Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with challenges to proteostasis
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Evaluation Summary:
The idea of individual aging trajectories of single cells is important and the authors provide sufficient evidence that there is some stochasticity that directs individual cells towards certain routes of aging - at least in budding yeast. Investigating the link between rDNA silencing and protein homeostasis, this study thus addresses an interesting and exciting question. The authors show how age-dependent loss of rDNA silencing might contribute to protein aggregation. Importantly, the paper furthers the understanding of distinct aging trajectories and raises important questions about how these processes might be relevant in multicellular organisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of Saccharomyces cerevisiae , a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.
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Evaluation Summary:
The idea of individual aging trajectories of single cells is important and the authors provide sufficient evidence that there is some stochasticity that directs individual cells towards certain routes of aging - at least in budding yeast. Investigating the link between rDNA silencing and protein homeostasis, this study thus addresses an interesting and exciting question. The authors show how age-dependent loss of rDNA silencing might contribute to protein aggregation. Importantly, the paper furthers the understanding of distinct aging trajectories and raises important questions about how these processes might be relevant in multicellular organisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
The idea of individual aging trajectories of single cells is important and the authors provide sufficient evidence that there is some stochasticity that directs individual cells towards certain routes of aging - at least in budding yeast. Investigating the link between rDNA silencing and protein homeostasis, this study thus addresses an interesting and exciting question. The authors show how age-dependent loss of rDNA silencing might contribute to protein aggregation. Importantly, the paper furthers the understanding of distinct aging trajectories and raises important questions about how these processes might be relevant in multicellular organisms.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Using single cell analysis, Paxman et al observe protein aggregation in aging yeast that is specific to cells with deregulated rDNA silencing. This is confirmed in sir2 mutant cells. The authors then investigate the mechanism by which silencing defects of the rDNA locus might be linked to a decline in protein homeostasis. Through a screen for aggregation of RNA binding proteins, they find that disruption of rDNA silencing leads to aggregation of those RNA binding proteins that are involved in rRNA processing. Overexpression of a subset of these rRNA binding genes consistently shorten the lifespan of mode 1 cells, presumably by contributing to their defects in protein homeostasis. This suggests that age dependent changes in rDNA silencing lead to the aberrant expression of rRNAs and the formation of rDNA …
Reviewer #1 (Public Review):
Using single cell analysis, Paxman et al observe protein aggregation in aging yeast that is specific to cells with deregulated rDNA silencing. This is confirmed in sir2 mutant cells. The authors then investigate the mechanism by which silencing defects of the rDNA locus might be linked to a decline in protein homeostasis. Through a screen for aggregation of RNA binding proteins, they find that disruption of rDNA silencing leads to aggregation of those RNA binding proteins that are involved in rRNA processing. Overexpression of a subset of these rRNA binding genes consistently shorten the lifespan of mode 1 cells, presumably by contributing to their defects in protein homeostasis. This suggests that age dependent changes in rDNA silencing lead to the aberrant expression of rRNAs and the formation of rDNA circles. Deletion of fob1 (resulting in a loss of rDNA recombination) indeed suppresses aggregation of Nop15 that is used for in-depth analysis. Of note, enhancing rRNA transcription or Nop15 expression leads to enhanced protein aggregation even in the absence of rDNA circles.
In all this study addresses an interesting and exciting question and is well executed. Importantly, it contributes to the understanding of distinct aging trajectories and raises important questions how these processes might be relevant in multicellular organisms. Given the fact that the paper focuses on rDNA silencing, I think that using the term "chromatin stability" is too broad and should be replaced with "rDNA silencing".
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Reviewer #2 (Public Review):
The reviewer is impressed by the microfluidics study which shows us the heterogeneity of cellular aging process regarding the appearance of different aging hallmarks. The authors tried to connect the chromatin instability in rDNA and proteostasis with a hypothesis that the overproduction of rRNA nucleates the aggregation of RBPs, somehow this drives the collapse of proteostasis reported by Hsp104 foci in the cytosol. This manuscript has strength on the techniques applied in the study, the data quality, and the great flow in writing. Although it is important to understand the crosstalk between hallmarks of aging and the data presented here are intriguing, the following issues have to be addressed.
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Reviewer #3 (Public Review):
The idea of individual ageing trajectories of single cells is important and the authors provide sufficient evidence that there is some stochasticity that directs individual cells towards certain routes of ageing - at least in budding yeast. Additionally, understanding the connection and dependence of various different processes that occur during ageing is critical and timely. However, despite the fact that the hypothesis laid out in the manuscript is tempting and the approaches taken might be the right way to tackle it, the results presented still fall short of connecting chromatin instability and protein aggregation. I have provided more detailed comments below, but in essence, I miss a clear experiment linking rRNA instability and the role of RBPs with protein aggregation and loss of proteostasis. All …
Reviewer #3 (Public Review):
The idea of individual ageing trajectories of single cells is important and the authors provide sufficient evidence that there is some stochasticity that directs individual cells towards certain routes of ageing - at least in budding yeast. Additionally, understanding the connection and dependence of various different processes that occur during ageing is critical and timely. However, despite the fact that the hypothesis laid out in the manuscript is tempting and the approaches taken might be the right way to tackle it, the results presented still fall short of connecting chromatin instability and protein aggregation. I have provided more detailed comments below, but in essence, I miss a clear experiment linking rRNA instability and the role of RBPs with protein aggregation and loss of proteostasis. All experiments that try to achieve this are either too unspecific (e.g. NAM as an inhibitor for Sir2, while it inhibits a wide variety of deacetylases) or do not show protein aggregation (e.g. Nop15-mNeon, which might simply stain a fragmented nucleolus).
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