Nucleolar stress caused by arginine-rich peptides triggers a ribosomopathy and accelerates aging in mice.

Nucleolar stress (NS) has been associated with age-related diseases such as cancer or neurodegeneration. To investigate how NS triggers toxicity, we used (PR)n arginine-rich peptides present in some neurodegenerative diseases as inducers of this perturbation. We here reveal that whereas (PR)n expression leads to a decrease in translation, this occurs concomitant with an accumulation of free ribosomal (r) proteins. Conversely, (PR)n-resistant cells have lower rates of r-protein synthesis, and targeting ribosome biogenesis by mTOR inhibition or MYC depletion alleviates (PR)n toxicity in vitro. In mice, systemic expression of (PR)97 drives widespread NS and accelerated aging, which is alleviated by rapamycin. Notably, the generalized accumulation of orphan r-proteins is a common outcome of chemical or genetic perturbations that induce NS. Together, our study presents a general model to explain how NS induces cellular toxicity and provides in vivo evidence supporting a role for NS as a driver of aging in mammals.

Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells.

The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC.

Increased Rrm2 gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice.

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.

A proteomic characterization of factors enriched at nascent DNA molecules.

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.

An extra allele of Chk1 limits oncogene-induced replicative stress and promotes transformation.

Replicative stress (RS) is a type of endogenous DNA damage that cells suffer every time they duplicate their genomes, and which is further boosted by oncogenes. In mammals, the RS response (RSR) is coordinated by ATR and Chk1 kinases. We sought to develop a mammalian organism that is selectively protected from RS. To this end, mice carrying an extra copy of the Chk1 gene were generated. In vitro, Chk1 transgenic cells are protected from RS-inducing agents. Moreover, an extra Chk1 allele prolongs the survival of ATR-Seckel mice, which suffer from high levels of RS, but not that of ATM-deficient mice, which accumulate DNA breaks. Surprisingly, increased Chk1 levels favor transformation, which we show is associated with a reduction in the levels of RS induced by oncogenes. Our study provides the first example where supra-physiological levels of a tumor suppressor can promote malignant transformation, which is a result of the protection from the RS found in cancer cells.