Abstract: Aging is a critically important process that impacts all organisms, but the mechanistic basis is unclear. The enlargement of the nucleolus, whose primary function is to house the rDNA and ribosome biogenesis, correlates with aging, cancer, progeria, and neurodegeneration. Meanwhile, treatments known to extend lifespan such as calorie restriction or TOR inactivation correlate with a smaller nucleolus. It has been shown that a larger nucleolus is more prone to rDNA instability which triggers genomic instability. This evidence has produced the “nucleolar aging hypothesis”, where nucleolar size is predicted to regulate the aging process of the cell. However, whether an enlarged nucleolus is correlative or causative for aging is unclear.

To determine whether artificially maintaining a small nucleolus is sufficient for lifespan extension, we engineered an artificial nucleolar tethering system in budding yeast - the leading model system for studying replicative aging. We generated a deactivated Cas9 (dCas9) protein fused to the nuclear membrane protein Heh1p (dCas9-Heh1) together with multiple guide RNAs directed to the rDNA, which we predicted would maintain a smaller nucleolus during aging. Using our microfluidics imaging platform of aging, we found that induction of dCas9-Heh1 extends replicative lifespan in yeast by 25 to 30%, which is equivalent to the lifespan extension achieved by calorie restriction - the gold standard for aging. We found that this extension in lifespan is accompanied by maintenance of smaller nucleolar size during aging and reduced loss of silencing of the rDNA during aging, as determined by live microscopic analysis of rDNA reporter gene expression during aging. Finally, we tested the “nucleolar aging hypothesis” by measuring live reporters of DNA damage during aging upon artificial tethering of the nucleolus to the nuclear membrane and found it reduced the generation of DNA double-strand breaks at early times during aging. Enlargement of the nucleolus starts early in lifespan, indicating that it could be a predictor of ultimate lifespan. Our work shows that nucleolar enlargement is a driver of aging, occurring due to loss of anchoring of the rDNA to the nuclear membrane, providing mechanistic understanding to the aging process and adding new targets in the search for therapeutics to extend lifespan.