Aging is a universal process characterized by cellular and molecular changes, including genome instability. A striking feature of aging across species is the enlargement of the nucleolus, a change in the cellular structure due to ribosomal deoxyribonucleic acid (DNA) instability.
In a recent study published in Nature Aging, researchers from Weill Cornell Medicine investigated the role of nucleolar size in regulating the replicative lifespan of yeast cells.
Through artificial restriction of nucleolar enlargement, the researchers explored its impact on genomic integrity and longevity.
The findings revealed that surpassing a specific nucleolar size threshold disrupts cellular homeostasis, triggering catastrophic genome instability. The study also uncovered a novel "mortality timer" mechanism linked to nucleolar expansion, which offered insights into the aging process.
Study: A mortality timer based on nucleolar size triggers nucleolar integrity loss and catastrophic genomic instability. Image Credit: syarifahaa/Shutterstock.com
Background
Aging is a natural process that is accompanied by distinctive cellular changes that are conserved across species, from yeast to humans. One of the key hallmarks of aging is the enlargement of the nucleolus, which houses the ribosomal DNA that is essential for protein synthesis.
Small nucleoli are often associated with increased lifespan and reduced genomic instability, as observed in various organisms and anti-aging conditions such as during dietary restriction and rapamycin treatment.
Conversely, nucleolar enlargement also correlates with aging-related genome instability and diseases such as cancer. Despite this, the underlying molecular mechanisms linking nucleolar size to aging remain unclear.
In yeast, ribosomal DNA regions are prone to double-strand breaks that are usually repaired by homologous recombination.
Aging cells show increased ribosomal DNA instability, possibly due to disrupted nucleolar organization. However, a clear understanding of how nucleolar size influences genome stability and lifespan is lacking.
About the Study
The present study used the model organism Saccharomyces cerevisiae to investigate how nucleolar size influences aging and genomic stability. The researchers engineered a system to anchor ribosomal DNA to the nuclear membrane and reduce nucleolar enlargement during the replicative lifespan of the yeast.
This system involved expressing deactivated clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCAS9) fused to the inner nuclear membrane protein Heh1. This dCas9-Heh1 complex was guided to specific ribosomal DNA loci to limit nucleolar expansion artificially. Subsequently, fluorescence microscopy was used to track nucleolar size changes, and the replicative lifespan was measured using microfluidic chips to monitor mother cell divisions.
Additionally, the researchers analyzed single-cell trajectories to identify the Nucleolar Size Threshold or NST, which is the size at which nucleolar enlargement accelerates. Beyond this threshold, nucleolar integrity is believed to diminish.
Furthermore, confocal microscopy was employed to examine the co-localization of nucleolar and repair proteins, which would also indicate the infiltration of the DNA repair and recombination protein Radiation Sensitive 52 or Rad52 into the enlarged nucleoli. Treatments with 1,6-hexanediol were also used to examine nucleolar integrity.
To assess genome stability, the study also evaluated ribosomal DNA loss using a marker-based assay and measured DNA repair defects by tracking Rad52 foci. Additionally, Western blotting and protein synthesis assays examined the broader impacts on cellular functions.
Major Findings
The results showed that nucleolar size directly impacts replicative lifespan and genomic stability. Restricting nucleolar enlargement through artificial anchoring of ribosomal DNA significantly extended the lifespan of the yeast, with cells showing delayed nucleolar expansion and reduced instability.
Furthermore, the lifespan extension occurred independently of changes in protein synthesis or ribosomal DNA silencing, which indicated that nucleolar size was the critical factor.
Additionally, the study identified a defined NST, beyond which nucleolar enlargement progressed rapidly, triggering loss of nucleolar integrity. This event was also found to be associated with the entry of Rad52 into the nucleoli, which disrupts ribosomal DNA stability.
Furthermore, the cells that surpassed the NST exhibited increased ribosomal DNA loss, persistent Rad52 foci, and catastrophic genome instability.
These changes were linked to inaccurate DNA repair processes within the nucleolus, which eventually led to chromosomal rearrangements and cellular death.
In contrast, the researchers also observed that the nucleoli smaller than the NST maintained the exclusion of repair proteins and exhibited stable ribosomal DNA organization.
Furthermore, experiments using 1,6-hexanediol also confirmed that nucleoli below the NST dissolved upon treatment, whereas the larger nucleoli resisted, indicating altered biophysical properties in aged cells.
These findings suggested that nucleolar enlargement disrupts the boundary of the nucleolar condensate, allowing the proteins that are normally excluded to infiltrate and destabilize the genome.
These results also indicated the role of nucleolar size as a "mortality timer” that could potentially predict the replicative lifespan remaining for a cell after surpassing the NST. Moreover, this mechanism was shown to operate independently of traditional aging pathways, emphasizing nucleolar integrity's pivotal role in cellular aging and genomic stability.
Conclusions
Overall, the study demonstrated that nucleolar enlargement beyond a critical threshold triggers genome instability and limits replicative lifespan.
By engineering smaller nucleoli, the researchers showed how the lifespan of the yeast cells was extended and how the ribosomal DNA organization was stabilized. These findings also highlighted the role of nucleolar size as a key regulator of aging and genomic stability, providing a potential target for interventions.
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