A single human cell can contain between 10 million to 10 billion ribosomes, a dense pool of molecular factories which drive nearly all cellular processes, sustaining life itself. Ribosomes are the site where protein synthesis happens. Messenger RNA (mRNA) slides into the ribosome’s core, to then be scanned, and let molecular adapters bring in the correct amino acids–protein building blocks–in a sequence dictated by mRNA. The ribosome then assembles full-length proteins by linking together each of the amino acids.
Ribosomes are initially built in the nucleolus–a specialised region within the nucleus–where ribosomal RNA (rRNA) is combined with several proteins to create a distinct two-part structure: a larger and a smaller subunit glued together. The small subunit binds to the mRNA, while the large subunit forms links between the amino acids. Once the subunits are complete, they exit the nucleus and finally unite in the cytoplasm, where they start the protein-building process.
Up to 60 percent of the cell’s total energy is dedicated to building ribosomes. Given the high energy and resources required to keep them active, the cell makes sure to tightly regulate the process, linking it to its needs of growth and proliferation. If the ribosome assembly line is compromised, so will be the overall health of the cell, potentially leading to disease.
While recent high-resolution studies have improved our understanding of ribosome assembly, there are still significant gaps. One major challenge is that the intermediate stages of this process are fleeting and difficult to capture, making it hard to fully understand how each step fits together. One approach to the problem is to stop the assembly process at selected stages using specific inhibitors. Researchers can then take snapshots of the immediate effects of these blocks to dissect the steps of ribosome biogenesis.
However, there are currently only a few known inhibitors of this kind, and scientists have been on a quest to find more molecules that can specifically disrupt ribosome production.
In 2019, researchers at the University of Graz led by Helmut Bergler evaluated more than a 1000 different molecules to identify potential inhibitors of ribosome biogenesis in a vast screening project. Among the most promising candidates they found the small molecule usnic acid, a natural compound derived from lichen, noted for its ability to potentially inhibit the maturation of eukaryotic ribosomes.
Recognising the potential of this compound, scientists from the IMP's Cryo Electron Microscopy Technology Platform led by David Haselbach, in collaboration with Bergler's team in Graz, set out to investigate its effects further. By combining biochemical data with advanced cryo-electron microscopy (cryo-EM) and artificial intelligence assisted data analysis, they now show where in the assembly process usnic acid impacts ribosome formation. The study expands the chemical toolbox of known inhibitors with potential applications in both basic and applied research. These findings are now published in the journal Nature Communications.
The scientists achieved this feat by establishing an innovative cryo-EM workflow, based on the algorithm cryoDRGN. This new software leverages neural networks and artificial intelligence to aid the analysis of cryo-EM images.
In cryo-EM, researchers capture thousands of 2D images of individual particles, which are then averaged to create detailed 3D reconstructions. CryoDRGN enhances this process by allowing them to compare and analyse multiple captures–or datasets–simultaneously, allowing the identification of the different structural states of particles in an unbiased and reproducible way.
With this tool, David Haselbach's team imaged the early stages of ribosome formation, starting with pre-ribosomes purified from yeast cells treated with usnic acid and untreated control cells. The data were then merged together and fed to CryoDRGN, and differences in the two datasets were automatically identified by the software.
This approach helped the team pinpoint where in the complex chain of events usnic acid disrupts ribosome assembly, providing valuable insights into its inhibitory mechanism.
“We could observe four sequential early stages of ribosome formation with this workflow, usnic acid blocking progression in the assembly line very early on,” explaines Lorenz Grundmann, co-first author and a student in the Vienna BioCenter PhD Program. “The inhibitor causes ribosomes to stall at the very beginning of the process, blocking any progression to later stages,” adds Grundmann.
“We now have a model of how usnic acid acts on early ribosome biogenesis, marking it as the first known inhibitor to target the nucleolar stage,” says David Haselbach. This discovery expands the toolbox of ribosome inhibitors and reveals more about usnic acid’s biological activity. Given its previously reported anti-tumour effects, this finding potentially opens new avenues for medical applications too. “Ribosome production is a highly conserved process from yeast and humans; the next steps will be to test it on human cells.”
Original publication
Lisa Kofler*, Lorenz Emanuel Grundmann*, Magdalena Gerhalter, Michael Prattes, Juliane Merl-Pham, Gertrude Zisser, Irina Grishkovskaya, Victor-Valentin Hodirnau, Martin Vareka, Rolf Breinbauer, Stefanie M. Hauck, David Haselbach# & Helmut Bergler# “The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation.” Nature Communications (2024). DOI: 10.1038/s41467-024-51754-3
*These authors contributed equally. #Corresponding authors.