An Intricate Network Involving the Argonaute ALG-1 Modulates Organismal Resistance to Oxidative Stress

Abstract

Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We show that upregulation of ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms' ability to withstand oxidative stress.

Fig. 1. ALG-1 levels are increased in long-lived mutants.

a alg-1 gene expression in long-lived mutants as determined by RNAseq. n = 4 per group. Values represent log₂(fold change of long-lived mutant/control), logFC. Adjusted P values and detailed information are described in Supplementary Data 1. b Representative images and c relative GFP fluorescence of the gfp::alg-1 reporter (PQ530) in different long-lived mutant backgrounds at day 1 of adulthood. Combined data of two independent replicates. n = 20, 20, 18, 18, 22 worms per condition. d Schematic of the RNAi screen used to find and validate alg-1 regulators. Two gfp::alg-1 reporter strains were used, CT20, a multicopy integrated alg-1p::gfp::alg-1 transgene (zals5) and, PQ530, a endogenous alg-1 allele (ap423) tagged with GFP. Created with BioRender.com. Relative GFP fluorescence of CT20 (n = 30, 27, 33, 31, 31, 28, 26 worms per condition) (e) and PQ530 (n = 32, 29, 31, 28, 28, 35, 36 worms per condition) (f) worms after RNAi exposure. Worms were grown on plates with control (ctrl) RNAi (empty vector), transferred to respective RNAi plates at day 0 of adulthood, and had their fluorescence measured on day 3. Combined data of three independent experiments. g gei-2/mep-1, nhr-28, nhr-77, R02D3.7 and skn-1 gene expression in long-lived mutants as determined by RNAseq. n = 4 per group. * Adjusted P values < 0.05, as described on Supplementary Data 1. c, e, f Bars represent mean ± SEM. Comparisons between controls groups versus experimental groups were made using one-way ANOVA with Dunnett’s post hoc test. ns – non-significant (P > 0.05), * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data and exact P values (whenever available) are provided in the Source Data file.

Fig. 2. alg-1 overexpression improves oxidative stress resistance.

Survival of day 3 adults after 8 hours on sodium arsenite of a CT20 (n = 21 wells with 10 worms each per condition) and b PQ535 (n = 16, 18, 16, 17 wells with 10 worms each per condition) strains in comparison with their controls. Combined data from three independent experiments. Survival on paraquat (4 mM) of c CT20 (n = 106 worms for ctrl; 128 worms for CT20) and d PQ535 (n = 122 worms for WT; 161 worms for PQ535) in comparison with their controls. Representative data from three independent replicates. e Survival of day 3 glp-1(e2144) and WT adult worms after 8 hours on sodium arsenite. n = 7, 5, 15, 15 wells with 10 worms each per condition. Combined data from two independent replicates. f Survival on paraquat (8 mM) of glp-1(e2144) and WT worms treated with alg-1 or control (ctrl) RNAi (empty vector) from day 0. n = 115 worms for WT on ctrl RNAi; 151 worms for WT on alg-1 RNAi; 131 worms for glp-1(e2144) on ctrl RNAi; 120 worms for glp-1(e2144) on alg-1 RNAi. Representative data from two independent replicates. Relative GFP fluorescence of day 3 worms expressing g sod-3p::gfp (muIs84) (n = 18, 20, 14, 18 worms per condition) or h gst-4p::gfp (dvIs19 III) (n = 19, 13, 20, 11 worms per condition) reporters on the WT or ALG-1/OE (PQ535) backgrounds. Combined data from two independent replicates. i Relative GFP fluorescence of day 3 worms expressing the sod-3p::gfp (muIs84) reporter under alg-1 RNAi or further carrying the ALG-1/OE transgene (PQ535). Worms were treated with 10 mM N-acetylcysteine (NAC) or vehicle (ctrl). n = 21, 25, 26, 22, 18, 18 worms per condition. Combined data from two independent replicates. c, d and f Data were compared using the log-rank test. ns – non-significant (P > 0.05), *P < 0.05, ****P < 0.0001. a, b, e, g–i Bars represent mean ± SEM. Comparisons were made using two-way ANOVA with Sidak’s post hoc test. ns – non-significant (P > 0.05), *P < 0.05, ****P < 0.0001. Source data and exact P values (whenever available) are provided in the Source Data file.

Fig. 3. Knockdown of R02D3.7 increases oxidative stress resistance.

a Survival after 8 hours of sodium arsenite exposure on day 3 adults. WT worms were treated with control (empty vector), gei-2 or R02D3.7 RNAi since day 0. n = 18, 17, 12, 18, 16, 14 wells with 10 worms each per condition. b Survival on paraquat (8 mM) of WT worms treated with control (empty vector), gei-2 or R02D3.7 RNAi since day 0 of adulthood. n = 98 worms for ctrl RNAi; 113 worms for gei-2 RNAi; 112 worms for R02D3.7 RNAi. c Survival after 8 hours of sodium arsenite exposure on day 3 adults. WT worms were treated with control (empty vector), R02D3.7, R02D3.7 + alg-1 or alg-1 RNAi since day 0. n = 18 wells with 10 worms each per condition. d Survival on paraquat (8 mM) of WT worms treated with control (empty vector), R02D3.7, R02D3.7 + alg-1 or alg-1 RNAi since day 0 of adulthood. n = 91 worms for ctrl RNAi; 99 worms for alg-1 RNAi; 117 worms for R02D3.7 RNAi; 78 worms for R02D3.7+alg-1 RNAi. e Survival on paraquat (8 mM) of WT and ALG-1/OE (PQ535) worms treated with control (empty vector) or R02D3.7 RNAi since day 0 of adulthood. n = 152 worms for WT ctrl RNAi; 133 worms for WT R02D3.7 RNAi; 124 worms for ALG-1/OE ctrl RNAi; 118 worms for ALG-1/OE R02D3.7 RNAi. f Lifespan under normal conditions of WT worms treated with control (empty vector), R02D3.7, R02D3.7 + alg-1 or alg-1 RNAi since day 0 of adulthood. n = 136 worms for ctrl RNAi; 132 worms for alg-1 RNAi; 127 worms for R02D3.7 RNAi; 135 worms for R02D3.7+alg-1 RNAi. a, c Bars represent mean ± SEM. Combined data of three independent replicates. Comparisons were made using two-way ANOVA with Sidak’s post hoc test. ns – non-significant (P > 0.05), *P < 0.05, ****P < 0.0001. b, d–f Representative data from three independent replicates. Data were compared using the log-rank test. ns – non-significant (P > 0.05), *P < 0.05, ***P < 0.001, ****P < 0.0001. Source data and exact P values (whenever available) are provided as a Source Data file.

Fig. 4. Increased miRNAs upon ALG-1 overexpression control resistance to oxidative stress.

Heatmaps of differentially expressed miRNAs (log₂ fold change > 1 or < −1) on a glp-1(e2144) mutants in comparison with WT worms, and b ALG-1/OE (CT20) strain in comparison with its control (ctrl) strain. Detailed information in Supplementary Data 3. Venn diagrams representing the miRNAs (c) upregulated or (d) downregulated on glp-1(e2144), ALG-1/OE (CT20), and in both. e Eleven miRNAs upregulated in both glp-1(e2144) mutants and ALG-1/OE worms. f Survival of mir-235(n4504) and WT worms after 8 hours of exposure to sodium arsenite on day 3 of adulthood. n = 22, 17, 24, 18 wells with 10 worms each per condition. Bars represent mean ± SEM. Combined data of three independent replicates. Comparisons were made using two-way ANOVA with Sidak’s post hoc test. **P < 0.01, ****P < 0.0001. g–i Survival on paraquat (4 mM) of mir-87(n4104), mir-230(n4535) or mir-235(n4504) mutants vs. WT. Experiments were performed together and share the same controls, which were split in different panels to allow better visualization. n = 119 worms for WT; 133 worms for mir-87; 110 worms for mir-230; 123 worms for mir-235. Data were compared using the log-rank test. *P < 0.05, ***P < 0.001, ****P < 0.0001. f–i Data represent one experiment of three independent replicates. Source data are provided as a Source Data file.

Fig. 5. Fine-tuning of the PDI pathway by miRNAs improves oxidative stress resistance.

a Schematic of the strategy used to find putative targets of the miRNAs upregulated in response to alg-1 overexpression and involved in oxidative stress resistance. b Approach used to validate pdi-2 as a target of miR-230-3p. Cel-miR-230-3p or cel-miR-85-3p (negative control) mimics were administrated in HEK293T cells transfected with luciferase reporter vectors containing the pdi-2 3’UTR (native or mutated – Firefly luciferase) or not (Renilla luciferase). Created with BioRender.com c Luminescence ratio of Firefly luciferase (potentially regulated by the presence of pdi-2 3’UTR) and Renilla luciferase (transfection control) of HEK293T cells. n = 12 independent cell pools per condition. d Survival of pdi-2(tm689) heterozygous (het) mutants and WT worms on paraquat (4 mM). n = 122 for WT and 85 for pdi-2(tm689). e Survival of pdi-2(tm689) heterozygous (het) mutants and WT worms after 8 hours of exposure to sodium arsenite on day 3 of adulthood. n = 22, 22, 28, 28 wells with 10 worms each per condition. f Survival on paraquat (8 mM) of WT worms exposed to control (ctrl, empty vector) or alg-1 RNAi since eggs. n = 91 worms for ctrl RNAi and 131 worms for pdi-2 RNAi. g Survival after 8 hours of exposure to sodium arsenite on day 3 of adulthood of WT worms exposed to ctrl (empty vector) or pdi-2 RNAi since eggs. n = 16, 16, 20, 20 wells with 10 worms each per condition. h Lifespan of WT worms exposed to ctrl (empty vector) or alg-1 RNAi since eggs. n = 142 worms for ctrl RNAi and 146 worms for pdi-2 RNAi. c, e, g Bars represent mean ± SEM. Combined data from two independent experiments. Comparisons were made using two-way ANOVA with Sidak’s post hoc test. ns – non-significant (P > 0.05), ****P < 0.0001. d, f and h Data were compared using the log-rank test. ****P < 0.0001. Representative data from three independent experiments. Source data and exact P values (whenever available) are provided as a Source Data file.

Fig. 6. An intricate mechanism involving the miRNA biogenesis machinery regulates oxidative stress resistance in C. elegans.

Arrows represent activation. Blunt-end represents inhibition. Continuous lines represent direct regulation. Dashed lines represent indirect regulation. Created with BioRender.com.