July 13, 2016
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.
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