothing else if glucose is available. All the genes necessary to metabolize other carbon sources are tightly turned off in the presence of glucose, a phenomenon termed glucose repression.

As Jarosz and coworkers studied this glucose repression, they stumbled upon the finding that contaminating bacteria could short circuit this process in yeast.  In other words, when yeast and these bacteria grew together, the yeast gained the ability to metabolize other carbon sources in the presence of glucose!  And even more surprisingly, that trait was passed on to the yeast’s future generations.

Here’s how this discovery unfolded.  The authors had plated yeast on medium containing glycerol as a carbon source, plus a small amount of glucosamine, which is a nonmetabolizable glucose analog. Wild-type cells cannot grow on this medium because the presence of the glucose analog makes it seem like glucose is present, causing glucose repression and preventing utilization of the glycerol.

However, there happened to be a contaminating bacterial colony on one plate, and the yeast cells immediately around this colony were able to grow on the glycerol + glucosamine medium.  When those yeast cells were re-streaked onto a fresh glycerol + glucosamine plate, with no bacteria present, they were still able to grow: they had undergone a heritable change. The ability to utilize glycerol in the presence of glucosamine was stably inherited for many generations, even without any selective pressure.

Although the first observation was serendipitous, this proved not to be an isolated phenomenon. The researchers were able not only to reconfirm it, but also to show that it could happen in 15 diverse S. cerevisiae strains. They identified the original bacterial contaminant as Staphylococcus hominis, but showed that some other bacterial species could also give yeast the ability to bypass glucose repression.

This group had previously found a way that yeast could become a nutritional generalist: by acquiring the [GAR+] prion. Prions are proteins that take on an altered conformation and can be inherited from generation to generation.  They usually confer certain phenotypes; one of the best known is bovine spongiform encephalopathy, or mad cow disease.

Luckily for the yeast, the [GAR+] prion is not nearly so devastating. Instead of a deteriorating brain, S. cerevisiae cells carrying the [GAR+] prion can grow on multiple carbon sources even in the presence of glucose.

Since this phenotype was suspiciously similar to that of the yeast that had been exposed to bacteria, Jarosz and colleagues tested them for the presence of the [GAR+] prion, and found by several different criteria that the cells had indeed acquired it. They looked to see if the yeast got other prions as well, but found that bacterial contact specifically induced only the [GAR+] prion.

The next step was to find out how the bacteria were communicating with the yeast.  Since active extracts could be boiled, frozen and thawed, digested with RNAse, DNAse, or proteases, or filtered through a 3 kDa filter without losing activity, the signaling molecule(s) was probably small.  But the researchers ended up with a complex mixture of small molecules, and more work will be needed to find which compound(s) are responsible for this effect.

In the case of animal friendships, it’s believable that intelligent animals are getting some emotional reward from their relationships (If you don’t believe it, the story of Tarra the elephant and Bella the dog in the video below may convince you!). We can’t exactly invoke this for microbes, so why would these organisms have evolved to affect each other in this way?  It seems there must be a “reward” of some kind.

The benefit to yeast cells from their bacterial friendship is that when they carry the [GAR+] prion, they can grow much better in mixed carbon sources and have better viability during aging.

Conversely, the bacteria benefit because [GAR+] yeast cells produce less ethanol than do cells without the prion. This makes a better environment for bacteria to grow, since too much ethanol is toxic. Interestingly, although the bacterial species that were the best inducers of [GAR+] are not phylogenetically closely related to each other, several of them share an ecological niche. They are often found in arrested wine fermentations, which are unsuccessful fermentations in which the yeast stop growing and bacteria take over.

So interspecies “friendships” can have more profound effects than just tugging at the heartstrings of viewers.  One example is the cat that acts as the eyes for that blind dog.  Another is this case, where bacteria can do yeast some permanent good and make a more hospitable environment for themselves in the process.

And this study reminds scientists of two important things. First, that the laboratory environment cannot tell us everything about biology. How often do yeast cells in nature grow as a monoculture on pure glucose, anyway?  And second, that sometimes accidental occurrences in the laboratory, in this case “contamination,” can broaden our findings…if we pay attention to them.  Just ask Alexander Fleming!

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

Categories: Research Spotlight

Tags: bacteria, glucose repression, prions, Saccharomyces cerevisiae

Now You Don’t See It, Now You Do!

September 25, 2014

One of the great joys of teaching can be found in the questions that students ask.  Because they are unconstrained by previous knowledge, they can think outside of the box and ask questions that force the teacher to see a problem in a new light.  Their unbiased questions often uncover aspects of a problem that a teacher didn’t think to look for or even consider.

The scientific enterprise can be very similar.  Sometimes an unbiased search of a process will uncover hidden parts scientists were completely unaware of.

This is exactly what happened in a new study in Science by Foresti and coworkers.  Using an unbiased proteomics approach they found a previously hidden part of the endoplasmic reticulum-associated degradation (ERAD) pathway in the inner nuclear membrane (INM) of the yeast Saccharomyces cerevisiae.  No one knew it existed before and, frankly, no one even knew to look!  By thinking outside of the box, these authors found that a novel protein complex in the INM targets certain proteins for degradation – both misfolded proteins, and some correctly folded proteins whose functions are no longer needed. 

Scientists already knew that the ERAD pathway uses different protein complexes to target proteins for degradation, depending on where those proteins are located.  For example, misfolded cytoplasmic proteins are targeted by a complex containing Doa10 (also known as Ssm4), while those in the membrane are targeted by the Hrd1 complex.  However, degradation of both sets of proteins requires ubiquitination by the shared subunit Ubc7. In addition to targeting misfolded proteins, both of these complexes also target certain functional proteins in response to specific conditions.

In the first set of experiments, Foresti and coworkers looked at the proteomes of strains deleted individually for Doa10, Hrd1, or Ubc7.  To their surprise, they found a set of proteins, including Erg11 and Nsg1, that are unaffected by the deletion of either Doa10 or Hrd1, but whose levels are increased in strains deleted for Ubc7.  This suggested there is a branch of the ERAD pathway that involves Ubc7 but is independent of Doa10 and Hrd1.  The authors set out to find this undiscovered third branch lurking somewhere within the yeast.

Some possible candidates for being part of the ERAD pathway were two paralog proteins Asi1 and Asi3,  and their associated protein Asi2.  Based on their sequences, Asi1 and Asi3 are putative ubiquitin-protein ligases like Doa10 and Hrd1.  Interestingly, all three Asi proteins localize to the inner nuclear membrane, which connects to the ER at nuclear pores.

When Foresti and coworkers deleted any one of the three Asi proteins, degradation of Erg 11 and Nsg1, both involved in sterol synthesis, was blocked. However deletion of Asi1, Asi2, or Asi3 didn’t affect all proteins involved in sterol biosynthesis, since Erg1 was unaffected.  Biochemical experiments confirmed that Erg11 binds to a complex composed of these three Asi proteins.

Since the ERAD pathway is important for degradation of misfolded proteins, the authors set out next to determine whether the Asi complex plays a role in this process as well.  That would be a somewhat surprising finding, since misfolded proteins aren’t generally found near the INM. But through a complicated set of experiments summarized below, Foresti and coworkers confirmed that the Asi complex does also have a role in this process.

They first tested several proteins that are known ERAD substrates, but mutations in the ASI genes had no measurable effect on them. Because some misfolded proteins are targeted by more than one ERAD complex, the authors next looked to see whether the Asi pathway contributed to either the Hrd1 or the Doa10 pathways. Testing the accumulation of several substrates in strains with different combinations of asi, hrd1, and doa10 mutations, they found that one mutant protein that misfolds, Sec61-2, had high steady state levels in a hrd1 knockout, but even higher steady state levels in a double knockout of hrd1 and asi1 or hrd1 and asi3.  So both the Asi and Hrd1 pathways appeared to work on this misfolded protein.

The researchers hypothesized that the Asi branch may target misfolded proteins for degradation as they travel through the inner nuclear membrane on the way to the ER.  To test this idea, they compared the steady state levels and localizations of two differently mutated versions of the Sec61 protein – one that localized to the inner nuclear membrane and one that did not, in both wild-type cells and a variety of deletion strains.

The bottom line from these experiments was that the mutant protein that was located at the inner nuclear membrane was more dependent on the Asi complex than the mutant that wasn’t.  Not only that, but the mutant Sec61 protein that was directed to the inner nuclear membrane changed its localization to the nuclear envelope in an asi1 deletion strain.  Both of these results are consistent with a role for the Asi complex in targeting proteins for degradation while they are in the inner nuclear membrane.

The final set of experiments confirmed the importance of the Asi complex in ER protein quality control.  Yeast responds to the presence of too many misfolded proteins in the ER with a signaling pathway called the unfolded protein response (UPR).  Strains in which this pathway is compromised, for instance by deleting IRE1, need a functional ERAD to thrive.  The authors found that deleting HRD1, IRE1, and ASI1 had a much more severe effect on viability than did just deleting HRD1 and IRE1.  This supports the idea that the Asi complex is important in ER protein quality control.

Foresti and coworkers have thus uncovered a previously undiscovered branch of the ERAD pathway in yeast by doing a broad, unbiased proteomics study.  The key proteins they identified, Asi1, Asi2, and Asi3, were originally discovered for their genetic effects on the transcriptional repression of amino acid permeases (hence their name, Amino acid Signaling Independent).  Their detailed biochemical functions were unknown until now.

A lesson here is that just because a process looks like it is pretty well locked down, this doesn’t mean that there aren’t hidden parts yet to be discovered. And just because a gene is implicated in one process, don’t assume it isn’t also involved in other processes as well. Looking from a different angle can allow you to see things you had missed before.

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

Categories: Research Spotlight

Tags: ERAD, inner nuclear membrane, Saccharomyces cerevisiae, ubiquitin-mediated degradation

Dealing With Alcohol, a Messy Business

September 16, 2014

Different people can respond to alcohol differently because of their genes.  For example, many Asians flush or even become ill from alcohol because of a mutation in their ALDH2 gene. (This is not just a minor annoyance—these unpleasant side effects come with a significant increase in esophageal cancer rates.)

This is a simple example where one gene has a significant effect. But of course, not everything to do with people and alcohol is so simple at the genetic level! 

For example, some people can drink you under the table while others are lightweights.  Some of this has to do with their lifestyle (how often they drink, how much they weigh, etc.), but a lot undoubtedly has to do with the variations they carry in multiple genes.

Well, it turns out this is also the case for yeast (our friend in the alcohol business). A new paper in GENETICS by Lewis and coworkers confirms that different strains of the yeast Saccharomyces cerevisiae tolerate high levels of alcohol differently because of their specific genetics. And at first the response seems…shall we say…incapacitatingly complex.

The results are interesting in that they help parse out how yeast responds to ethanol, but the implications are more far-reaching than that.  This analysis helps to form the framework for investigating how natural variation in gene expression can affect the traits of individuals and their responses to certain environmental stimuli.

Lewis and coworkers used three strains in their study: a lab strain that came from everyone’s favorite workhorse S288c, the strain M22 from a vineyard, and the oak soil strain YPS163.  They had previously shown that thousands of genes in each strain responded differently to 5% ethanol.  In this study they set out to find out what was behind these differences.

First off they wanted to confirm their previous results.  Using six biological replicates, they found that 3,287 genes out of a total of 6,532 were affected in at least one strain when treated for 30 minutes with 5% ethanol.  This is over half the genes in the genome!

To try to get a handle on what is causing such widespread effects, they next performed eQTL mapping in 45 F2 crosses of S288c X M22 and S288c X YPS163 (these particular matings were chosen because much of the variation they saw was in S288c).  This analysis was designed to try to find “hotspots” in the genome:  loci that affected many different transcripts, or that could account for all the variation they saw.

When they did this analysis they found 37 unique hotspots. Each hotspot represented 20-1,200 different transcripts, with a median of 37 transcripts.  Of these, 15 were seen in both crosses, 12 in just the S288c X M22 and 10 in the S288c X YPS163 matings. No silver bullet, but 37 is certainly easier to work with than 3,287!

Lewis and coworkers next set out to find the key gene(s) in the hotspots responsible for affecting multiple transcripts in the presence of ethanol.  Some were easy to find.  For example, HAP1 in S288c and CYS4 in M22 X S288c.  But the big prize in this analysis probably goes to MKT1, which affected over 1,000 transcripts in this study.

Now MKT1 is not one of the usual suspects, in that it is not a transcription factor.  However, MKT1 has been implicated in lots of observed differences between strains, including alcohol tolerance in one Brazilian overproduction strain.  Given this, the authors set out to explore whether there were any differences in Mkt1p activity in response to ethanol in the different strains.

This analysis revealed that Mkt1p localizes to P-bodies upon ethanol stress in S288C but not YPS163. And this wasn’t some general defect in Mkt1p, since it is known to colocalize with P-bodies in both strains in response to hypo-osmotic stress.

With this discovery, things were starting to make a bit more sense!  Since P-bodies are involved in mRNA turnover, it follows that a P-body component might affect so many transcripts. One potential explanation might be that Mkt1p serves as a regulator by translationally silencing specific mRNAs at P-body loci. This would be consistent with its known role in translational regulation of the HO transcript.

This study reveals how difficult it is to get to the bottom of determining exactly how massive differences in gene expression lead to differences in traits.  But it also shows that while daunting, it is doable.  And perhaps yeast can show us how best to interrogate our own differences in gene expression to help figure out why we are the way we are—not only in terms of whether we dance on the tables or fall to the floor after a few drinks, but in many other respects as well.

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

Categories: Research Spotlight

Tags: eQTL mapping, ethanol response, Saccharomyces cerevisiae, transcription regulation

Pseudouridine: Not Just for Noncoding RNA Anymore

September 11, 2014

If you think back really hard to your basic molecular biology classes you can probably remember that weird nucleotide pseudouridine (ψ). You probably learned that it is found in lots of tRNAs and rRNAs but never in mRNA. You also may remember that while its function is still a bit unclear, it may have something to do with RNA stability and/or helping aminoacyl transferases interact with tRNAs.

If a new paper in Nature holds up, one of those things we learned is almost certainly wrong. In this study, Carlile and coworkers show pretty convincingly that ψ is also found in mRNA. And not only that, but it may be doing something important there.

The authors used a sensitive high-throughput technique called Pseudo-seq to look for ψ in all the RNA in a yeast cell. The first step in this technique is to treat the RNA with a chemical called CMC.* This chemical reacts with ψ in such a way as to create a block to reverse transcriptase. In other words, reverse transcriptase can only convert RNA into DNA up to the point of a ψ. The next step is to analyze the products and to determine where reverse transcriptase has been halted. 

Carlile and coworkers first validated their technique by looking at RNAs known to have ψ’s. They showed that their technique had an estimated false discovery rate of 5% for highly expressed genes and 12.5% for poorly expressed genes. They were now ready to tackle mRNA to see what they could find.

They first looked at the mRNA of the yeast Saccharomyces cerevisiae during post-diauxic growth (after log phase) and found 260 ψ’s in 238 protein coding transcripts. This is 260 more ψ’s than had been found before.

The next step was to try to get a feel for whether or not these changes were important. To do this, they decided to compare pseudouridylation (we promise not to use that word again!) in log phase and post-diauxic growth. They found that 42% of the sites modified after log phase were not modified during log phase. In other words, it looks like the level of mRNA modification is different depending upon the growth rate. 

Uracils are modified to ψ by a surprisingly large number of enzymes. One enzyme, Cbf5p, uses snoRNA guide sequences to find the right uracils to modify. Cbf5p may not be that important for converting U’s to ψ’s in mRNA , however, since only 3/260 of the sites identified by the authors appeared to be targeted by this enzyme.

The other nine known enzymes in yeast all have the rather unfortunate acronym “PUS,” for PseudoUridine Synthase. Carlile and coworkers tested the effects of individually deleting eight of these on their newly identified ψ sites in mRNA and found that deleting PUS1 affected the highest number of mRNAs.  Interestingly, many of the Pus1p target sites were modified more often during post-diauxic growth than during exponential growth. Deleting the other PUS genes had similar, if smaller, effects. 

The authors next confirmed that something similar happens in human cells. Using very strict criteria, they identified 96 ψ’s in 89 human mRNAs and found that some of these were regulated by growth conditions (serum starvation), just as in yeast. So, modification of mRNAs with this interesting residue appears to happen in people too (or at least in HeLa cells).

Finding ψ’s in mRNA is a big contradiction to everything we’ve been taught!  The next step is to figure out what they are doing there, and there are lots of possible answers.

One possibility is that the newly discovered mRNA modifications make possible a whole new set of translated proteins. Adding a ψ to mRNA changes codon usage at that position in vitro. For example, one study found that converting the stop codons UAA and UGA to ψAA and ψGA, respectively, changed them from stop codons into sense codons both in vitro and in vivo. So ψ’s in mRNAs could cause a whole slew of new alleles to appear under certain conditions – at the RNA level instead of the DNA level. A proteomics study should help determine whether this is happening or not.