rs17294019

183788157

P(A)0.80 082 0.84 0.86 0.88 0.90rs17294019100-105Age 106-119pia)0.640.601s3788157100-105 106-119Age

Figure 5. Age related trend of allele frequencies. The two barplots show the age related trend of allele frequencies of SNPs rs17294019 (ADARB2, SNP # 98 in Table 1) and rs3788157 (ADARB1, SNP # 135 in Table 1) in the NECS (n=1,023). The frequencies of the common allele for both SNPs were stratified in the age groups 90–99; 100–105, 106 and higher. Trends of allele frequencies for increasing age groups are consistent with a strong correlation between genotype and phenotype that results in substantial enrichment of protective alleles in older subjects.

are unlikely to have influenced our lifespan measurements, since the adult worms were made sterile using FudR and were not transferred during the course of the lifespan assay.

Eggs were isolated from gravid worms and synchronized by hatching overnight in the absence of food. The synchronized L1

Fraction alive0.25 0.sO 0.750.00ensaSRse  b)1.000.75Fraction alive0.SO0.25 

Figure 6.ADARmediated decline in lifespan,daf-2influence, andrde-1rescue. a) Lifespan using mutant strains for adr-1;adr-2 in the context of dsRNA mediated gene inactivation of daf-2. Synchronized worms at the larval stage 4 (L4) were sterilized with FudR and allowed to feed on bacterial lawns that contained dsRNA for daf-2. Note: adr-1; adr-2 double mutant (red solid), adr-1; adr-2 double mutant with dsRNA for daf-2 (red hatched), N2 wild type (blue solid), N2 with dsRNA for daf-2 (blue hatched). Note decline in lifespan due to adr-1; adr-2 compared with N2 wildtype. Also note increases in lifespan of both N2 and adr-1; adr-2 in the presence of dsRNA for daf-2. The 50% survival time in the adr-1; adr-2 mutant animals was 10 days (95% limits 9 and 12 days) compared with 20 days (95% limits 18 and 20 days) for N2 wild-type control worms. RNAi to daf-2 increases lifespan to 34 days (95% limits 32 and 40 days), compared with 20 days for the wild type (N2 worms fed empty vector (RNAi)). daf-2 gene inactivation, in the background of the adr-1 and adr-2 null mutations also restored lifespan to 18 days (95% limits 16 and 20 days), compared with 10 days for the adr-1;adr-2 double mutant strain. b) Lifespan using mutant strains for adr-1;adr-2 (solid red), N2 wildtype (solid blue), rde-1 (grey hatched), adr-1; adr-2; rde-1 (grey solid) demonstrate declines in lifespan using mutant strains and full rescue of lifespan in an RNAi defective (rde-1) background. The adr-1; adr-2 mutant was again about half as long lived as wild-type (median survival time 9 days for adr-1;adr-2 strain (95% limits 9 and 11 days), and median survival time 21 days (95% limits 18 and 21 days), for N2 wild-type worms. The survival distribution of the triple mutant adr- 1;adr-2; rde-1 is median lifespan 21 days (95% limits 18 and 21 days), which is significantly different from adr-1;adr-2, with a median lifespan of 9 days (95% limits 9 and 11 days). The lifespan of rde-1 was modestly reduced compared with the wild-type N2, as was reported previously29. Inset boxes displays 50% survival (days) for each condition and demonstrates that daf-2 gene inactivation increases lifespan, in both wild type and in adr-1;adr-2 mutant strains (a) and that RNAi knockout (rde-1) restores lifespan.

allowed to develop to L4-stage larvae at 20C. The L4-stage larvae were washed thoroughly, and placed either on Escherichia coli HT115 with empty RNAi vector or Escherichia coli HT115 expressing double-stranded RNA (dsRNA) for daf-2. Briefly, dsRNA -expressing bacteria were grown overnight in LB with 50 ug/ml ampicillin and then seeded onto RNAi NGM plates containing 5 mM isopropylthiogalactoside (IPTG). The RNAi bacteria were induced overnight at room temperature for dsRNA expression. About 30 synchronized L4-stage animals were added to each well and allowed to develop to adults, followed by the addition of FudR. Worms were kept at 20C, and their lifespan was monitored. Worms feeding on bacteria carrying the empty vector were used as a negative control. Log-rank test in the R package survival was used for the statistical analysis.