New & Noteworthy

RNase P, Unmasked

August 28, 2014

Masks for a masquerade party come in a dazzling array of shapes and sizes.  And yet they all pretty much serve the same purpose — they hide the identity of the wearer.

Biology sometimes has its own dazzling array of cellular machines all doing the same thing.  One of the best examples of this is RNase P.   This enzyme trims tRNA precursors into mature tRNAs and has pretty much been around in one form or another since there were cells.  And yet, despite this common heritage and its one apparent job, it seems that no two are exactly alike.

In bacteria, RNase P is a piece of RNA that serves as the enzymatic component, complexed with a single protein.  Most Archaea and eukaryotes kept the RNA and added a varying number of protein subunits to make some wildly complex enzymes.  But in a few eukaryotes, the RNA has been dropped completely and a single protein substituted to provide the enzymatic activity. 

A new study out in PLOS Biology by Weber and coworkers shows that, despite this structural diversity, all the different forms of RNase P pretty much do the same thing.  Just like someone can hide who they are with any old mask, a cell can trim its tRNA precursors with any old RNase P.  Well, at least the simple RNase P of Arabidopsis thaliana, comprised of a single enzymatic protein subunit, can replace the enzymatic RNA and at least one protein subunit from the much more complex RNase P of our friend Saccharomyces cerevisiae!

This suggests that evolution has done something weird here.  It took what most likely started out as an RNA enzyme and made various changes to it over time.  Despite these changes, the enzyme kept doing the same thing: trimming tRNA precursors.  It is as if the enzyme went through a bewildering set of evolutionary changes and ended up at nearly the same place doing the same thing.   

How did Weber and coworkers arrive at this startling finding? Yeast RNase P consists of nine protein subunits and an RNA component that comes from the RPR1 gene.  The first thing Weber and coworkers did was to show that the lethal phenotype of a rpr1 knockout could be rescued by the single-subunit RNase P from either the plant Arabidopsis thaliana or the trypanosome Trypanosoma brucei.  The RNase P in these beasts consists of only a single polypeptide.

The authors next integrated the RNase P gene of A. thaliana into the genome of a yeast cell lacking both RPR1 and one of the protein subunits of RNase P, Rpr2p, and put it through a set of rigorous tests.  To their surprise, they found that this strain does a perfectly fine job of processing tRNA precursors.  There was no buildup of intermediates and, if anything, the A. thaliana RNase P proved to be a bit more efficient at trimming these tRNA precursors.

Of course just because the simpler RNase P can substitute for the RNA subunit of the more complex RNase P, that does not mean the two do the exact same thing.  It could be that the more complex form of RNase P has a broader set of functions, but that the only function absolutely required for life is the trimming of tRNA precursors.  But this does not appear to be the case.

Previous research showing that unprocessed forms of other RNAs accumulate at the restrictive temperature in an rpr1-ts mutant had suggested that yeast RNase P also processes a number of other RNAs besides tRNAs.  Since Weber and coworkers didn’t see these unprocessed forms accumulating in their strain, either the simple A. thaliana RNase P was able to process those other RNAs, or they’re actually not RNase P substrates. 

By analyzing the phenotypes of several different RNase P mutants, they showed that the other RNAs aren’t RNase P substrates; apparently their accumulation in the rpr1-ts mutant is an indirect effect.  All in all, these results show that the added complexity of yeast RNase P did not arise so that the enzyme could also process these other RNAs.

The authors next set out to see if there was any subtle difference between the two strains.  In other words, does replacing the RNA component of yeast RNase P with the catalytic protein subunit from A. thaliana have any effect on the yeast whatsoever? 

Weber and coworkers tested this by comparing the growth of the two strains under a wide range of conditions.  They saw no significant effects in any of the over 30 conditions tested.  If the yeast RNase P has any added features over the A. thaliana one, they are very, very subtle.

Pushing to see if they could find any differences, they even set the two up in direct competition to see which was the best suited for survival.  They did this by adding GFP to one or the other strain so that they could follow it, putting the two strains together, and growing them for many generations to see if one routinely outcompeted the other.  Neither did…it was a draw.  There appears to be no advantage to having the yeast RNase P despite its complexity!

This is weird.  It is almost like round trip evolution.  RNase P starts out as a single RNA that processes tRNA precursors.  Then as it moves around the tree of life, it picks up various bells and whistles and occasionally is even replaced by a protein.  And yet in the end, all RNase P’s are strangely equivalent.  As if all of that evolving was for naught!

Obviously there are still plenty of unanswered questions.  Why did yeast build up this complexity if there is seemingly no advantage?  And is the protein subunit superior to the RNA subunit?  If so, this last question would at least explain why a few beasts evolved away from the RNA catalytic subunit to the protein one – but still wouldn’t answer why all those proteins are glomming onto the perfectly adequate RNA that probably predates proteins.  More studies in yeast may help us “unmask” the answer to this fundamental question.

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

New Sequence, Chromosome, and Contig pages

August 25, 2014

New Sequence pages are now available in SGD for virtually every yeast gene (e.g., HMRA1 Sequence page), and include genomic sequence annotations for the Reference Strain S288C, as well as several Alternative Reference Genomes from strains such as CEN.PK, RM11-1a, Sigma1278b, and W303 (more Alternative References coming soon). Each page includes an Overview section containing descriptive information, maps depicting genomic context in Reference Strain S288C (as shown below) and Alternative Reference strains, as well as chromosomal and relative coordinates in S288C.

The sequence itself includes display options for genomic DNA, coding DNA, or translated protein.

Also available on each Sequence page are links to redesigned S288C Chromosome pages, links to new Contig pages for Alternative Reference Genomes, and a Downloads menu for easy access to DNA sequences of several other industrial strains and environmental isolates. The new Sequence, Chromosome, and Contig pages make use of many of the features you enjoy on other new or redesigned pages at SGD, including graphical display of data, sortable tables, and responsive visualizations. The Sequence pages also provide seamless access to other tools at SGD such as BLAST and Web Primer. Please explore these new pages, accessible via the Sequence tab on your favorite Locus Summary page, and send us your feedback.

Special Delivery for Cytotoxic Proteins

August 21, 2014

Say you want to send a letter to your friend on the other side of the country. First off you’ll need to put the right address and postage on the envelope. Then you’ll need the U.S. Postal Service (USPS) to take your letter and deliver it to the right person. The stamp tells the USPS to deliver the letter, and the address indicates where it should be delivered (unimpeded by snow nor rain nor heat nor gloom of night, of course!).

It turns out something similar happens in human cells with aggregated proteins. Aggregated proteins are “stamped” by attachment of the small protein ubiquitin and “addressed” to the Atg8 protein. Atg8p triggers the aggregated proteins’ incorporation into autophagosomes for eventual degradation in the lysosome.

And just as it can be devastating if your mail doesn’t get to where it needs to go, so too can it be devastating for these aggregates to accumulate instead of being properly delivered. A buildup of these aggregates is a big factor in Alzheimer’s and Huntington’s diseases.

Enter the cellular USPS. Just as is the case for a prepared letter, the human cell has a service that delivers the ubiquinated proteins to the autophagosome, in the form of the protein adaptor p62 (SQSTM1) and its relative, NBR1.

These adaptor proteins can act as a postal service because they recognize both the aggregated proteins’ stamp (ubiquitin) and their addressee (Atg8p). Specifically, they each possess an ubiquitin-conjugate binding domain (UBA) and an Atg8-interacting motif (AIM). The protein p62 in particular has been shown to associate with protein aggregates linked to neurodegenerative diseases like Huntington’s disease.

In a new paper published in Cell, Lu et al. asked whether there is a link between the ubiquitin and autophagy systems in yeast. If so, yeast might provide some clues about diseases like Huntington’s. Proteins stamped with ubiquitin are known to be addressed to the proteasome for degradation in yeast, but no link between ubiquitination and autophagy had previously been seen, even though many central components of autophagy were actually first described in yeast.

Indeed, the authors showed that cells specifically deficient in the autophagy pathway (atg8∆, atg1∆, or atg7∆), accumulated ubiquitin conjugates under autophagy-inducing conditions. This suggests that the ubiquitin and autophagy pathways are connected in yeast, as is the case for humans.

Next, the researchers looked to see if there is an adaptor in yeast analogous to p62 in humans. When they pulled down proteins that bind yeast Atg8p under starvation conditions, they found ubiquitin conjugates and, using mass spectrometry, further identified peptides from a few other proteins – one of which was Cue5p.

Could Cue5p, like p62 in humans, be the postal service that recognizes both stamped ubiquitin conjugates and the addressee Atg8p in yeast? Strikingly, Cue5p had both a CUE domain that binds ubiquitin and an Atg8p-interacting motif (AIM). The authors confirmed in vivo that Cue5p binds ubiquitin conjugates and Atg8p using these domains, particularly under starvation conditions. They also showed that it acts specifically at the stage of ubiquitin-conjugate recognition and on aggregated proteins, without affecting the process of autophagy itself.

Given that Cue5p functions similarly to p62 and p62 is known to associate with protein aggregates involved in neurodegenerative disease, Lu et al. were quick to look for Cue5p substrates. Analyzing ubiquitin-conjugated proteins that accumulated in cue5 mutant cells, they identified 24 different proteins. Although these 24 Cue5p substrates had diverse functions, the common thread was that many had a tendency to aggregate under certain conditions such as high temperature.

Could Cue5p then actually facilitate removal of cytotoxic protein aggregates in neurodegenerative diseases? Indeed, the authors showed that CUE5 helped clear cytotoxic variants of the human huntingtin protein (Htt-96Q) when it was expressed in yeast, and that Htt-96Q is ubiquitinated in yeast.

These experiments started with an observation in human cells that prompted discovery of an analogous system and adaptor protein in yeast. Now the authors turned the tables and used yeast to look for new adaptor proteins in human cells. Using bioinformatics, they identified a human CUE-domain protein, Tollip, which, although different in its domain organization from Cue5p, contains 2 AIM motifs.

To make a long story (and a lot of work!) short, they showed that Tollip binds both human Atg8p and ubiquitin conjugates and clears cytotoxic variants of huntingtin in human cells. Expressed in yeast, it similarly binds ubiquitin conjugates and Atg8p and suppresses the hypersensitivity of cue5∆ cells to the variant huntingtin protein Htt-96Q. So Tollip is a newly defined adaptor protein and functional homolog of Cue5p!

Letter carriers of one sort or another have been around for as long as human civilization has existed, from homing pigeons to FedEx. Now we know that for even longer, cells from yeast to human have been using similar ways to recognize stamped proteins and deliver them to the right address. And once again, yeast has helped us understand the inner secrets of human cells.

by Selina Dwight, Ph.D., Senior Biocurator, SGD

Pinpointing Peroxisomes

August 14, 2014

One way to think about the cell is that organelles float around in it like those globs in a lava lamp.  This is obviously a simplification, but it’s also true that organelles aren’t locked into place.  As usual, the real picture lies somewhere in between these two extremes.

What we know about the architecture of the cell has mostly been discovered using classical cell biology and genetic techniques. But in a paper published in Molecular BioSystems, Cohen et al. uncovered some very interesting small details using a very large-scale approach.

The authors were interested in peroxisomes, where a lot of critical metabolic reactions happen (or fail to happen, in several human diseases). The researchers were able to see that peroxisomes not only interact with other organelles, but they contact the endoplasmic reticulum (ER) and mitochondria in a way that could be extremely important for cellular metabolism. And surprisingly, it was by combining a variety of different high-throughput techniques that Cohen and colleagues could uncover this fine structure.

The first step was to set up two reporter constructs to look for genes involved in two different peroxisomal processes.

One reporter was a red fluorescent protein, mCherry, modified to carry a peroxisomal targeting signal and show whether import into peroxisomes was normal. Another reporter, a peroxisomal membrane protein (Ant1p) tagged with green fluorescent protein (GFP), would show whether peroxisomal membranes were normal.

The reporters were crossed into mutant collections, creating one strain for each gene in the genome that had either a complete deletion (for nonessential genes) or a knock-down allele (for essential genes), plus both reporters. Now the researchers could systematically test for genes that, when mutated, affected one or both of these aspects of peroxisomal biogenesis.

To visualize the mutant phenotypes, they used a sophisticated technique termed “high-content screening.” This is an automated way to analyze micrographs that both pinpoints the intracellular location of a fluorescent reporter and measures its quantity. Screening the mutant collection in this way showed that 56 strains had altered distribution of the two different reporter proteins.  Some had a reduction in peroxisomal protein import (mCherry fluorescence), while some had fewer or no peroxisomes and some had peroxisomes that were smaller than normal (GFP fluorescence).

One result that caught the researchers’ eyes was that one of the strains with smaller peroxisomes had a mutation in the MDM10 gene. Mdm10p is part of the ERMES (ER-Mitochondria Encounter Structure) complex that tethers mitochondria to the ER, and this wasn’t previously known to have any connection with peroxisomes. Strains that were mutant in other ERMES subunits had the same phenotype, confirming that the complex has something to do with peroxisome structure.  Other results from the screens added weight to the idea of a three-way connection between peroxisomes, the ER, and mitochondria, and the authors went on to show that peroxisomes often sit at the ERMES complex where mitochondria contact the ER.

Next, to test whether mitochondria might have specific subdomains where peroxisomes interact, the authors used yet another large-scale screen. In the C-terminal GFP fusion library, where each yeast open reading frame is C-terminally tagged with GFP, 96 strains showed a punctate pattern of the fluorescent signal – meaning that the protein was concentrated in spots, rather than evenly distributed.  They labeled the mitochondria with a red fluorescent marker protein in these strains and, again using the high-content screening system, identified protein spots that co-localized with mitochondria. The most intense hit was for Pda1p, a subunit of the mitochondrial enzyme pyruvate dehydrogenase (PDH), and a similar result was obtained for another PDH subunit. So PDH isn’t distributed uniformly in the mitochondrion, but is instead concentrated in clusters.

Looking more closely using the various reporter constructs in their collections, the authors found that peroxisomes and the ERMES complex most often co-localized with those mitochondrial globs of PDH. It would make metabolic sense for peroxisomes to hang out near PDH on mitochondria because this could increase the local concentration of metabolites that they both use.

Intriguingly, Cohen et al. also found that mitochondria and peroxisomes co-localized in mammalian cells. Given that many diseases are linked to peroxisomal metabolism, this is an important avenue to investigate.

So while organelles don’t float around in the cell quite as fluidly as the globs in a lava lamp, the data generated from large-scale approaches boiled down to learning some very fine-grained detail about cellular architecture. We think that’s, like, groovy.

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