New & Noteworthy

The Latest Buzz on Stressed-Out Mitochondria

September 30, 2015

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Stinging wasps get our attention, and with good reason—getting stung hurts a lot! If you see wasps going into a nest within the walls of your house, you’ll likely try to block their access.

But this could backfire: instead of being able to peacefully go into their nest, a swarm of angry wasps could be buzzing around looking for trouble. It might get so bad that you’ll need to call in an exterminator to take care of the problem.

Mitochondrial proteins might seem a lot less scary than wasps, but it turns out that they can also cause trouble if they can’t get into mitochondria. In two new letters to Nature, Wrobel and colleagues and Wang and Chen used complementary approaches to ask what happens when dysfunctional mitochondria aren’t able to import all of the proteins that are waiting to get in. 

What they both found was that these piled up proteins cause real problems for the cells. In desperation, the cells slow down protein synthesis to reduce the excess, and also turn to their own exterminator, the proteasome, to keep these proteins under control.

This is a paradigm shift in thinking about how poorly functioning mitochondria cause disease. In the past, almost everyone focused on how damaged mitochondria couldn’t make enough energy for a cell. Now it looks like there are other ways for a nonworking mitochondria to do a cell in. And new targets for scientists to go after in treating mitochondrial disease.

As we all know, mitochondria are the powerhouses of the cell, where energy is generated, and they’re also the site of many other essential biochemical reactions. They’re composed of about 1,000 proteins, and nearly all of those are synthesized in the cytoplasm and then imported into mitochondria by an intricate system of transporters.

In order to find out what happens when these 1,000 proteins don’t get into mitochondria as efficiently as they should, both groups created strains whose mitochondrial import was impaired.

Wrobel and colleagues used the temperature-sensitive mia40-4int mutation, affecting an essential component of the mitochondrial import system. Wang and Chen started with the aac2-A128P mutation in PET9 (which is also known as AAC2), an ADP/ATP carrier of the mitochondrial inner membrane. Overexpression of the aac2-A128P allele causes mitochondrial dysfunction and eventual cell death.

Wrobel and colleagues decided to get a comprehensive look at what happens in the mia40-4int mutant by assaying its transcriptome and proteome, using RNA-seq and stable isotope labelling by amino acids in cell culture (SILAC), respectively. Surprisingly, one of the biggest differences from wild type that they saw in the import-defective mutant was a decrease in cytoplasmic translation. Whether they looked at the mRNAs encoding ribosomal proteins, the proteins themselves, or the polysome content and translational activity of the cells, everything pointed to down-regulation of translation. And at the same time, the proteasome—the molecular machine that breaks down unwanted proteins—was activated.

To verify that what they were seeing wasn’t peculiar to the mia40-4int mutant, Wrobel and colleagues slowed down mitochondrial import in several other ways: using different mia40 mutant alleles, other import mutants, or treatment with a chemical that destroys the mitochondrial membrane potential required for import. Under these different conditions causing the accumulation of mitochondrial precursor proteins in the cytoplasm, they still saw decreased cytoplasmic translation and increased proteasome activity.

Wang and Chen took a different approach, looking to see whether over-expression of any other genes could compensate for the lethality of overexpressing the aac2-A128P allele. The researchers transformed the mutant with a library of yeast genes on a multicopy plasmid, and found 40 genes whose expression could keep it alive.

The suppressor genes found by Wang and Chen were all involved in some aspect of synthesis or degradation of cytoplasmic proteins, just like the genes found by Wrobel and colleagues whose expression was altered in the mia40 mutant. And Wang and Chen also verified that these suppressors weren’t specific to the aac2-A128P mutation: they suppressed a variety of other mutations that decreased import.

Both groups observed precursors of mitochondrial proteins accumulating in the cytosol of the mutant strains they studied. Wang and Chen saw a couple other very interesting proteins increase in abundance: Gis2 and Nog2. These proteins are involved in regulating ribosome function, and the researchers speculate that their stabilization during this stress response contributes to the translational down-regulation. Intriguingly, their human orthologs are implicated in neuromuscular degenerative disease.

So, using orthogonal approaches, the two groups converged on the same model: a newly discovered cellular pathway that regulates cytosolic translation and protein degradation in order to deal with the stress of inefficient mitochondrial import. Wrobel and colleagues have named it UPRam, for Unfolded Protein Response activated by mistargeting of proteins, while Wang and Chen call it mPOS, mitochondrial Precursor Over-accumulation Stress.

Before this work, it was unknown whether cytosolic pathways were even affected by mitochondrial dysfunction. Now we know that the cell has a specific response when mitochondrial precursor proteins begin swarming in the cytosol, unable to get into their home: it slows down the production of those proteins and calls in the proteasome exterminator to take care of them.

We usually think of mitochondrial disease symptoms as being caused by the reduced energy generation of sick mitochondria, or by the lack of other key events that happen in mitochondria—for example, the synthesis of the iron-sulfur clusters that some vitally important enzymes need. Now, these findings raise the possibility that proteostatic stress on the cell caused by the accumulation of mitochondrial precursors could also lead to impaired cell function and disease.

Perhaps drugs that inhibited cytoplasmic translation, or activated the proteasome exterminator, would be helpful in reducing the buzzing swarm of mitochondrial precursor proteins. Wouldn’t it be wonderful if this knowledge suggested new avenues of treatment to take some of the sting out of human mitochondrial disease?

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

A Scientist Sees Transcription

September 23, 2015

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In the classic Dr. Seuss tale Horton Hears a Who, the elephant Horton thinks he hears voices coming from a speck of dust. He gets into all sorts of trouble over this until all the Whos in Whoville prove they are alive when they all shout at once. Now Horton’s jungle compatriots believe him and Horton can hang out with his new friends.

Horton’s companions never get to hear an individual Who. They are not blessed with Horton’s big elephant ears and so have to just hear all the Whos shouting at once.

Up until recently, we have been in the same situation as the kangaroo and everyone else in the jungle when it comes to transcription in a cell. We can use all sorts of tools to get at what goes on when RNA polymerase II (pol II) gets ready and then starts to transcribe a gene, but we can only get an aggregate picture of lots of cells where it is happening. We can’t hear the Mayor of Whoville amidst all of the other Who voices.

In a new study out in Nature, Fazal and coworkers use the equivalent of elephant ears, optical tweezers, to study the initiation of transcription by purified pol II machinery from Saccharomyces cerevisiae on single molecules. And what they find is that at least for one part of the process, our having looked at things in the aggregate may have fooled us about how the process worked. It was important that we be able to pick out individual voices from the cacophony of the crowd.

Not surprisingly, transcribing a gene is tricky work. It is often split into three steps: initiation, elongation, and termination. And each of these can be subdivided further.

Fazal and coworkers focused on transcription initiation. Previous work had suggested that the process goes something like this:

Top image via Wikimedia Commons

Basically, an alphabet soup of general transcription factors and pol II sit down on a promoter. This complex then pries open the DNA and looks for a signal in the DNA to start transcribing. The polymerase then transcribes short transcripts until it shifts into high gear when it escapes the promoter and enters elongation phase.

This theory comes from the study of transcription in bulk. In other words, it derives from looking at many cells all at once or many promoter fragments in a test tube.

Fazal and coworkers set out to look at how well this all holds up when looking at single genes, one at a time. To do this they used a powerful technique called optical tweezers.

Optical tweezers can “see” what is going on with moving enzymes by measuring the change in force that happens when they move. For this study, the preinitiation complex bound to a longish (2.7 kb) piece of DNA was attached to one bead via pol II, the moving enzyme. The other end of the DNA was attached to a second bead. Each bead is then immobilized using lasers (how cool is that!) and the DNA is stretched between the two beads. Watch this video if you want more details on the technique.

Depending on where you attach the DNA to the bead, you can either track polymerase movement or changes in DNA by precisely measuring changes in the forces keeping the beads in place. Using this technique the researchers found that the bulk studies had done pretty well for most every step. Except for the initial transcribing complex.

The earlier studies had suggested that an open complex of around 15 nucleotides was maintained until elongation began. This study showed that in addition to the 15 base pairs, an additional 32 to 140 base pairs (mean of about 70 base pairs) was also opened before productive elongation could begin. And that this whole region was transcribed.

This result paints a very different picture of transcription initiation. Rather than maintaining a constant amount of open DNA, it looks like the DNA opens more and more until the open DNA collapses back down to the 12-14 base pair transcription bubble seen during elongation.

It turns out that this is consistent with some previous work done in both yeast and fruit flies. Using KMnO₄, a probe for single stranded DNA, scientists had seen extended regions of open DNA around transcription start sites but had interpreted it as a collection of smaller, opened DNA. In other words, they thought they were seeing different polymerases at different positions along the DNA.

These new results suggest that they may have actually been seeing initial transcribing complexes poised to start processive elongation. Seeing just one complex at a time changed how we interpreted these results.

Fazal and coworkers were also able to see what happened to some of the 98% of preinitiation complexes that failed to get started. Around 20% of them did end up with an extensive region of open DNA of around 94 +/- 36 base pairs but these complexes were independent of transcription, as they didn’t require NTPs.

But since this opening did require dATP, they propose that it was due to the general transcription factor TFIIH, a helicase. It looks like in these failed complexes, TFIIH is opening the DNA without the polymerase being present.

A clearer picture of what might be going on at the promoter of genes starts to emerge from these studies. Once around 15 base pairs of DNA are pried open to form the appropriately named open complex, TFIIH unwinds an additional 70 or so base pairs. The polymerase comes along, transcribing this entire region. The whole 85 or so base pairs stays open during this process.

Eventually the polymerase breaks free and the opened DNA collapses back down to around 12-14 base pairs. Now the polymerase can merrily elongate to its heart’s content. Until of course something happens and it stops…but that is another story. 

Recent network issues resolved

September 15, 2015

The SGD website was experiencing some network issues over the past few days. You may have noticed that some pages were sluggish to load, while others were completely unavailable. We apologize for this inconvenience, and appreciate your patience while we performed several rounds of network optimization. All seems to be fine now, but please let us know if you notice anything unusual. We are very happy to be back!

New SGD Help Video: What is GO?

September 14, 2015

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The Gene Ontology (GO) is an integral part of modern biology. It provides a common language that unifies the description of gene products from all organisms, structured in a way that allows very detailed information to be captured while at the same time facilitating broad categorizations. 

Watch our new video for a brief refresher course on GO: what it is, how it’s structured, and why it’s important.