Nucleoli disappear during mitosis but burst back into existence in daughter cells. Mechanisms governing this feat have been hypothesized, but remain unproven. Now, a team of scientists reporting in the journal Genes and Development have found that only two things are needed to make a new nucleolus: a protein called UBF and transcription of ribosomal DNA.
Following mitosis, new nucleoli form in Nucleolar Organizing Regions (NORs), which contain DNA encoding ribosomal RNA and so-called secondary constrictions, regions where the DNA remains under-condensed, appearing as an unstained gap on chromosomes during cell division.
Although secondary constrictions were first implicated in nucleolar formation by Emile Heitz and Barbara McClintock in the 1930s, no one knew how they formed or what role they played in regenerating nucleoli. Fast forward to today where Brian McStay at the National University of Ireland in Galway and his colleagues decided to explore the role a common rDNA binding protein called UBF might play in this process. They used RNAi to first knock out UBF in human cells and then watched as, during mitosis, the rDNA in these UBF-free cells condensed just like the rest of the DNA instead of forming a secondary constriction as would be seen in normal cells. The nucleolus did not form around this region either, proving that this so-called “mitotic bookmarking” by UBF is necessary for nucleolar formation.
The team next took their experiments one step further -- creating a synthetic nucleolus by inserting several UBF-binding site repeats and rDNA into chromosomes. The inserts caused a secondary constriction identical to those seen in normal NORs and recruited factors that allowed transcription of the rDNA genes by Pol I. In addition, the synthetic nucleoli produced mature rRNAs that assembled with ribosomal proteins to make functional ribosomes. Regardless of their location on the chromosome, these inserts created functional nucleoli.
The researchers determined that as long as UBF is present on the rDNA, the rDNA is ready for transcription and the nucleolus quickly re-forms after mitosis ends. “You don’t have to unwrap the genes from chromatin, associate the transcription factors, go through all those chromatin hoops to reactivate transcription. It’s just like flipping a switch,” explained McStay.
McStay’s studies could have important clinical applications as one of the standard assays for cancer proliferation is staining with an antibody against a nucleolar protein called KI67.
“It’s been known for well over 100 years that the nucleolus is dramatically altered in cancer,” he said, noting how nucleoli become enlarged and irregular in shape, reflecting upregulated ribosome biogenesis.
“There’s an increasingly popular view that the nucleolus is a good target for anticancer drugs,” he added. “So it’s against that background that I think these basic questions need to be addressed.”
Grob A, Colleran C, McStay B. Construction of synthetic nucleoli in human cells reveals how a major functional nuclear domain is formed and propagated through cell division. Genes Dev. 2014 Feb 1;28(3):220-30.