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