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With cryo-EM, scientists determine structure of key factor in RNA quality control

Met cryo-EM bepalen wetenschappers de structuur van de sleutelfactor in RNA-kwaliteitscontroleCell (2022). DOI: 10.116/j.cell.2022.04.016″ width=”800″ height=”530″/>

Graphic abstract. Credit: Cell (2022). DOI: 10.1016/j.cell.2022.04.016

In biology, getting rid of stuff can be just as important as creating it. A buildup of cells, proteins, or other molecules that are no longer needed can cause problems, so living things have developed several ways to clean the house.

A good example is the RNA exosome. RNA molecules perform many roles in cells. Some of them are translated into proteins; others make up a cell’s protein-building machinery. The RNA exosome is a cellular machine that breaks down RNA molecules that are defective, harmful, or no longer needed. Without this microscopic Marie Kondo to prune what brings no joy, our cells would become dysfunctional hoarders, unable to function.

“RNA surveillance and degradation pathways exist in all forms of life,” explains Christopher Lima, chair of the Structural Biology Program at the Sloan Kettering Institute. “From bacteria to humans, all living things have mechanisms to control the quality of RNA and break it down on purpose.”

For a long time, says Dr. Lima, these trails, like housework, were considered boring. But it turns out that these degradation pathways are highly regulated, controlling everything from embryonic development to cell cycle progression.

In addition, errors in these pathways can lead to many types of diseases, from cancer to neurodegeneration.

In a new article published on June 9, 2022 in Cell, present Dr. Lima and M. Rhyan Puno, a postdoctoral researcher in the Lima lab, report findings that help explain how the RNA exosome locates the RNA to be broken down. Using cryogenic electron microscopy (cryo-EM), an advanced type of imaging technology, the scientists were able to decipher the structure of a protein assembly called Nuclear Exosome Targeting (NEXT) Complex, which is an important part of the breakdown machinery.

“We knew that NEXT targets and delivers RNA to the exosome, but biochemically and structurally we had no idea what it looks like or how it works,” says Dr. puno.

Now, using cryo-EM, the scientists have obtained the first clear pictures of NEXT bound to RNA. These pictures, coupled with associated biochemical and biological experiments, offer hints about how RNA molecules are transferred to the exosome for destruction.

Crawling closer to a structure

Several years ago, Dr. Puno to study the structure of NEXT using the then gold standard approach to X-ray crystallography. In this method, proteins are first made into crystals, with the proteins all aligned in 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 could crystallize the NEXT protein, the resulting X-ray diffraction pictures were not good enough to see 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.”

Visualizing proteins in motion

Cryo-EM works by capturing many different images of a frozen but non-crystallized sample of a protein and then using computer methods to align them into a final sharp image.

“It’s almost like taking a lot of pictures of a bird in flight,” says Dr. Lima. “There are all kinds of confusing movements and the bird’s wings can appear blurry. But if we can find parts of the wing in all those different photos, then we can align the photos to reconstruct what the bird’s wings look like and define how they work.”

From the cryo-EM photos, the scientists were able to see that the NEXT proteins form a very flexible dimer — meaning two copies of NEXT proteins come together as a functional unit.

“That was really puzzling,” says Dr. Puno, and he notes that dimer formation has not been visualized for these types of proteins before.

“From biochemical experiments we’ve performed, we know that dimerization is somehow important for degradation,” he continues. “But it’s still a mystery to us what role the dimer plays in directing RNA to the exosome.”

To help solve the mystery, they hope to capture the NEXT complex interacting at different steps in the degradation process and then visualize these conformations with cryo-EM.

RNA degradation and disease

Great interests are at stake. An indication of how important RNA degradation is comes from the long list of diseases that result from defective or poorly controlled degradation. Perhaps the best-known example is cystic fibrosis. In this case, the messenger RNA encoding a protein that transports ions across cell membranes is broken down by RNA decay pathways. As a result, the protein is not present in the mucous membranes of the lungs, leading to a buildup of mucus there and resulting in severely impaired breathing.

“It’s a famous example of RNA quality control with poor results,” says Dr. Lima.

But defects in RNA degradation pathways also play a role in several cancers. Two of the genetic mutations that MSK’s genetic testing platform, MSK-IMPACT, tests for are even found in genes related to the RNA exosome pathway, including a protein in NEXT.

And it’s not just messenger RNA that needs good quality control, explains Dr. Lima out.

“The reality is if you have faulty RNA quality control pathways, your ribosomes don’t work, your transfer RNAs don’t work, your spliceosomes don’t work.” The list goes on.

The wide range of functions RNA performs explains why defective RNA degradation pathways can have such escalating disease-causing effects.

Understanding these effects requires a deeper and more comprehensive understanding of not only the RNA exosome itself, but also the “upstream” proteins, such as NEXT, that help monitor RNA and decide when an RNA is defective or no longer available. is needed.

“The dream is to start the RNA degradation reaction, place the sample in the cryo-EM and see all the possible confirmations as it does its job,” says Dr. Lima. “As structural biologists, we want to be able to see processes in action and then put them back together.”

Researchers prove that protein synthesis and mRNA degradation are structurally linked

More information:
M. Rhyan Puno et al, Structural basis for RNA surveillance by the human nuclear exosome targeting (NEXT) complex, Cell (2022). DOI: 10.1016/j.cell.2022.04.016

Journal information:

Provided by Memorial Sloan Kettering Cancer Center

Quote: Using cryo-EM, scientists determine the structure of the key factor in RNA quality control (2022, 9 June), retrieved 10 June 2022 from https://phys.org/news/2022-06-cryo-em-scientists-key -factor -rna.html

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