Old cells lose chromosomes

A) Cells carrying labeled chromosome II (Chr 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 and minichromosome labels used in this study. C) Chromosome loss frequency 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 frequency of chromosome loss in a cohort of ∼50 cells. Unpaired T-test (*<0.05, **<0.005, ***<0.0005).

Old cells lose chromosomes by asymmetric sister chromatid partitioning

A) Images of anaphase cells carrying the labeled chromosome II as in Fig. 1A. Red arrows mark the old SPBs. B) Frequency 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 mis-segregation in the cohort of ∼50 anaphases C) Fraction of anaphase mis-segregation events that lead to TetR-GFP foci being present only in mother or daughter cell (n=66, cohorts of 10). D) Graphical schematic of all observed anaphase mis-segregation 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) Frequency 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) Chromosome II loss frequency per CBE in strains of indicated genotype (divided into categories of 5xCBE)(N, n(cells/divisions) wt=458/8600, sir2Δ=158/1779, fob1Δ=127/2681, sgf73Δ=142/3950 mlp1Δ=302/4270).Loss frequencies in the same age category are compared to wt. Unpaired T-test (*<0.05, **<0.005, ***<0.0005). B) Replicative lifespan of listed genotypes (n>120 cells, Log-rank (Mentel Cox) test *<0.05, **<0.005, ***<0.0005).

Introns drive asymmetric chromatid partitioning 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) Chromosome II loss frequency 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 the frequency of chromosome loss in a cohort of ∼50 cells. Unpaired T-test (*<0.05, **<0.005, ***<0.0005). C) Frequency of anaphase mis-segregation 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 mis-segregation frequency in a cohort of ∼50 anaphases. Loss frequencies in the same age category are compared to wt. 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) Frequency of anaphase mis-segregation of chromosome II in cells of indicated genotypes (n>2500 cells). Each data point represents anaphase mis-segregation frequency in a cohort of >300 cells. C) Fraction of chromosome II mis-segregation with old (red) or new SPB (pink) in anaphase cells of indicated genotype (n=30 cohorts of ∼10 mis-segregation events) Unpaired T-test (*<0.05, **<0.005, ***<0.0005) D) Chromosome II loss frequency in indicated genotype as a function of CBE (categories of 5xCBE) (N, n(cells/divisions) wt=458/8600, mlp1Δ=302/4270,3xΔi=469/9974 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).

Non-centromeric DNA is sufficient to drive aging and chromosome loss

A) Schematic of transformation of chromosome II reporter strains with YRp17 (-CEN plasmid) and the accumulation of non-centromeric plasmids. B) Replicative lifespan of strains with and without YRp17 plasmid. Red stars represent p-value between the YRp17 and YRp17 3xΔi strain. Orange stars represent the p-value between YRp17 3xΔi and wt (n>200 cells, Log-rank (Mentel Cox) test, *<0.05, **<0.005, ***<0.0005) C) Chromosome II loss frequency in indicated genotypes as a function of CBE (divided into categories of 5xCBE) (N, n(cells/divisions) wt=458/8600,3xΔi=469/9974,+YRp17 =199,2097, + YRp17 3xΔi=200,3134). D) Replicative lifespan of sir2Δ and sir2Δ3xΔi strain. Stars represent p-value between sir2Δ and sir2Δ3xΔi (n>200 cells, Log-rank (Mentel Cox) test, *<0.05, **<0.005, ***<0.0005). E) Chromosome II loss frequency in indicated genotype as a function of CBE (divided into categories of 5xCBE) (N, n(cells/divisions) wt=458/8600,3xΔi=469/9974, sir2Δ=351,3500, sir2Δ3xΔi=350,3840).

Pre-mRNA leaks to the cytoplasm of old cells

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). D) Images of the pre-mRNA translation reporter in cells (right panel) of indicated genotype (top) at indicated ages (bottom). E) Ratio of mCherry/GFP signal in the same cells at 0h and after 24 hours of aging. 119/146 cells have a ratio greater than “1” F) 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 mCherry/GFP intensity of cells at 12 or 24h (*<0.05, **<0.005, ***<0.0005).

Pre-mRNA leakage is sufficient to drive asymmetric chromatid partitioning

A) Chromosome II mis-segregation in cells of indicated genotypes (n=2774,3000,5269,2650,1607). Each data point represents anaphase mis-segregation frequency in the cohort of >300 cells B) Fraction of chromosome II mis-segregation with old (red) or new SPB (pink) in anaphase cells of indicated genotype (n=50,30 cohorts of ∼10 mis-segregation events). Unpaired T-test (*<0.05, **<0.005, ***<0.0005).

Introns affect chromosome loss, but not whole nuclei mis-segregation in aging

A) Stills of a whole nuclear mis-segregation event, with both SPBs ending up in the daughter cell. B) Quantification of the frequency of whole nuclear mis-segregation events in wt and 3xΔi (n=450 cells, cohorts of 50 cells, Unpaired T-test. C) 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.D) Frequency 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).

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) Frequency of chromosome II, IV and GFP minichromosome loss (See Fig 1B) in wt and upon removal of intron of GLC7 at 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).

The 3xΔi removal does not alter mitotic timing, growth or budding in old age due to GLC7Δi duplication.

A) Stills depicting the method of mitotic timing quantification in young cells, measuring the time between SPB alignment and SPB separation, marking anaphase onset B) Mitotic timing in wt and 3xΔi cells (n>50, unpaired T-test). C) 10-fold serial dilution spot assay for different intron removal strains. YPD, two days of growth, 30°C. D) Measuring the timing between the emergence of the bud and cytokinesis in the final completed division of the replicative lifespan in wt and 3xΔi cells(n>50, unpaired T-test). E) Map of the GLC7 locus on chromosome V, the flanking Ty1 elements, and the result of Ty1 transposition. F) The result of tagging the duplicated locus with C-terminal ye-GFP tag. Upon tagging the GLC7-Δi but not the wt GLC7 locus, the locus duplication becomes evident.

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).