Cryo-EM Helps Visualize Key Cellular Housekeeping Machine

Researchers at New York’s Memorial Sloan Kettering Cancer Center have used cryogenic-electron microscopy (Cryo-EM) to determine the structure of a protein assembly vital to RNA quality control—a process that, if faulty, can lead to multiple serious diseases.

Without RNA exosome—a microscopic cellular “housekeeper” that degrades RNA molecules that are faulty, harmful, or no longer needed—cells would become dysfunctional hoarders unable to function, according to the study authors. “RNA surveillance and degradation pathways exist in all forms of life,” says Christopher Lima, Chair of the Structural Biology Program in the Sloan Kettering Institute. “From bacteria to humans, all living things have mechanisms to monitor the quality of RNA and to purposely degrade it.”

For a long time, these pathways were overlooked. But it turns out that these degradation pathways are highly regulated and control everything from embryonic development to the progression of the cell cycle. Errors in these pathways can also lead to many types of disease, from cancer to neurodegeneration.

In a paper published recently in Cell, Lima and postdoctoral fellow M. Rhyan Puno, report how the RNA exosome locates the RNA that needs to be degraded. Using cryo-EM, an advanced type of imaging technology, they were able to decipher the structure of a protein assembly called Nuclear Exosome Targeting (NEXT) Complex, which is a key part of the degradation machinery. “We knew that NEXT targets and delivers RNA to the exosome, but biochemically and structurally, we didn’t have a clue what it looks like or how it works,” Puno says.

Cryo-EM also yielded the first clear pictures of NEXT bound to RNA. These pictures, in conjunction with accompanying biochemical and biological experiments, offer hints about how RNA molecules are handed over to the exosome for destruction. Lima and Puno were able to see that the NEXT proteins form a very flexible dimer—meaning that two copies of NEXT proteins join together as a functional unit.

“That was really, really puzzling,” Puno says, noting that dimer formation hasn’t been visualized before for these types of proteins. “From biochemical experiments we performed, we know that dimerization is somehow important for degradation,” he says. “But it’s still a mystery to us what role the dimer plays in guiding RNA to the exosome.”

In future work, they hope to capture the NEXT complex interacting at different steps in the degradation process and then visualize these conformations with cryo-EM.

“The dream is to initiate the RNA degradation reaction, put the sample in the cryo-EM, and actually see all the possible confirmations as it’s doing its job,” Lima says. “As structural biologists, we want to be able to see processes in action and then be able to reassemble them.”