New & Noteworthy
Sometimes You Need Half a Trillion
June 01, 2016
Image courtesy of The Internet Speculative Fiction Database.
In the Foundation series by Isaac Asimov, Hari Seldon invented a field of study called psychohistory which was able to “make general predictions” about human history. It only worked because the population of the Galactic Empire was in the quadrillions. You need huge numbers to get useful predictions.
Sometimes real life biologists need lots of individuals to see what they are looking for too. In the case of a recent paper in PNAS by Lee and Stevens, they needed to sift through half a trillion yeast to find what they were looking for. And boy was it worth it!
They saw an intron jump from one gene to another. Twice. In 500 billion tries.
This is the first example where we have caught an intron in the act of moving from one place to another. This has important implications for the study of evolution where over time introns spread or recede. It might even help us better understand cancer where intron loss can play a role.
Seeing something as rare as this means making a very specific, complicated reporter. You don’t want to manually sort those 500 billion colonies…or at least I wouldn’t want to.
Here is a “simplified” schematic of the reporter they came up with:
It is as complicated as it looks but it got the job done! Let me walk you through what everything is and how it works.
The GU and AG sequences are splice site junctions. Sequences between a GU and an AG can be spliced out.
The red gene is the S. pombe his5+ gene that has a promoter driving its expression shown with the red arrow. It is in the reverse orientation compared to the eGFP gene which is under the control of the promoter represented by the blue arrow.
The S. pombe his5+ gene works fine in S. cerevisiae his3 mutants to make up for histidine auxotrophy. But it won’t work from this construct.
See, the his5+ gene has an intron that keeps it from being translated correctly unless the intron is spliced out. But this intron can’t be spliced out when the gene is transcribed using the red promoter because the splice junctions GU2 and AG1 are in the wrong orientation. Any transcripts from the red promoter will not work because the intron cannot be spliced out.
You also can’t get His5 from the blue promoter. Even with splicing (which will work in that orientation), the gene can’t be translated because it is in the wrong orientation.
To get any his5+ transcript, the yeast needs to take the spliced RNA from between GU1 and AG2 and get it into DNA. It also needs to get rid of the intron in the middle of his5+ gene before it happens.
So what they are looking for are yeast that glow green and can survive in the absence of histidine. There are a couple of ways this can happen.
The more common way results in the his5+ gene recombining back into the plasmid such that the intron is missing from its middle. Basically the RNA between GU2 and AG1 is spliced out and the resulting RNA is reverse transcribed into DNA. This DNA then undergoes homologous recombination with the plasmid DNA resulting in a working his5+ gene. They got over 10,000 of these in their experiment.
The much less common way involves the intron being inserted into a gene within the genome. This way uses some of the same steps with one extra one—a reverse splicing event.
As I said, they got two of these with one ending up in the RPL8B gene and the other in the ADH2 gene.
Here’s how they think this happened…
The spliced out RNA from the plasmid was left for a brief time in the spliceosome (or what remained of it after the splicing reaction). During this time, a second mRNA arrived for splicing while the intron from the reporter was still there.
Then something called reverse splicing happened which basically replaced the native intron of the RPL8B or the ADH2 gene with the spliced out intron from the reporter. Next this was turned into DNA with reverse transcriptases and then this construct ended up in the genome through homologous recombination.
Only with yeast can we sift through 500,000,000 cells to find the two introns that have moved to new genes. Image from Needle in a Haystack, on Tumblr.
No wonder this was so rare! Reverse splicing is thought to be really uncommon in vivo as is reverse transcription of DNA. Add in a loitering intron on the remnants of a spliceosome and you can see why this was a 1 in 250 billion shot.
So there you have it, one way a transposon can hop. It is no transposon, but occasionally an intron can move to a new gene.
And of course we turned to the awesome power of yeast genetics to help us figure this out. Only with yeast can we sift through half a trillion cells to find the two that show us how intronogenesis, the introduction of an intron to a new site, might happen. #APOYG!
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: mobile intron, reverse splicing