In biology, getting rid of things can be just as important as making them happen. An accumulation of cells, proteins or other molecules that are no longer needed can cause problems, so living things have evolved in different ways to clean the house.
A great example is the RNA exosome. RNA molecules play many roles in cells. Some of them are translated into proteins; others form the protein-making machinery of a cell. RNA exosome is a cellular machine that degrades RNA molecules that are defective, harmful, or no longer needed. Without this microscopic Marie Kondo to prune what does not arouse joy, our cells would become dysfunctional accumulators, unable to function.
“RNA surveillance and degradation pathways exist in all forms of life,” says Christopher Lima, president of the structural biology program at the Sloan Kettering Institute. “From bacteria to humans, all living things have mechanisms to monitor the quality of RNA and degrade it on purpose.”
For a long time, says Dr. Lima, these paths were considered, like housework, to be a bit boring. But it turns out that these degradation pathways are highly regulated and control everything from embryonic development to cell cycle progression.
Furthermore, errors in these pathways can lead to many types of diseases, from cancer to neurodegeneration.
In a new article published on 9 June 2022 a Cell, Dr. Lima and M. Rhyan Puno, a postdoctoral fellow in the Lima laboratory, present results that help explain how the RNA exosome locates the RNA that needs to be degraded. With the help of cryogenic electron microscopy (cryo-EM), an advanced type of imaging technology, scientists were able to decipher the structure of a protein assembly called the Nuclear Exosome Targeting (NEXT) complex, which is a key part of machinery degradation.
“We knew that NEXT targets and delivers RNA to the exosome, but biochemically and structurally we had no idea what it was like or how it works,” says Dr Puno.
Now, with cryo-EM, scientists have obtained the first clear images of NEXT bound to RNA. These images, along with the accompanying biochemical and biological experiments, offer hints on how RNA molecules are delivered to the exosome for destruction.
Closer and closer to a structure
Several years ago, Dr. Puno began studying the structure of NEXT using the then gold standard approach of X-ray crystallography. In this method, the proteins are first transformed into crystals, with the proteins all aligned with the same way. Then, X-rays are passed through the crystals and the pattern of X-rays hitting a detector can be interpreted to determine the structure of the protein.
Although Dr. Puno was able to crystallize the NEXT protein, the resulting X-ray diffraction images were not good enough to see the details of the structure.
“But then came the cryo-EM revolution,” he says. “Cryo-EM helped us visualize what this protein looks like and how it binds to its RNA substrates.”
Visualization of proteins in motion
Cryo-EM works by capturing many different images of a frozen but uncrystallized sample of a protein and then using computational methods to align them into a sharp, finalized image.
“It’s almost like capturing a bunch of photos of a bird in flight,” says Dr. Lima. “There are all kinds of fuzzy movements and the bird’s wings can look blurry. But if we can find parts of the wing in all these different images, then we can line up the images to reconstruct the look of the bird’s wings and determine how it operates. “
From the cryo-EM images, the scientists were able to see that the NEXT proteins form a very flexible dimer, which means that two copies of the NEXT proteins come together as a functional unit.
“It was really, really baffling,” says Dr. Puno, noting that dimer formation hasn’t been shown before for these types of proteins.
“From the biochemical experiments we performed, we know that dimerization is somehow important for degradation,” he continues. “But it is still a mystery to us what role the dimer plays in driving RNA to the exosome.”
To help solve the mystery, they hope to capture the NEXT complex interacting at different stages of the degradation process and then visualize these conformations with cryo-EM.
RNA degradation and disease
There are big stakes. An indication of the importance of RNA degradation comes from the long list of diseases that result from defective or poorly controlled degradation. Perhaps the most famous example is cystic fibrosis. In this case, the messenger RNA that codes for a protein that carries ions across cell membranes is degraded by the RNA decay pathways. As a result, the protein is not present in the mucous membranes of the lungs, which leads to a buildup of mucus there and causes severely impaired breathing.
“It’s a famous example of RNA quality control with negative results,” says Dr. File.
But defects in RNA degradation pathways also play a role in several types of cancer. In fact, two of the genetic mutations tested by MSK’s genetic testing platform, MSK-IMPACT®, are found in genes related to the RNA exosome pathway, including a protein in NEXT.
And it’s not just messenger RNA that needs proper quality control, explains Dr. Lima.
“The reality is that if you have faulty RNA quality control pathways, your ribosomes aren’t working, your transfer RNAs aren’t working, your spliceosomes aren’t working.” The list could go on and on.
The breadth of functions performed by RNA explains why faulty RNA degradation pathways can have such disease-causing cascade effects.
Making sense of these effects will require a deeper and broader understanding of not only the exosome of the RNA itself, but also of the “upstream” proteins, such as NEXT, which help monitor RNA and decide when an RNA it is defective or no longer needed.
‘The dream is to initiate the RNA degradation reaction, insert the sample into the cryo-EM and actually see all possible confirmations while it is doing its job,’ says Dr Lima. “As structural biologists, we want to be able to see processes in action and then be able to reassemble them.”