Old cells lose chromosomes.

A) Cells carrying labeled Chromosome II and imaged at different ages on the microfluidic chip. Time of acquisition is indicated on the left. Fluorescent markers are indicated on top. Replicative age of imaged cells (completed budding event – CBE) is indicated in merged images. Scale bar (upper right panel) is 5µm. Green arrow marks the TetR-GFP foci B) Schematic representation of the chromosome labels used in this study. C) Chromosome loss fraction of indicated chromosomes after 0h (∼0-3 CBE) and 24h (∼18-22 CBE) of imaging. The mode of chromosome visualization is indicated in parenthesis, i.e., TetR-GFP (green), TetR-mCherry (red) or mini-chromosome encoded GFP (GFP exp). Chromosome loss is defined as the absence of the label in the mother cell in G1 shortly after the final cell cycle, or the final anaphase. Each data point represents a fraction of chromosome loss in a cohort of ∼50 cells. Unpaired T-test (*<0.05, **<0.005, ***<0.0005).

Old cells lose chromosome by asymmetric sister chromatid partition.

A) Images of anaphase cells carrying the labeled chromosome II as in Fig. 1A. Red arrows mark the old SPBs. B) Fraction of anaphase mis-segregation events where chromosomes are visible only in the mother or daughter cell (n>150) Bars are labelled as in Fig 1C. Each data point represents frequency of anaphase missegregation in the cohort of ∼50 anaphases C) Fraction of anaphase missegregation events that lead to TetR-GFP foci being present in the mother or the daughter cell (n=66, cohorts of 10). D) Graphical schematic of all observed anaphase missegregation events and their frequency. E) Fraction of anaphase mis-segregation events biased towards the old (red) or new SPB (pink) for mini-chromosome (n=30) and Chr II with normal (Daughter(D)) and inverted segregation of the old SPB (Mother (M)) (n=60 and 30). Cohorts of 10-20 missegregating anaphases F) Fraction of Chromosome II loss in wt, Ipl1-321, at 0h (∼0-3 CBE), 12h (∼8-12 CBE) and 24h (∼18-22 CBE) at 27°C. Each data point represents loss frequency in a cohort of ∼50 cells. Unpaired T-test (*<0.05, **<0.005, ***<0.0005).

ERC formation and NPC remodeling drive chromosome loss and aging.

A) Loss fraction of Chromosome II in strains of indicated genotype as a function of CBE (divided into categories of 5xCBE)(N, n(cells/divisions) wt=458/8600, sir2Δ=158/1779, fob1Δ=127/2681, sgf73Δ=142/3950 mlp1Δ=302/4270) B) Replicative lifespan of listed genotypes (n>120 cells, Log-rank (Mentel Cox) test *<0.05,**<0.005,***<0.0005).

Introns drive asymmetric chromatid partition and chromosome loss in aging.

A) A list of genes encoding kinetochore-associated proteins. Adapted from Biggins et al 2013. Intron containing genes are marked in red. B) Fraction of Chromosome II loss at indicated aging time (top) in cells of indicated genotype. 3xΔi stands for GLC7-Δi MCM21-Δi NBL1-Δi triple mutant. Each data point represents a fraction of chromosome loss in a cohort of ∼50 cells. Unpaired T-test (*<0.05, **<0.005, ***<0.0005). C) Fraction of anaphase missegregation of Chromosome II at indicated aging time (top) in cells of indicated genotype. 3xΔi stands for GLC7-Δi MCM21-Δi NBL1-ΔI triple mutant. Each data point represents missegregation frequency in a cohort of ∼50 anaphases. Unpaired T-test (*<0.05, **<0.005, ***<0.0005). D) Replicative lifespan upon intron removal (n>200 cells, Log-rank (Mentel Cox) test, *<0.05, **<0.005, ***<0.0005).

Basket displacement causes intron-dependent chromosome loss in young and old cells.

A) Stills of symmetric and asymmetric anaphases in young cells. Arrow marks the old SPB. Scale bar (upper right panel) is 5µm B) Anaphase mis-segregation of Chromosome II in cells of indicated genotypes (n=2774,3000,2700). Each data point represents anaphase missegregation fraction in the cohort of >300 cells. C) Fraction of Chromosome II missegregation with old (red) or new SPB (pink) in anaphase cells of indicated genotype (n=30 cohorts of ∼10 missegregation events) Unpaired T-test (*<0.05, **<0.005, ***<0.0005) D) Chromosome II loss probability in indicated genotype as a function of CBE (divided into categories of 5xCBE) (N, n(cells/divisions) wt=458/8600, mlp1Δ=302/4270, mlp1Δ 3xΔi=295/4757) E)Replicative lifespan of listed genotypes. Red and green stars represent p-values between the wt and the corresponding genotype. Black stars represent the p-value between mlp1Δ and mlp1Δ 3xΔi (n>300 cells, Log-rank (Mentel Cox) test, *<0.05, **<0.005, ***<0.0005).

Pre-mRNA leaks to the cytoplasm in aging.

A) Images of RNA FISH targeting three long introns (GLC7, YRA1, DBP2) in young and old cells. B) Quantification of GLC7 intron RNA FISH foci localization in young and old cells (n=295, cohorts of ∼100 cells) C) Principle of the pre-mRNA translation reporter (left panel) adapted from (Sorenson et al. 2014 49). D) Images of the pre-mRNA translation reporter in cells (right panel) of indicated genotype (top) at indicated ages (bottom). E) Log2 ratio of mCherry/GFP in cells of indicated genotype as a function of age, normalized to wt median at 0h (n=60, 35, 32). Unpaired T-test comparing the log2 ratio of mCherry/GFP intensity of cells at 12 or 24h (*<0.05, **<0.005, ***<0.0005).

Pre-mRNA leakage is sufficient to drive asymmetric chromatid partition.

A) Anaphase mis-segregation of Chromosome II in cells of indicated genotypes (n=2774,5269,2650,1607). Each data point represents anaphase missegregation fraction in the cohort of >300 cells B) Fraction of Chromosome II missegregation with old (red) or new SPB (pink) in anaphase cells of indicated genotype (n=50,30 cohorts of ∼10 missegregation events). Unpaired T-test (*<0.05, **<0.005, ***<0.0005).

Introns inhibit mitotic error correction in aging.

A) Replicative lifespan of wt, GLC7Δi, Ipl1-321 and Ipl1-321 GLC7Δi cells grown at 27 Celsius (n=300), Log-rank (Mentel Cox) test *<0.05,**<0.005,***<0.0005.B) Fraction of Chromosome II loss in wt, Ipl1-321, Ipl1-321 3xΔi (GLC7-Δi MCM21-Δi NBL1Δi) at 0h (∼0-3 CBE), 12h (∼8-12 CBE) and 24h (∼18-22 CBE) at 27°C. Each data point represents loss frequency in a cohort of ∼50 cells. Unpaired T-test (*<0.05, **<0.005).

Chromosome loss in aging is ERC and NPC remodeling dependent.

A) Fraction of Chromosome II loss (POA1:TetO /TetR-GFP) in wt, fob1Δ,sir2Δ, sgf73Δ and mlp1Δ mutant cells at 0h (∼0-3 CBE),12h (∼8-12 CBE) and 24h (∼18-22 CBE). Each data point represents a fraction of loss in a cohort of ∼50 cells. Stars correspond to p-values of wt vs genotype of listed color at the corresponding timepoint. Unpaired T-test (*<0.05, **<0.005, ***<0.0005).

The effect of introns on chromosome loss is not chromosome-specific.

A) Replicative lifespan of wt, mad1Δ, mad2Δ cells. n>100 cells, Log-rank (Mentel Cox) test. B) Fraction of Chromosome II, IV and GFP minichromosome loss (See Fig 1B) upon removal of intron of GLC7 0h (∼0-3 CBE) and 24h (∼18-22 CBE). Each data point represents a fraction of loss in a cohort of ∼50 cells. Unpaired T-test (*<0.05, **<0.005, ***<0.0005).

smRNA FISH probes are intron specific.

A) Images of smRNA FISH experiments in wt and GLC7-Δi cells B) Quantification of smRNA FISH foci per cell in wt and GLC7-Δi cells C) Quantification of smRNA FISH foci per cell in wt cells at 0 and 28h of aging (n>300, cohorts of 100+ cells)