New & Noteworthy

Like People, Prions Need Intimate Contact to Spread

June 19, 2014

In the Matrix Trilogy, the delicate balance of a virtual world is upset by a rogue computer program that goes by the name of Agent Smith.  This program finds and touches other agent programs, converting them into copies of itself.  Eventually, all the agent programs are copies of Agent Smith and only the hero Neo can save humanity in an epic battle within the virtual world of the Matrix.

Prions are the Agent Smiths of cells. They convert healthy proteins into prions just by touching them. Image by Marcin Wichary

A new study out in GENETICS by Li and Du provides additional evidence that prions in the yeast Saccharomyces cerevisiae work similarly to Agent Smith, in that they spread through a direct contact model.  These prions are proteins that have entered a rogue conformation, and they end up converting all copies of the same protein into a similar rogue conformation.  The proteins change from a hardworking Agent Smith trying to do its job into something that mucks up the working of a cell.  And the results, at least in humans, can be as catastrophic for the cell as Agent Smith was for the Matrix. 

Mad cow disease, for example, is caused by prions converting the prion protein (PrP) in the brain cells of people from a useful conformation to a dangerous one that spreads.  As the conformation spreads throughout the cell, these prions form amyloid fibrils that eventually kill the cell.  When enough brain cells are killed, the person dies.

The authors chose to work in yeast because unlike in people, there are multiple examples of proteins in yeast that can go prion.  The list includes Sup35p, Ure2p, Rnq1p, Swi1p, Cyc8p, Mot3p, Sfp1p, Mod5p and Nup100p.  As you might guess from the sheer number of these prion-ready proteins, prions actually do more than kill a cell in yeast; they can serve useful functions. Scientists have yet to identify any useful functions for the prion form of PrP in people. 

Having multiple prions in a cell allowed Li and Du to perform some experiments to try to distinguish between two models of prion conformation spreading.  In the first, called the cross-seeding model, the prion acts very much like Agent Smith in that it needs to contact a “healthy” protein to convert it into a prion.  In the second model, the titration model, factors in the cell that prevent prion formation are titrated out when prions form.  As the factors are taken out of commission, prions are free to form.

The main evidence in this study that supports the cross-seeding model has to do with the localization of pre-existing prions during the de novo formation of a new prion.  Li and Du found that the prion [SWI+] localized to newly forming [PSI+] prions but not to already formed [PSI+] prions.  This is not the result we would expect if prion formation were due to titrating out of inhibitors of prion formation.  If that were the mechanism, then there would be no reason for [SWI+] to colocalize with newly forming [PSI+]. These experiments are like having a google map of the Matrix where we could see Smiths converting other agents by touch and then moving on and touching other agents.

Work like this is important for helping to find treatments for prion associated diseases and, perhaps, other amyloid fibril forming diseases like Huntington’s or Alzheimer’s.  Scientists need to focus on the amyloid fiber forming proteins themselves instead of trying, for example, to ramp up the activity of factors that inhibit formation.  Scientists probably need to eliminate Agent Smith to prevent the destruction of the Matrix and all of mankind.

This is how prions turn other proteins into copies of themselves:

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.

The little Dutch boy used his finger, but yeast can stop the mighty torrent of translation using only a tiny noncoding RNA. Image from Wikimedia Commons

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!



Saddling Mom with the Burden of Old Age

June 5, 2014

In A World Out of Time by Larry Niven, people live forever by teleporting the unfolded protein aggregates associated with aging out of their cells.  Turns out that our very clever yeast Saccharomyces cerevisiae can do the same thing and it doesn’t need a machine.  

Yeast cells don’t need a teleporter to eliminate age-related protein bundles. They just leave them with mom and use a caspase to chew up the rest. Image by Chris Radcliff

Instead of teleporting the aggregates away, yeast saddles the mother cell with them when it buds.  The daughter has now regained her youth and the mother is left to struggle with old age.

In a new study in Science, Hill and coworkers show that the yeast metacaspase gene MCA1 is critical in this process.  But it doesn’t look like it is involved in segregating these bundles to the mother cell.  Instead, it appears to help clear away many of the bundles left in the daughter.   If it were in Niven’s original story, Mca1p might be a little nanobot that chewed up any aggregates the teleporter missed.  

This all makes sense given caspases’ role in multicellular beasts.  There, these executioner proteases chew up cellular proteins during apoptosis, the process of programmed cell death that is a critically important part of development and growth. 

Although apoptosis has been observed in yeast and Mca1p is involved in the process, it has always been a bit of a mystery why a single-celled organism needs a mechanism for suicide.  This study now suggests that yeast’s only caspase, Mca1p, has a role as a healer as well as an executioner. It saves the daughter by degrading and proteolytically clearing away the aggregated bundles clogging up her cell. 

Scientists already knew that HSP104 was a key player in making sure that aggregates stayed with mom.  Hill and coworkers used this fact and performed a genetic interaction screen using HSP104 to identify MCA1 as required to keep protein aggregates out of the daughter in response to a heat shock.  Follow up work confirmed this result by showing that overexpressing MCA1 led to more efficient segregation of aggregates and that deleting it led to poor segregation of aggregates.

Digging deeper, these authors found that this poor segregation was because Mca1p was not eliminating aggregates in the daughter, as opposed to affecting the segregation itself.  They also showed that the protease activity of Mca1p was needed for this effect.

In the final set of experiments we’ll discuss, the authors looked to see what effect MCA1 has on the life span of a yeast cell.  They saw little effect of deleting MCA1 unless a second gene was also deleted: YDJ1, which encodes an HSP40 co-chaperone.  The double deletion mutant yeast were able to divide fewer times before petering out.  Consistent with this, overexpressing MCA1 led to increased life span and this effect was enhanced in the absence of YDJ1.

Finally, cells lived for a shorter time if just the active site of the Mca1p protease was compromised in a ydj1 deletion background.  This again confirms that proteolysis is key to MCA1’s effects on aging. 

So yeast attains eternal youth by both dumping its age-related aggregates on its mother and by using Mca1p to destroy any aggregates that managed to get into the daughter.  The daughter gets a reset until she builds up too many aggregates, in which case she gets saddled with them.

Yeast may be showing us another way to live a longer life.  If we can specifically degrade our aggregates without causing our cells to commit mass suicide, maybe we can extend our lives.  And we don’t even need fancy teleporting machinery; we just need to adapt the molecular machinery yeast is born with.  Feel free to use this idea for a new science fiction story!

Professor Jure Piskur, Ph.D. (1960 – 2014)

June 4, 2014

Jure Piskur, 1960-2014

Dr. Jure Piskur, Professor and Carlsberg Foundation Chair in Molecular Food Microbiology at Lund University, sadly passed away on May 18, 2014. Dr. Piskur worked on yeast early in his scientific career, including postdoctoral research in yeast molecular biology at Carlsberg Brewery. From there, he studied Drosophila genes involved in the metabolism of nucleic acid precursors as well as yeast biodiversity and mitochondrial genetics. Most recently, his research focused on genes involved in the metabolism of nucleic acid precursors and “the evolution and molecular mechanisms which reshaped the modern enzymes and yeast genomes.” Dr. Piskur published many scientific papers, many of them represented in SGD. He was also a FEMS Microbiology Reviews Editor and Yeast Research Editorial Board Member. For more information on Dr. Piskur, please view his Lund University profile page.

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