New & Noteworthy

Not Lost Without Translation

January 22, 2015

Sometimes, important information gets lost during translation. But new research shows that translation isn’t even necessary for adding a certain kind of sequence information to proteins. Image by Michael Cote via Flickr

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

Categories: Research Spotlight

Tags: ribosome quality control , Saccharomyces cerevisiae , stress response

How To Remember Without a Brain

October 02, 2012

Single celled beasts like the yeast S. cerevisiae can “remember” previous insults and so respond better to environmental changes in the future. For example, a yeast cell treated with 0.7M NaCl will respond better in the future to hydrogen peroxide. Not only that but so will its daughters, granddaughters and even its great, great granddaughters.

Who needs a brain to remember things?

In a new study out in GENETICS, Guan and coworkers show at least a couple of ways that this can happen. One is what anyone in biology might expect these days (although with an interesting twist). The NaCl treatment causes a rewiring of the regulatory network at an epigenetic level and this affects future responses to environmental insults.

But fancy epigenetic changes aren’t the only way that yeast remembers things.  No, it also uses a simple, elegant solution—protein stability.
Long Live The Protein!

The researchers did a set of experiments that showed that a yeast’s memory of a salt treatment did not rely on new protein synthesis and that it slowly faded with each generation.  One possible explanation was that the salt induced a stable factor that was divvied up and diluted with each passing generation. Guan and coworkers found that this was the case and that at least one of these factors was the cytosolic catalase 1 protein, Ctt1p.

The cytosolic catalase 1 or CTT1 gene is induced by salt but quickly returns to normal levels when the salt is removed.  However, Ctt1p is so long lived that it hangs around for at least six hours.  In that time the yeast has budded off multiple daughters, all of which are still better at dealing with hydrogen peroxide than their untreated sisters.

What a marvelously simple way to adapt!  Just make something that hangs around a long time and you and your kids will do better when the next insult comes.  The elegance of evolution.

This explains in part how yeast cells can remember the salt treatment of their ancestors, but a single long-lived protein isn’t the whole story.  No, there is something a bit more complicated going on at the nuclear pore too.

Attached for Quick Access

Guan and coworkers looked at the gene expression pattern of salt stressed and naïve yeast when exposed to hydrogen peroxide.  They found that 449 genes responded more quickly to hydrogen peroxide treatment if the cell had been pretreated with salt.  Importantly, 51 of these hadn’t reacted previously to the salt treatment, meaning that previous activation wasn’t required.

One idea is that these genes are more accessible to transcription because they are associated with the nuclear pore.  The idea is that faster response happens because the gene is closer to the nuclear envelope and/or because it has been looped near some sort of activator.

This is what has been proposed with inositol starvation and it looks like it may be true here too.  In both cases, eliminating Nup42p, a nuclear pore protein, eliminates the more rapid response to hydrogen peroxide.

So in this case it looks like cells can remember a previous insult with just a long-lived protein and a bit of genetic rewiring.  It will be interesting to see how universal these sorts of mechanisms are for cell memory.

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

Categories: Research Spotlight

Tags: cellular memory , Saccharomyces cerevisiae , catalase , stress response