The RNA transcripts of virtually all human genes must be processed by one cellular machine: the spliceosome. The mind-boggling array of proteins and RNAs that make up the spliceosome allow it to control how genes are expressed. Many researchers are interested in the dynamic interplay between these components of the spliceosome and how they evolved, likely from a parasitic RNA that invaded eukaryotic cells at around the same time eukaryotic and bacterial cells became distinct from one another approximately two billion years ago.
The spliceosome is also a reason that complex organisms like animals and humans can exist. Through splicing, the cell is able to get several different transcripts out of just one gene, meaning a more diverse array of proteins can be made from a relatively small amount of DNA. This creates evolutionary diversity much more rapidly than other mechanisms, allowing complex organisms to evolve more quickly.
Researchers chip away at the spliceosome and its dynamic components by several approaches. One approach, facilitated by recent advances in cryo-electron microscopy (cryo-EM), involves determining three-dimensional structures of fully assembled spliceosomes. Another approach involves studying smaller sub-complexes, which allows more detailed views of protein-RNA interactions and also provides tractable in vitro systems for testing specific hypotheses about function.
The laboratories of biochemistry professor Sam Butcher and biomolecular chemistry professor David Brow at the University of Wisconsin–Madison recently took the latter approach to hone in on a protein called Prp24 and a seven-protein ring called the Lsm2-8, both of which recruit a critical RNA called U6 into spliceosomes. In their newest study published in Nature Communications on May 1, 2018 they reported for the first time the structure of these eight proteins interacting with the U6 RNA.
The structure solved by the researchers. It depicts a protein called Prp24
and a seven-protein ring called the Lsm2-8 interacting with the U6 RNA.
Photo courtesy of Eric Montemayor.
“This is an important piece of the puzzle for trying to understand how the spliceosome assembles,” Butcher says. “The spliceosome is a molecular machine with a lot of moving parts, and this particular part has never been seen before.”
One of their most interesting findings, they say, was that the structure revealed that the Prp24 protein and Lsm ring are much closer in three-dimensional space than previously thought.
“There were some interesting surprises here,” says Eric Montemayor, an associate scientist in the Butcher and Brow labs and first author on the study. “We knew that the ring helped Prp24 to pair U6 with another spliceosomal RNA called U4, but we did not know how. It turns out there are protein-protein contacts between Prp24 and Lsm2-8 that place the ring right on top of the ‘active site’ of Prp24. We had no idea this would be the case.”
They also found that the ring recognized the 3’ (three prime) end of U6 RNA with a very specific modification, which is conferred by an enzyme called Usb1. This exact type of modification was identified by Allison Didychuk (a recent biophysics Ph.D. graduate of UW–Madison) in a related study published in Nature Communications last summer.
Eric Montemayor, associate scientist in the labs of
biochemistry professor Samuel Butcher and
biomolecular chemistry professor David Brow. Photo
by Robin Davies.
Montemayor says knowing the exact modification was key to moving his study forward because they were not able to make stable samples for biochemical assays without that knowledge.
“The spliceosome was discovered 33 years ago and understanding its structure and mechanism has been an intriguing challenge,” Brow says. “This study brings us another step closer to that goal.”
After seeing the interaction between the ring and the modified end of U6, the researchers tested the biological implications of “breaking” that interface and found that several targeted mutations were lethal in vivo.
“This means that the ring and Usb1 and U6 are all co-evolved to work together,” Montemayor explains. “You can’t change one without changing the other. We know this because humans and yeast have different Usb1 enzymes that leave different modifications, and the corresponding rings have tailored affinities for the different modifications.”
Montemayor says the spliceosome has always interested him, especially from an evolutionary biology perspective. He explains that the leading theory behind how the spliceosome originated involves parasitic RNAs — also called retroelements — that invade the genome and find a new home as introns nestled between two exons.
“Think about it, a few billion years ago a eukaryote was invaded by one of these things, and through co-evolution the ‘invader’ was retooled to work as a master regulator of human gene expression,” he says. “And this process is still in action. We see it in the complex between Prp24, Lsm2-8 and U6 and how the ring is recognizing this modification that is different between yeast and humans.”
Support for this research and data collection was provided by the Department of Energy, National Institutes of Health, and National Science Foundation.
Read more stories about spliceosome research taking place in the UW–Madison Department of Biochemistry: