New & Noteworthy
Keep Only What You Need
July 13, 2016
Like yeast with its introns, Aragorn kept only what he needed to try to rescue Merry and Pippin from Saruman’s orcs. Image from deviantart.net.
In the Lord of the Rings trilogy, the evacuees from Edoras are warned to take only what they need. Aragorn, Gimli and Legolas do the same thing when they chase down the orcs who kidnapped Merry and Pippin. And Sam and Frodo get rid of all nonessentials so they can make it to Mount Doom.
They all need to do this because if they are weighed down they won’t make it to their goal or maybe even die. If Sam and Frodo had kept all of their equipment, they would have died on the Plateau of Gorgoroth before reaching Mount Doom and saving Middle Earth.
In some ways, the hurly burly world of yeast is a bit like these characters in Middle Earth. If yeast cells are weighed down by slightly deleterious or even nonessential items, they will not survive. They will be outcompeted by their leaner, less burdened peers. The orcs would have made it to Isengard if Aragorn, Gimli, and Legolas had been slowed down by too much extra stuff.
One place where we can see this is with introns. Unlike many other eukaryotes, S. cerevisiae has gotten rid of almost all of its introns—only around 5% of its genes have them. This suggests that the ones that have stuck around are doing something important.
In a new study out in GENETICS, Hooks and coworkers explore the idea that at least some of the remaining introns have hung around because they play an important role as untranslated RNAs with specific secondary structures. In fact, they provide evidence that the secondary RNA structure of an intron in the GLC7 gene in critical for the cell’s ability to respond to salt stress.
The first step was to identify introns with a conserved secondary structure. They compared 36 fungal genomes using three different RNA structure prediction tools and found that all three programs were able to identify structures for 14 of the introns. They also found 3 introns that scored very well with at least two of the programs. With the exception of known snoRNAs, none of these matched any other noncoding RNAs.
Next, Hooks and coworkers used RT-PCR as well as re-analysis of deep sequencing data of total RNA to figure out which of these introns might actually be a real noncoding RNA. They found that six of the introns remained intact in the cell much longer than is typical for excised introns and that noncoding RNAs were further processed in two of them, an intron from GLC7 and one from RPL7B.
They set out to determine if the predicted secondary structure of the RNA of the intron in GLC7 really did anything important in the cell. GLC7 was a good choice as it has been previously reported that this intron is involved in a cell’s response to high salt. So if the structure is important, than if it is disrupted, the cell should not respond as well to high salt.
Like Gimli and his axe, or Sam (pictured here) and Frodo with the Phial of Galadriel, the yeast genome has kept only what it needs. Images from lapetitebrique.com.
They used a couple of different mutants to get at this question. The first mutant, the GLC7 ncRNA deletion mutant, simply deleted the predicted noncoding RNA from the intron. The second mutant, the GLC7 ncRNA insertion mutant, inserted 139 base pairs in the middle of the predicted noncoding sequence. The researchers found that neither responded as well to a high salt concentration, 0.9 M NaCl, as did a wild type or a negative control deletion that removed part of the intron that did not overlap with the predicted noncoding RNA sequence.
They also found that this loss in response could not be rescued with the noncoding RNA being expressed in trans from a separate, constitutive promoter. The secondary structure of this intron plays an important role in dealing with the stress of high salt in cis.
While deletion of the predicted noncoding RNA had little effect on GLC7 expression at low salt, the same was not true at higher salt. At 0.9 M NaCl, GLC7 mRNA levels were about half of that of the wild type or the negative control deletion mutant. It looks like under high salt conditions, this intron is important for getting enough GLC7 made to deal with the stress.
So, like Gimli’s axe or Sam’s water bottle, yeast has maintained this intron because it plays an important role in survival. It will be interesting to see why other introns have been maintained and if they too play their roles as noncoding RNAs.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: introns, ncRNA, RNA structure
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!
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
Categories: Research Spotlight
Tags: ncRNA, osmotic stress, Saccharomyces cerevisiae, translation