New & Noteworthy
January 22, 2015
We all learned in biology class that nucleotides get added to an mRNA using a DNA template. And that amino acids get added to proteins using an mRNA template. But as with most everything in biology, there are exceptions.
For example, a long string of A’s gets added to mRNAs in eukaryotes without a DNA template of T’s. And now, in a new study published in Science, Shen and co-workers have shown that in certain cases amino acids can be added to proteins without translating an mRNA template.
Specifically, these authors showed that threonines and alanines that are not encoded by mRNA can be added to polypeptide chains stalled on the ribosome and that a key protein in this process is Rqc2p. It makes sense that Rqcp is on the spot to do this job, as this protein is part of the ribosome quality control (RQC) complex whose job it is to ubiquinate proteins stalled at the ribosome to target them for destruction.
These added amino acids aren’t the result of some glitch of a misbehaving cell. They appear to be critical for the cells to mount a response to a situation where ribosomes are failing to complete normal translation.
Working with our favorite model organism, S. cerevisiae, the researchers started out to investigate the ribosome quality control (RQC) complex. This complex is like a cleanup crew for stalled ribosomes. If something goes wrong during translation and the ribosome stops elongating the nascent protein chain, the RQC complex steps in and tags the partially synthesized protein with ubiquitin, marking it for degradation.
The scientists were hoping to figure out how the RQC complex finds and recognizes stalled ribosomes. So they immunoprecipitated RQC and used cryo-electron microscopy to look at the structure of the complex bound to stalled ribosomes.
After a ribosome stops translating, it falls apart into its large and small subunits. Shen and colleagues found that one component of the RQC complex, Rqc2p, binds to the large (60S) ribosomal subunit after dissociation. They also found something unanticipated: tRNAs were present in the 60S ribosomal subunit at the A and P sites. This is where the tRNAs normally reside during translation, but translation obviously couldn’t be happening, since there was no mRNA present.
The presence of a tRNA at the ribosomal A site was especially surprising because tRNAs don’t bind there stably; they need mRNA and elongation factors to stabilize the interaction. It turned out that what was keeping the tRNAs on the ribosomal large subunit was Rqc2p, which bound to both of them and stabilized them. The researchers used a new thermostable reverse transcriptase and deep sequencing to find that the bound tRNAs were two specific alanine and threonine tRNA species. Why these specific tRNAs?
Pursuing this question, Shen and colleagues made another unexpected discovery: nascent polypeptide chains from stalled ribosomes were smaller in the rqc2 null mutant than in wild type. This suggested that Rqc2p was adding something extra to the unfinished, stalled proteins.
Putting these observations together, the researchers formulated the hypothesis that Rqc2p mediates the addition of extra alanine and threonine residues to the C termini of proteins whose translation has been stalled. They created an ingenious set of reporter constructs to test this hypothesis.
The basic reporter contained the green fluorescent protein (GFP) gene fused to a coding sequence containing multiple “difficult” codons that would cause the ribosome to stall. The researchers found that most of the strains that were mutant for different subunits of the RQC complex contained a smear of variably sized protein products, the size of GFP and larger. But the rqc2 mutant only contained unmodified GFP; it failed to add anything. This confirmed the earlier suggestion that Rqc2p was responsible for adding the mysterious extra mass.
Next, they added a protease cleavage site at various locations in the reporter gene, and found that the extra mass was added at or downstream of the stalling sequence. In other words, it was added to the C terminus of the nascent polypeptide.
To be completely sure that translation was not involved, the researchers put stop codons in every frame after the stalling sequence. They had no effect, so the extra mass couldn’t be attributed to translation in any frame.
Finally, the scientists analyzed the C-terminal extensions by total amino acid analysis, Edman degradation, and mass spectrometry. They found that the extensions consisted of between 5 and 19 alanine and threonine residues, in no defined sequence. They named them Carboxy-terminal Ala and Thr extensions, or CAT tails.
This is all very cool, but do the tails actually do anything? Yes, it looks like they do!
When translation stalls, the cell responds to this stress using the transcription factor Hsf1p. By mutating three conserved residues in the Rqc2p NFACT nucleotide-binding domain, Shen and colleagues were able to generate a protein that could still recognize the plugged-up ribosome, but couldn’t add the alanines or threonines. It still worked fine to clean up stalled proteins, but the stalled proteins had no CAT tails. And sure enough, there was no Hsf1p-mediated heat shock response either.
So the CAT tails are part of the signal that tells the cell it had better start a stress response because things aren’t looking too good at the ribosome. It’s still not obvious exactly how the CAT tails participate in this process. But this isn’t some peculiarity of yeast: the genes are conserved, and mutations in homologs of some of the RQC genes and other genes involved in translation quality control cause neurodegeneration in mice.
The ribosome is one of the best-studied molecular machines, and you might have thought we already knew just about everything there was to know about it. This work reminds us that no matter how familiar something seems, there is always more to learn when we pay attention to unexpected results.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
January 13, 2015
Influencing mitochondrial import to treat disease:
Bounce houses are a great way for kids to burn off their excess energy. They can bounce off the floor and walls and scream to their hearts’ content.
Of course, adults need to keep an eye on how many kids are in the house at any one time, to keep things safe. And if one child starts to push and kick the others, it might be easier to restore calm if the adults are careful about how many kids, and which ones, they allow inside.
The yeast mitochondrion is actually a lot like a bounce house. It’s full of energy, and it has multiple gatekeepers—protein complexes in the mitochondrial membrane that imported proteins must pass through on their way in.
And, just like a bounce house, things can go very wrong inside the mitochondrion if its proteins don’t behave properly. The end result isn’t just an upset child with a black eye, either. Genetic diseases that affect mitochondrial function are among the most severe and the hardest to treat.
Now, described in a new paper in Nature Communications, Aiyar and colleagues have used a yeast model of human mitochondrial disease to discover both a drug and a genetic means to regulate a mitochondrial import complex. Surprisingly, tweaking mitochondrial import slightly by either of these methods mitigated the disease symptoms in both yeast and human cells. They found a gatekeeper who can make sure there is the right number of kids in the bounce house and that they’re all behaving properly (at least, as well as they can!).
The researchers were interested in mitochondrial disorders that affected ATP synthase. This huge molecular machine in the mitochondrial inner membrane is responsible for generating most of the cell’s energy, so if it doesn’t work properly it can be a disaster for both yeast and human cells.
Aiyar and coworkers used a genetic trick to create a yeast model that had lower amounts of functional ATP synthase. This mimics many mitochondrial disorders.
They were able to reduce the amount of functional ATP synthase by using an fmc1 null mutant. Fmc1p is involved in assembly of the complex, so the fmc1 null mutant has lower amounts of functional ATP synthase and a reduced respiration rate.
First, they looked for a drug that would mitigate the effects of the fmc1 mutation. They tested the drugs in a collection that had already been FDA approved—a drug repurposing library—to see if any would improve the mutant’s respiratory growth.
The one candidate drug that emerged from the screen was sodium pyrithione (NaPT), which is used as an antiseptic. Not only did it improve the respiration of the yeast fmc1 mutant, it also improved the respiratory growth of a human cell line carrying the atp6-T8993G mutation found in patients with neuropathy, ataxia and retinitis pigmentosa (NARP, one type of ATP synthase disorder).
Aiyar and colleagues wondered exactly what was being affected by the NaPT. To figure this out, they used the S. cerevisiae genome-wide heterozygous deletion mutant collection. This is a set of diploid strains, each heterozygous for a null mutation of a different gene, that has been an incredibly useful resource for all kinds of studies in yeast.
They tested the effect of NaPT on each of the mutant strains and found that strains with mutations in the TIM17 and TIM23 genes were among the most sensitive. And, when they checked the data from previous chemogenomic screens, they saw that these two mutants were much more sensitive to NaPT than to any other drug, showing that the effect was specific.
TIM17 and TIM23 are both subunits of the Tim23 complex in the mitochondrial inner membrane that acts as a gate for many of the proteins that end up in mitochondria. The researchers found that NaPT specifically inhibited the function of this mitochondrial gatekeeper complex in an in vitro mitochondrial import assay, confirming its selectivity.
So, Aiyar and coworkers had found a drug that alleviates the effects of an ATP synthase disorder by modulating the function of a mitochondrial gatekeeper. This in itself was a huge advance: the discovery that a potentially useful, already-approved drug has a specific effect on this disease phenotype.
However, the scientists took things a step further by looking to see whether a genetic therapy could accomplish the same thing as the drug. It was already known that overexpressing Tim21p, a regulatory subunit of the Tim23 complex, could modulate the function of the complex similarly to the effects they had seen for NaPT.
So the researchers tested whether overexpressing Tim21p would improve respiratory growth of the fmc1 mutant. Sure enough, it did. Consistent with this, assembly of the respiratory enzyme complexes of the mitochondrial inner membrane was more efficient when Tim21p was overexpressed.
Most importantly, overexpression of Tim21p in the fmc1 mutant cells caused their total ATP synthesis to more than double. And even more exciting was the discovery that overexpressing TIMM21, the human ortholog of TIM21, in the NARP disease human cell line improved survival of those cells.
So, just like a parent deciding how many kids should be in a bounce house so that everyone has a good time, the Tim23 complex can be made to “decide” which proteins, or perhaps how many proteins, get into mitochondria, with the end result that ATP synthesis happens as efficiently as possible. The exact mechanism of this effect is still unclear, but it is clear that modulating import in this way can improve mitochondrial health even when disease mutant proteins are present.
The next step will be to translate this discovery into therapies that will help mitochondrial disease patients. People with various mitochondrial disorders may finally be able to turn their mitochondria into safe, fun places.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
January 8, 2015
What’s for dinner tonight? For many of us the answer will be “pasta with tomato sauce”, even if we don’t have Italian roots.
But as you know, there isn’t any single recipe for homemade tomato sauce. Onions and garlic, or just garlic? Pork, beef, or no meat at all? How many bay leaves go in the pot? Every cook will use a slightly different combination of ingredients, but all will end up with tomato sauce.
When it comes to combining different allele variants to survive a lethal challenge, yeast is a lot like those cooks. In a new paper in GENETICS, Sirr and colleagues used divergent yeast strains to generate a wide range of allelic backgrounds and found that there is more than one way to survive a deadly mutation in the GAL7 gene. Just as there is more than one way to make a delicious tomato sauce…
This isn’t just an academic exercise either. GAL7 is the ortholog of the human GALT gene, which when mutated leads to the disease galactosemia. And just like in yeast, people with different genetic backgrounds may do better or worse when both copies of their GALT gene are mutated.
GAL7 and GALT encode an enzyme, galactose-1-phosphate uridyl transferase, that breaks down the sugar galactose. If people with this mutation eat galactose, the toxic compound galactose-1-phosphate accumulates; this can cause serious symptoms or even death. The same is often true for yeast with a mutated GAL7 gene.
Ideally we would want to be able to predict how severe the symptoms of galactosemia would be, based on a patient’s genetic background. So far, though, it’s been a challenge to identify comprehensively the whole set of genes that affect a given human phenotype. Which is why Sirr and colleagues turned to our friend S. cerevisiae to study alleles affecting the highly conserved galactose utilization pathway.
The researchers started with two very divergent yeast strains, one isolated from a canyon in Israel and the other from an oak tree in Pennsylvania. Both were able to utilize galactose normally, but the scientists made them “galactosemic” by knocking out the GAL7 gene in each.
After mating the strains to create a galactosemic diploid, the researchers needed to let the strain sporulate and isolate haploid progeny. But its sporulation efficiency wasn’t very good, only about 20%. And they needed to have millions of progeny to get a comprehensive look at genetic backgrounds.
To isolate a virtually pure population of haploid progeny, Sirr and coworkers came up with a neat trick. They added a green fluorescent protein gene to the strain and put it under control of a sporulation-specific promoter.
Cells that were undergoing sporulation would fluoresce and could be separated from the others by fluorescence-activated cell sorting (FACS). The FACS technique also allowed sorting by size, so they could select complete tetrads containing four haploid spores and discard incomplete products of meiosis such as dyads containing only two spores.
After using this step to isolate tetrads, the researchers broke them open to free the spores and put them on Petri dishes containing galactose—an amount that was enough to kill either of the parent strains. One in a thousand spores was able to survive the galactose toxicity. Recombination between the divergent alleles from the two parent strains somehow came up with the right combination of alleles for a survival sauce.
Sirr and colleagues individually genotyped 247 of the surviving progeny, using partial genome sequencing. They mapped QTLs (quantitative trait loci) to identify genomic regions associated with survival. If they found a particular allele in the survivors more often than would be expected by chance, that was a clue that a gene in that region had a role in survival.
We don’t have the space here to do justice to the details of the results, but we can summarize by saying that a whole variety of factors contributed to the galactose tolerance of the surviving progeny. They had three major QTLs, regions where multiple alleles were over- or under-represented. The QTLs were centered on genes involved in sugar metabolism: GAL3 and GAL80, both involved in transcriptional regulation of galactose utilization genes, and three hexose transporter genes (HXT3, HXT6, and HXT7) that are located very close to each other.
It makes sense that all of these genes could affect galactosemia. Gal3p and Gal80p are regulators of the pathway, so alleles of these genes that make galactose catabolism less active would result in less production of toxic intermediates. And although the hexose transporters don’t transport galactose as their preferred substrates, they may induce the pathway by allowing a little galactose into the cell. So less active alleles of these transporters would also result in less galactose catabolism.
Another event that occurred in over half of the surviving progeny was aneuploidy (altered chromosome number), most often an extra copy of chromosome XIII where the GAL80 gene is located. The same three QTL peaks were also seen in the disomic strains, though, leading the authors to conclude that the extra chromosome alone was not sufficient for survival of galactosemia.
And finally, some rare non-genetic events contributed to survival of the progeny. The authors discovered this when they found that the galactose tolerance of some of the progeny wasn’t stably inherited. This could result from differences in protein levels between individual cells. For example, if one cell happened to have lower levels of a galactose transporter than other cells, it might be more resistant to galactose.
The take-home message here is that there are many different ways to get to the same phenotype. The new method that they developed allowed the researchers to see rare combinations of alleles in large numbers of individual progeny, in contrast to other genotyping methods where progeny are pooled and only the average can be detected.
For any disease or trait the ultimate goal is to identify all the alleles of all the genes that influence it. Imagine the impact on human health, if we could look at a person’s genotype and accurately predict their phenotype!
So far, it’s been a challenge to identify these large sets of human genes in a comprehensive way. But this approach using yeast could provide a feast of data to help us understand monogenic diseases like galactosemia, cystic fibrosis, porphyria, and many more, and maybe even more complex traits and diseases. Now that’s an appetizing prospect for human disease researchers. Buon appetito!
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
December 18, 2014
Yeast need translation and organelle tethering to sequester misfolded proteins away from the cytoplasm:
Sometimes parents feel like they’re constantly nagging their kids to clean things up. Eventually, some parents just give in and do it themselves. It turns out that yeast cells aren’t all that different.
And it is even more important for cells to clean up their cellular trash than it is for that sullen teenager. If trash like misfolded or damaged proteins is not sequestered from the rest of the cytoplasm, it can cause other proteins to change their conformation or interfere with metabolic processes. This is obviously much worse than having a smelly room!
One way that cells bin their trash is by compacting it into globs, or aggregates. While this works in the short run, as cells age these aggregates build up, and in human cells, they’re hallmarks of diseases like ALS and Alzheimer’s.
In a new study published in Cell, Zhou and colleagues looked at the process of protein aggregation in S. cerevisiae. A number of other studies had already suggested that just like human parents, mother yeast cells clean up trash for their daughters. The researchers confirmed this and also made a couple of very surprising findings.
Zhou and coworkers discovered that although aggregates are mostly composed of previously translated proteins, they don’t form without new translation. And they found that aggregates aren’t littered around the cytoplasm, but instead are collected in very specific trash bins located on the surface of cellular organelles.
To do these studies, the scientists assembled a toolkit of ways to induce and visualize protein aggregation. They used stresses like heat shock or various chemicals to stimulate aggregate formation.
They visualized the aggregates by using GFP-labeled Hsp104p, which binds to them specifically. They also had some thermally unstable reporter proteins fused to different colored fluorescent markers that helped them track aggregation. And they created time-lapse videos to watch the whole process.
They first looked in detail at aggregate formation, creating two different kinds of cellular trash (aggregates of distinct sets of marker proteins) at different times to ask whether they would all end up in the same aggregates or in separate ones. These experiments showed that in general, newly aggregated proteins will join existing aggregates rather than creating new ones. And intriguingly, these results also hinted that new translation was needed to start the aggregation process.
Zhou and coworkers tested this directly by adding cycloheximide, an inhibitor of translation, to their aggregation experiments. Sure enough, cycloheximide prevented all of the treatments from causing aggregation, and other treatments and conditions that blocked translation did the same thing.
So just as the threat of taking away the car keys may get that sullen teenager off the couch to start cleaning up, newly synthesized polypeptides are the inducer for the cellular clean-up. Without them, all the trash just stays littered around the cell.
The researchers guessed that if aggregation starts at sites where translation is occurring, it might be concentrated at the surface of the endoplasmic reticulum (ER), where a large proportion of ribosomes are bound. They used several different sophisticated microscopy techniques to confirm that most aggregates were in fact associated with the ER.
But they also got a surprise: in addition to the ER, many aggregates were associated with the mitochondrial surface and some even formed directly on it.* The authors also observed aggregates that formed at the ER but migrated along it to ER-mitochondrial contact points and eventually to mitochondria. So, cells don’t litter their trash just anywhere; they start specific collection points on the surfaces of organelles. And much of the trash seems to end up attached to mitochondria.
The researchers noticed something else when they looked at aggregates in cells that were dividing. When a bud forms, mitochondria are actively transported into it. However, the aggregates that were on mitochondria didn’t go into buds. They stayed on mitochondria that remained in the mother cell. Mom protects her daughter from any trash that mom created!
To figure out what controls this asymmetric segregation, Zhou and colleagues tested a panel of 72 mutant strains, each with a deletion in a mitochondrial outer membrane protein. One strain, the fis1 null mutant, was markedly defective: aggregates often went into the bud. Fis1p is known to be involved in mitochondrial fission, but this result suggests it may have an entirely separate role in making sure that trash-bearing mitochondria stay in the mother cell.
And finally, the authors saw that as mother cells got older, they got less and less able to keep aggregates out of their daughters’ cytoplasm. Towards the end of the mothers’ lives, aggregates were distributed more or less randomly between mother and daughter.
Trash build up is a big problem in teenagers’ rooms and in cells. Just like mom, the cell packs the trash away into bins where it will do less harm. Unfortunately, as the cell (and mom) get older, this gets harder and harder to do. In both cases, the daughter is saddled with more and more trash as mom struggles to keep up. And this is bad for the daughter as well as for the mom.
So, there’s actually a very good reason behind all that nagging to clean up your room. The secret to a long life is to always pick up your trash!
*New data from the Weissman lab, described here in a recent blog post, dovetail nicely with this finding since they establish that a lot of translation takes place on the mitochondrial surface.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD