New & Noteworthy

Ribosomes Caught in the Act

November 24, 2014

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If you want to see what animals really do out in the wild, first you need to hide a camera and a trip-wire so well that the jungle seems totally undisturbed. Then, if you’re lucky, you’ll be able to catch them in the middle of the night as they pass by. Now you can surprise that tiger and find out what he is doing at that specific spot.

In two companion Science articles from the Weissman group at UCSF, Jan et al. and Williams et al. did essentially the same thing to S. cerevisiae ribosomes. They hid a molecular tag and the enzyme that recognizes it at various interesting places within yeast cells, so cleverly that the cells had no idea anything was different. Instead of a flash of light, they used a pulse of the small molecule biotin to find out which mRNAs were being translated at specific locations in the cell.

What they found was that when ribosomes are translating proteins that are targeted to a particular organelle, they hang around the surface of that organelle—way more frequently than was previously thought. And the exquisite specificity of this technique, allowing them to pinpoint one particular mRNA within the cell, uncovered a fascinating case of dual protein localization.

Pinpointing Translation Locations

The researchers needed to develop a technique for catching ribosomes in the act of translation. One part of this had already been worked out in the same group: ribosomal profiling, a method that allows you to map very precisely the positions of ribosomes on mRNAs.

Briefly, cells are lysed and translating ribosomes are treated with nucleases that nibble away mRNAs, except for the 30 nucleotides or so that are protected within the ribosome. Then those protected fragments are analyzed by deep sequencing. This shows, at the single nucleotide level, where ribosomes are sitting on each individual mRNA.

Ribosomal profiling tells us where translating ribosomes are in relation to mRNAs, but not where they are in relation to the rest of the cell. To get this location information, the researchers came up with a clever tagging strategy.

They started with a bacterial gene, E. coli BirA, that encodes a biotin ligase—an enzyme that can attach biotin to specific acceptor peptides. They fused BirA to various yeast genes in order to target biotin ligase to different places in the cell.

Next they tagged ribosomes by putting a biotin acceptor, called the AviTag, on ribosomal proteins such that the tag would be sticking out on the ribosomal outer surface. They tested both the BirA and AviTag fusions to make sure that they didn’t interfere with the functions of any proteins. Just like the camera hidden in the jungle, the tags didn’t perturb yeast cells in the least.

Now the researchers were set to surprise ribosomes with a pulse of biotin. Any ribosomes that were close to BirA would become biotinylated. The tagged ribosomes could then be isolated, and the mRNA sequences being translated in those ribosomes could be identified. The method as a whole is termed proximity-specific ribosomal profiling.

Jan and coworkers set up and validated this method in their paper, and used it to look at translation of secretory proteins at the surface of the endoplasmic reticulum (ER), while Williams and colleagues used the method to look closely at translation at the mitochondrial surface. Import into both of those organelles has previously been studied intensively, but often in vitro and mostly for just a few model protein substrates. In contrast, proximity-specific ribosomal profiling gives us the ability to look at translation of the entire proteome in vivo.

While it was known before that proteins targeted towards a certain organelle tended to be translated near that organelle, these researchers found that it was much more common than previously believed. For example, they found that most mitochondrial inner membrane proteins were translated at the mitochondrial surface and imported cotranslationally, in contrast to the previous view that mitochondrial import is predominantly posttranslational.

Both studies discovered many more details than we can summarize here. But the comparison between the ER and mitochondrial studies led to a special insight about one protein.

Osm1p Goes Both Ways

Osm1p, fumarate reductase, was thought to be a mitochondrial protein (although results from a few high-throughput studies had hinted at a link to the ER). But proximity-specific ribosomal profiling showed very clearly that it was translated at both the ER and mitochondrial surfaces. Williams and coworkers went on to confirm by fluorescence microscopy of an Osm1p-GFP fusion that Osm1p is indeed present in both ER and mitochondria.

Both of these organelles have pretty strict criteria for the signal sequences of proteins they import, so how could it be possible that the same protein goes to both locations? The researchers found that in fact, it’s not! They repeated the ribosomal profiling on the OSM1 mRNA, this time adding the drug lactimidomycin which makes ribosomes pile up at translational start sites. This showed that OSM1 actually has two start codons and produces two different proteins targeted to the two locations.

The OSM1 methionine codon currently annotated as the start would produce a protein with an ER targeting signal. Ribosomes piled up there, but also at another methionine codon 32 codons downstream. Starting translation at this codon would produce a protein with a mitochondrial targeting signal. Williams and colleagues confirmed this idea by showing that mutating the first Met codon made all of the Osm1p go to mitochondria, while mutating the Met codon at position 32 sent all of it to the ER.

The mutant form of Osm1p that couldn’t go to the ER conferred an intriguing phenotype: the inability to grow in the absence of oxygen. Osm1p generates oxidized FAD, which is necessary for oxidative protein folding, and it also interacts genetically with ERO1, which is involved in this process. Taken together, this all suggests that Osm1p activity drives oxidative protein folding in the ER.

The traditional ways of determining where a protein is in the cell, microscopic visualization or physical fractionation, can both be difficult and imprecise. Proximity-specific ribosomal profiling gets around those challenges, and gives a very precise picture of exactly where proteins are being created and how ribosomes are oriented with respect to organelles.

The example of Osm1p localization gives just a hint of the insights that are waiting for scientists who exploit this technique further. And we’re not just talking about yeast: the authors tested and validated the method in mammalian cells. Just like that tiger, surprised ribosomes in many different cell types will be giving up their secrets about where they roam and what they do.

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

Categories: Research Spotlight

Tags: endoplasmic reticulum, mitochondria, protein targeting, Saccharomyces cerevisiae, translation

Shmoos Lost in Translation

August 07, 2014

To mate, the yeast Saccharomyces cerevisiae needs to shmoo — to generate a projection that reaches out to a nearby yeast of the opposite sex, until the yeast cell is shaped like the Al Capp cartoon character.  And to shmoo yeast needs, among other things, polyamines like spermidine.

Spermidine is important for one of the most interesting proteins in the world, the translation initiation and elongation factor eIF5A.  Not only is this protein pretty much conserved in just about every living thing, but it is also the only protein to have the unique amino acid hypusine.  And to make things even more fascinating, there are two other conserved proteins whose only job is to convert a single lysine residue of eIF5A into hypusine, using polyamines like spermidine.  Simply mind boggling.

In a new study in GENETICS, Li and coworkers provide compelling evidence that spermidine is important in yeast shmooing because of its involvement in the hypusinylation of eIF5A.   They also found that one reason eIF5A is so important in this process is that it is necessary for translating Bni1p, a formin needed to organize the actin cables of the shmoo.  Without these actin cables, the shmoo can’t form.

It looks like yeast needs eIF5A to translate Bni1p because of the long stretches of prolines found in this protein.  This suggests that like its bacterial ortholog EF-P, a key job for eIF5A is to help the cell deal with polyproline stretches in proteins.

To show this the researchers made a set of targeted mutations to check whether hypusinylation of eIF5A is necessary for shmooing.  When they knocked out LIA1, one of the enzymes that uses spermidine to convert lysine to hypusine, the resulting yeast failed to shmoo.  Since the only known target of the Lia1 protein is eIF5A, this suggests that hypusinylation of eIF5A is critical to its function in shmooing.

They also used temperature sensitive mutants of eIF5A to show that this gene (HYP2, also known as TIF51A) is involved in shmooing.  At the nonpermissive temperature, only 7.7% of yeast with the less severe mutant allele, tif51A-1, shmooed, while none of the yeast with the more severe mutation, tif51A-3, were able to shmoo.  These two results taken together establish the importance of eIF5A in shmooing.

Because eIF5A was known to be important for translating polyproline regions, the researchers looked for yeast proteins with such stretches, with the idea that their failure to be translated may be behind the need for eIF5A in shmooing.  They found 549 such proteins, and a comparison of their Gene Ontology (GO) annotations showed four overrepresented categories including “mating projection” (shmoo).  They focused on a protein from this group, Bni1p, because it was known to be involved in shmoo formation and it was one of only two proteins with ten or more prolines in a row. 

Bni1p is important for organizing the actin cables that are needed to make a shmoo.  Li and coworkers showed that the temperature sensitive mutants of eIF5A and bni1 mutants had similar phenotypes in terms of actin organization in the shmoo.

So the idea here is that yeast need eIF5A to shmoo because they need eIF5A to translate Bni1p, and Bni1p is needed to set up the actin framework of the shmoo.  In this hypothesis, it is the indirect action of eIF5A that prevents the shmooing. To test this hypothesis, the authors generated a bni1 mutant that lacked the polyproline regions. 

They compared the transcript levels of wild type BNI1 and the mutant lacking the polyproline stretches using RT-qPCR and found that the presence of eIF5A didn’t matter much.  The transcript levels of the mutant and wild type BNI1 were pretty much the same.

It was a different story for the protein levels.  Using Western blots Li and coworkers saw very little wild type Bni1p, but lots of the mutant protein.  The yeast cells struggled to translate wild type Bni1p but had no trouble with the mutant.  The easiest explanation is that eIF5A is needed to help the yeast translate polyproline regions of proteins, including Bni1p. 

Finally, to confirm the eIF5A and Bni1p connection, they showed that additional Bni1p could partially overcome the shmoo defect of the temperature sensitive mutants of eIF5A.  Since this suppression was only partial, and since the mutant phenotype of the eIF5A mutant is more severe than that of the bni1 mutant, there are probably other proteins involved in shmooing that require eIF5A for translation. Some likely candidates are those proteins containing polyproline stretches that are annotated to the GO term “mating projection”.

Although a connection between oddly-shaped yeast cells and human fertility and/or disease may not seem obvious, there might indeed be one. It turns out that eIF5A is so highly conserved  that human eIF5A works just fine when expressed in yeast, and mammalian formins, like Bni1p, are also proline-rich. Formins are necessary for polarized growth, which is a feature of both reproductive cell and cancer cell growth, and spermidine is required for fertilization.

Hard to believe that yeast channeling a cartoon character can teach us so much about the most fascinating of proteins, eIF5A.  And maybe even shed light on our own fertility. 

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

Categories: Research Spotlight

Tags: eIF5A, hypusine, Saccharomyces cerevisiae, translation

Holding Back the Translation Torrent

June 12, 2014

We all know the story of the little Dutch boy who stuck his finger into a hole in a dike to keep his village from being flooded.  Now, a new study out in Molecular Cell by Pircher and coworkers has identified a novel regulatory mechanism involving a small 18-nucleotide RNA that behaves similarly to this boy.

A big difference in our story is that unlike the broken dike, the “water flow” in yeast cells is usually a good thing.  It is the continuous stream of protein translation that goes through the ribosome. 

When it’s under stress, though, yeast needs to slow down translation in order to make sure that it is making and folding each protein correctly. This gives it a better shot at surviving the stress.  Once the stress is gone, translation can ramp up again. This is where that 18-nt RNA comes in.

This group identified this 18-nt RNA as a ribosome binding RNA in a previous study. Because there are only a couple of known cases where a noncoding RNA (ncRNA) regulates the ribosome directly, Zywicki and colleagues had wanted to see whether this happens in yeast.  They found about 20 ncRNAs that bound to the ribosome, with the most abundant being an 18-nt fragment that corresponded to part of the coding sequence of the TRM10 gene that encodes a tRNA methyltransferase.

In the current study Pircher and coworkers reconfirmed that in yeast cells about 80% of this 18-nt RNA is associated with ribosomes. To verify whether it really bound to the ribosome rather than to the mRNA being translated, they broke apart polysomes with the chelating agent EDTA. This separated the large and small ribosomal subunits from each other and from mRNA.

All of the 18-mer stayed with the large subunit, showing that it really does interact with the ribosome. The researchers also found that under normal conditions it is bound to nontranslating ribosomes, while in stressed cells it shifts to actively translating polysomes.

The mutant phenotype of the trm10 null mutant suggested that the 18-mer might have a role in adapting to stress conditions. This mutant looks normal under standard conditions, but grows slower than wild type when under osmotic stress.

Pircher and colleagues used a clever strategy to find out whether this phenotype was due to the absence of Trm10p or to the absence of the 18-mer. First, they added a stop codon into the TRM10 gene, outside the region encoding the 18-mer. This mutation blocked production of Trm10p, but didn’t affect the 18-mer. The mutant looked just like wild type under osmotic stress conditions, showing that Trm10p isn’t involved in the stress response.

Second, to see directly whether the 18-mer is important, they mutated its sequence by changing some of the codons within it to other, synonymous codons encoding the same amino acid. So the Trm10p derived from this gene was wild-type, although the 18-mer sequence was different.

A couple of mutants of this type both showed the same phenotype of slow growth under osmotic stress. So production of the 18-mer is in fact important for maintaining growth rate under stress conditions. These mutant 18-mers also failed to bind to ribosomes. 

To find out what this little RNA actually does, they used electroporation to load up each cell with about 200,000 molecules of the 18-mer. This was about the same as the number of ribosomes per cell. Translation was almost completely inhibited. When they did the same experiment with an 18-mer with a scrambled sequence, it had no effect.

Further in vitro experiments confirmed the inhibitory effect of the 18-mer on translation, and showed that the inhibited step is translation initiation. It’s not completely clear why slowing down translation promotes cell growth during stress, but the authors speculate that it leads to more accurate translation and protein folding, which improves protein homeostasis and adaptation to stress. It also remains to be determined whether the 18-mer is created by processing of the TRM10 mRNA or is transcribed independently.

This regulatory mechanism is surprising and relatively novel: there are just a couple of known cases of ncRNAs regulating the ribosome directly. But it makes sense that regulating translation in this way allows the cell to react very quickly to changing environmental conditions, without needing to synthesize any new molecules.

Small ncRNAs like microRNAs or small interfering RNAs are emerging as big players in regulation in many organisms. However, miRNAs and siRNAs are not found in S. cerevisiae. But as this study shows, this does not mean that yeast doesn’t use small RNAs for regulation.  And one of the most surprising things about this story is that such a tiny scrap of RNA can regulate the ribosome, with its 5.5 kb of rRNA and 80 proteins. The little Dutch boy’s finger is immense by comparison!

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

Categories: Research Spotlight

Tags: ncRNA, osmotic stress, Saccharomyces cerevisiae, translation

The Rhythm of Ribosomes

February 13, 2013

We all know that some people march to the beat of a different drummer. But now we’re finding out that mRNAs also have their own particular rhythms as they move along the ribosome.

It’s long been known that some codons just work better than others. They are translated faster and more accurately mostly because they interact more strongly with their tRNAs and because there are more of their specific tRNAs around. So why hasn’t evolution gotten rid of all the “slow” codons? With only optimal codons, translation could move at a marching beat all the time.

One idea has been that a few pauses every now and then are a good thing. For example, maybe slowing down translation at the end of a stretch coding for a discrete protein domain gives that domain time to fold properly. This would make it less likely for the polypeptide chain to end up tangled, or misfolded. Great thought, but even when researchers looked in multiple organisms, they couldn’t find a consistent correlation between codons used and protein structure. Until now, that is.

In a recent study published in Nature Structural and Molecular Biology, Pechmann and Frydman took a novel approach to this question. They derived a new formula to measure codon optimality. Using it they found that codon usage was highly conserved between even distantly related species, and that this conservation reflected the domain structure of the particular protein a ribosome was translating.

First, the authors came up with a more accurate way of classifying codons as optimal or non-optimal. They took advantage of the huge amount of data available for S. cerevisiae and included a lot more of it in the calculation, such as the abundance of hundreds of mRNAs and their level of ribosome association. They also took into account competition between tRNAs based on supply and demand, something that the previous studies had not done.

Once they developed this new translational efficiency scale, they applied it to ten other yeast species – from closely related budding yeasts all the way out to the evolutionarily distant Schizosaccharomyces pombe. The authors found that positions of optimal and non-optimal codons were indeed highly conserved across the yeasts. And codon optimality was highly correlated with protein structure.

One of the better examples of this is alpha helices. These protein domains form while still inside the ribosomal tunnel. The authors found that the mRNA regions coding alpha helices use a characteristic pattern of optimal and non-optimal codons to encode the first turn of the helix. They theorize that this sets the rhythm for folding the rest of the helix. Other structural elements are coded by distinct codon signatures too.

This isn’t just interesting basic research. It has some far-reaching practical implications too.

When using yeast to make some sort of industrial product, the thought has been to use as many optimal codons as possible. This has not always worked out, and now we may know why. A gene that tailors the codon usage to the rhythm of the protein structure is probably the best way to make a lot of correctly folded protein.

And the factory isn’t the only place where this kind of information will come in handy. Protein misfolding is the known or suspected culprit in a whole slew of human neurodegenerative diseases such as Alzheimer’s, ALS, Huntington’s chorea, and Parkinson’s disease. A better understanding of its causes might give us insights into managing those diseases.

Who knew in 1971 that translation actually is a rhythmic dance?

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

Categories: Research Spotlight

Tags: evolution, protein folding, ribosome, Saccharomyces cerevisiae, translation

It Takes More Than an AUG to Translate a Gene

August 02, 2012

Translating a gene is easy, right?  Hop on the end of an mRNA and start translating at the first AUG.

Of course nothing in biology is that simple!  Not all AUGs in the beginning of mRNAs serve as the starts of translation and occasionally translation will start at a codon other than AUG.  There is obviously more to a translation start than an AUG.

In a recent study, Kochetov and coworkers set out to better define what makes a ribosome sit down and start translating.  They used a dataset compiled from S. cerevisiae in 2009 that included a wide range of translation starts ranging from the traditional to the barely recognizable.

The researchers focused on three classes of translation starts:

1)      Traditional yeast gene start sites

2)      AUG-containing uORFs

3)      uORFs that lack an AUG

The last two sets are translation starts that happen upstream of traditional genes (hence the name upstream open reading frame or uORF).  These tend to be weaker than traditional translation starts, have very short associated ORFs, and are thought to play a regulatory role in the translation of the “real” gene.

When Kochetov and coworkers analyzed the data, they confirmed some previous studies that showed that strong translation starts have an AUG, upstream RNA that is predicted to be unfolded and to be A-rich between nucleotides -6 and -1, and downstream RNA that is predicted to form a hairpin.  Most of the traditional yeast genes possessed most of these attributes.  The uORF translation starts were a different matter though.

The uORFs that had an AUG lacked the other features of a strong translation start.  They tended to have fewer A’s in the upstream region and their RNA was structured in all the wrong ways.  The uORFs that lacked an AUG apparently made up for it by having all of the other features of a strong translation start.  They were A-rich between -6 and -1, had an unstructured RNA upstream and a hairpin downstream of the translation start.  The thought is that translation starts that lack an AUG make up for it with all of the rest of the translation context being exceptionally strong. 

These kinds of studies will make the tough job of identifying genes a bit easier.  Which can only be a good thing as more and more genomes come on line.     

How translation worked at Stanford in the 70’s

Categories: Research Spotlight

Tags: AUG, ribosome, translation, translation start

More Going on in the Ribosome Than Expected

January 20, 2012

As scientists peer ever more deeply into a cell, the picture of how things work becomes more and more complicated.  This was true when scientists took a hard look at transcription and gene regulation and found lots of little RNAs scurrying around the cell, regulating genes.  And it now appears to be true for what is being translated and how translation is regulated.

In a new study, Brar and coworkers used ribosome profiling to explore what happens in yeast cells during meiosis at the level of translation.  What they found was that a whole lot more was being translated (or at the very least gumming up the translation machinery) than anyone expected. They also found that translation is as finely regulated as is transcription. 

And this doesn’t just happen in yeast.  The same group has also generated similar findings in mice embryos as well.  Results with human cells should be right around the corner…

Ribosome Profiling

In ribosome profiling, scientists determine what RNAs are contained in a ribosome at a given time point.  The basic idea is that they isolate ribosomes, treat them with nucleases and then harvest the associated 30-35 nucleotide long mRNAs.  They then sequence all of the isolated RNAs and identify where they came from.

Like lots of biology these days, this technique has only become possible with the advent of cheap, robust sequencing.  In fact, the size of these sequences is ideal for modern sequencing techniques.

Researchers in the Weissman lab are finding all sorts of interesting things using this new tool.  For example, in meiosis they were better able to determine which proteins are involved at various stages of meiosis, to see how involved “untranslated” mRNA leaders are in translation, and to identify smaller, previously ignored transcripts associated with ribosomes.  In this post we’ll just focus on the last point but encourage the reader to learn about the study’s other findings here.

Of Shorter ORFs

Ribosome profiling has revealed that a lot more is being translated in yeast than the standard set of genes identified in the Saccharomyces Genome Database (SGD).  For example, Brar and coworkers found that the mRNA of many open reading frames (ORFs) shorter than the usual 250 or so base pairs were associated with the ribosomes.  Shorter ORFs like these aren’t routinely thought of as genes and so have not been extensively studied.

However, given how many of these ORFs were associated with ribosomes, scientists probably should start paying more attention now.  Even before meiosis, 5% of the ribosomes tested in yeast contained RNAs from these shorter ORFs.  Once meiosis kicked in, the number went up to an astonishing 30%.

Since scientists have only just started to focus on them, it isn’t surprising they don’t know how many of these smaller ORFs are translated into smaller peptides.  Or what any of these peptides that do get translated might be doing in a cell.

In a recent study, Kondo and coworkers have shown that one of these ORFs is translated into a peptide and proposed it affects how the transcription regulator Shavenbaby works in Drosophila.  Work similar to this will need to get underway before we have a good handle on what exactly is going on with these shorter ORFs.

Whatever they turn out to do, these small ORFs will probably change what we consider to be a gene.  Again.  The cell just keeps getting more and more complicated! 

Lengthy but informative lecture on ribosome profiling.

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

Categories: Research Spotlight

Tags: regulation, ribosome, ribosome profiling, translation, untranslated leader, UTR

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