Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans

Abstract

Protein synthesis is a regulated cellular process that links nutrients in the environment to organismal growth and development. Here we examine the role of genes that regulate mRNA translation in determining growth, reproduction, stress resistance and lifespan. Translational control of protein synthesis by regulators such as the cap-binding complex and S6 kinase play an important role during growth. We observe that inhibition of various genes in the translation initiation complex including ifg-1, the worm homologue of eIF4G, which is a scaffold protein in the cap-binding complex; and rsks-1, the worm homologue of S6 kinase, results in lifespan extension in Caenorhabditis elegans. Inhibition of ifg-1 or rsks-1 also slows development, reduces fecundity and increases resistance to starvation. A reduction in ifg-1 expression in dauers was also observed, suggesting an inhibition of protein translation during the dauer state. Thus, mRNA translation exerts pleiotropic effects on growth, reproduction, stress resistance and lifespan in C. elegans.

Fig. 1

Inhibition of either rsks-1 or ifg-1 reduces the rate of protein translation. (a) Reduction in rates of protein synthesis measured by ³⁵S-methionine incorporation upon inhibition of ifg-1and rsks-1. Age-matched adults were placed in ³⁵S-methionine-labelled OP50 for 0, 2, 4, and 8 h. Radioactivity was measured in the TCA precipitated protein extract and standardized for protein concentration. Protein synthesis rate was evaluated by measuring the slope of the curves during incorporation. The following slopes were observed: N2 on L4440 (control RNAi), 23.5 (cpms (counts per minute) μg^-1) h; rsks-1(ok1255) on L4440 (control RNAi), 15.5 (cpms μg^-1) h; N2 on ifg-1(RNAi), 11.4 (cpms μg^-1) h; rsks-1(ok1255) on ifg-1(RNAi), 8.3 (cpms μg^-1) h. Each data point represents the mean of three biological replicates. Similar results were obtained in two separate experiments. (b,c) Polysomal profiles were generated by measuring absorbance profiles at 260 nm of worm extracts separated on 10-50% sucrose gradients. (b) The N2 strain on control RNAi and ifg-1(RNAi) and from (c) N2 and rsks-1(ok1255). 40S, 60S, and 80S labels show the position of ribosomal subunits and whole ribosomes. There was an increase in the 60S and the 80S peaks upon inhibition of ifg-1 accompanied by a decrease in the polysomal peaks suggesting a significant reduction in ribosomes that were involved in mRNA translation but an increase in free ribosomes (b). Maximum absorbance in N2 was detected in the sixth ribosome peak (A) vs. the fifth (B) in the rsks-1(ok1255) (c). Results are representative of three experiments performed.

Fig. 2

Lifespan extension by inhibiting ifg-1 and rsks-1 and their interaction with other longevity genes. (a) Mean lifespan was 16.4 days (n = 100) for N2 on control RNAi; 20 days (n = 116) for rsks-1(RNAi); and 22.3 days (n = 164) for ifg-1(RNAi) (P < 0.0001). (b) Mean lifespan was 19.2 days (n = 83) for N2 on control RNAi; 29.8 days (n = 83) for N2 on 1: 1 ifg-1(RNAi) and control RNAi; 21.2 days (n = 87) for 1: 1 let-363(RNAi) and control RNAi; and 32 days (n = 59) for N2 on 1: 1 ifg-1(RNAi) and let-363(RNAi) (P < 0.0001). (c) Mean lifespan was 19.2 days (n = 83) for N2 on control RNAi; 21 days (n = 78) for rsks-1(ok1255) on control RNAi; 30.6 days (n = 67) for rsks-1(ok1255) on 1: 1 ifg-1(RNAi) and control RNAi (P < 0.0001); and 16.1 days (n = 67) for 1: 1 let-363 (RNAi) and control RNAi. (d) daf-2(e1370) on control RNAi, rsks-1(RNAi), and ifg-1(RNAi). Mean lifespan was 30.8 days (n = 115) for daf-2(e1370) on control RNAi; 38.1 days (n = 119) for rsks-1(RNAi); and 52.3 days (n = 55) for ifg-1(RNAi) (P < 0.0001). (e) Mean lifespan was 12 days (n = 144) for daf-16(mu86) on control RNAi; 13.7 days (n = 136) for rsks-1(RNAi); and 15.2 days (n = 138) for ifg-1(RNAi) (P < 0.0001). (f) Mean lifespan was 19.6 days (n = 128) for clk-1(e2519) on control RNAi; 22.1 days (n = 95) for rsks-1(RNAi); and 24.1 days (n = 127) for ifg-1(RNAi) (P < 0.0001). (g) Mean lifespan was 19.5 days (n = 138) for eat-2(ad456) on control RNAi; 21.7 days (n = 133) for rsks-1(RNAi); and 30.1 days (n = 153) for ifg-1(RNAi) (P < 0.0001). (h) Mean lifespan was 15.2 days (n = 95) for sir-2.1(ok424) on control RNAi; 20.2 days (n = 105) for rsks-1(RNAi); and 29.3 days (n = 75) for ifg-1(RNAi) (P < 0.0001). Similar results were obtained in at least two separate experiments for each panel.

Fig. 3

Pleiotropic effects on growth, development, reproduction and stress resistance upon inhibition of ifg-1 and rsks-1. (a) ifg-1 expression is reduced during dauer state. Quantification of relative levels of ifg-1 mRNA in N2 animals at different developmental stages at 20 °C using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). pmp-2 expression was used for internal reference. Mean and standard error of the relative ifg-1/pmp-2 ratios based on three real-time RT-PCR trials are shown. Emb, embryos; PD, post dauer; 6 h, 6 h after dauer larvae were exposed to food. (b) rsks-1 regulates rate of development in C. elegans. Average length of 20 worms was measured every 24 h till 96 h after egg lay in the N2 vs. rsks-1(ok1255) strain. Similar results were obtained in five trials. (c,d) Reduction of fecundity by inhibition of ifg-1 and rsks-1. Progeny were counted every 24 h in (c) N2 on control RNAi and ifg-1(RNAi) (d) N2 and rsks-1(ok1255).

Fig. 4

Effect of inhibition of ifg-1 and rsks-1 on resistance to various stresses. (a) One-day-old adult N2 and rsks-1(ok1255) worms were exposed to control RNAi, ifg-1(RNAi), and daf-2(RNAi) for 48 h in 20 °C before heat stressed at 35 °C. N2 on control RNAi (n = 43) N2 ifg-1(RNAi) (n = 44); N2 on daf-2(RNAi) (n = 21) rsks-1(ok1255) on control RNAi (n = 43), rsks-1(ok1255) ifg-1(RNAi) (n = 44), rsks-1(ok1255) daf-2(RNAi) (n = 30). Similar results were obtained in three experiments. (b) Adult worms were exposed to control RNAi, rsks-1(RNAi), and ifg-1(RNAi) for 48 h in 20 °C before being transferred to NGM agar plates containing 240 μm juglone. Mean survival was 3.5 h (n = 81) for N2 on control RNAi; 1.7 h (n = 66) for rsks-1(RNAi) (P < 0.0001); and 3.6 h (n = 74) for ifg-1(RNAi). (c) Worms treated with RNAi bacteria for 48 from the L4 stage were exposed to 2000 J m^-2 of UV via a Stratalinker® 1800. Mean survival was 3.7 days (n = 47) for N2 on control RNAi; 3.4 days (n = 43) for rsks-1(RNAi); and 4.5 days (n = 57) for ifg-1(RNAi) (P < 0.0005). (d) N2 and rsks-1(ok1255) were both exposed to control RNAi, rsks-1(RNAi), and ifg-1(RNAi) for 48 h before being starved in S-basal. Standard deviations were calculated from five biological replicates. Between 50 and 100 worms per replicate were scored each day. Median survival was 6.4 days for N2 on control RNAi; 8.2 days for rsks-1(RNAi); and 10 days for ifg-1(RNAi). Similar results were obtained in at least three experiments.