Ribosomes produce proteins, yet they are also made up of proteins. Each ribosome is a large complex consisting of approximately 80 different proteins and several RNA molecules encoded by ribosomal DNA. The imaging tool, named GapR-GFP, consists of green fluorescent protein (GFP) fused to a protein that binds to DNA where genes are highly active, particularly those within ribosomal DNA during ribosome production.

Previous studies have examined how changes in ribosome generation correspond to the appearance and structure of the nucleolus, the specialized compartment of the cell nucleus where ribosomes are assembled. Some changes, such as enlargement of the nucleolus, have been identified as predictive indicators for human cancers, which require increased protein production and thus more ribosomes to grow and divide.

Portions of chromosomes containing ribosomal DNA are responsible for creating the nucleolar compartment. These regions contain multiple copies of the genes encoding the RNA components of ribosomes to help meet the cell’s demand. The researchers wanted to determine how the spatial organization of ribosomal DNA is related to its function and to nucleolar structure, a question previously limited by the lack of methods to visualize ribosomal DNA organization.

After introducing GapR-GFP into fission yeast, the researchers conducted proof-of-principle experiments by activating or inhibiting ribosome generation. They found that activation led to visual expansion of both ribosomal DNA and the nucleolar compartment, whereas inhibition resulted in ribosomal DNA condensing and retracting from the nucleolus and compaction of the nucleolus.

“Using GapR-GFP, we saw that ribosomal DNA arrays show visual changes based on how many ribosomes the cell needs to make,” said Cockrell.

Next, the researchers introduced GapR-GFP into hundreds of yeast strains having unique genetic mutations. A group of the mutants that showed increased ribosomal DNA size compared to control cells were missing genes for building the ribosome complex.

Further genetic experiments showed that in these ribosomal protein mutants, different genes are activated, indicating a response by the cells to compensate for the reduction of ribosome proteins. Identifying the genes involved in the compensation response gives researchers new modulators to explore in the context of ribosomal DNA regulation and ribosome production.

“Our work presents the first broad investigation for regulators of ribosomal DNA spatial organization in any organism,” said Cockrell. “Importantly, we have shown that when cells have difficulty producing ribosomes, they may attempt to compensate by using alternative tactics.”

“Our findings have potential implications for human health,” said Gerton. “Ribosomal protein insufficiency occurs in several human disease contexts. A compensation response could contribute to disease processes in cancer and developmental disorders known as ribosomopathies where ribosomal protein insufficiency occurs.”

Additional authors include Jeffrey J. Lange, Ph.D., Christopher Wood, Ph.D., Mark Mattingly, Scott M. McCroskey, William D. Bradford, Juliana Conkright-Fincham, Ph.D., and Lauren Weems from the Stowers Institute, and Monica S. Guo, Ph.D., from the University of Washington School of Medicine, Seattle.

This research was funded by a training grant from National Institutes of Health (NIH) (award: F30GM140716 to AJC), a grant from the National Institute of General Medical Sciences of the NIH (award: R00GM134153 to MSG), and institutional support from the Stowers Institute for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.