MicroRNAs both promote and antagonize longevity in C. elegans

Abstract

Background: aging is under genetic control in C. elegans, but the mechanisms of life-span regulation are not completely known. MicroRNAs (miRNAs) regulate various aspects of development and metabolism, and one miRNA has been previously implicated in life span.

Results: here we show that multiple miRNAs change expression in C. elegans aging, including novel miRNAs, and that mutations in several of the most upregulated miRNAs lead to life-span defects. Some act to promote normal life span and stress resistance, whereas others inhibit these phenomena. We find that these miRNAs genetically interact with genes in the DNA damage checkpoint response pathway and in the insulin signaling pathway.

Conclusions: our findings reveal that miRNAs both positively and negatively influence life span. Because several miRNAs upregulated during aging regulate genes in conserved pathways of aging and thereby influence life span in C. elegans, we propose that miRNAs may play important roles in stress response and aging of more complex organisms.

Figure 1. Novel miRNAs identified in aged C. elegans

(A) Secondary structures of putative precursor hairpins corresponding to miRNAs identified in a pool of RNA enriched for aged C. elegans. The predicted mature sequence is highlighted in red. (B) Conservation of novel miRNAs miR-A, miR-B, miR-C and miR-I to known miRNAs. Identical nucleotides are highlighted in black. C) Validation of expression of candidate novel miRNAs. The expression of novel miRNAs mir-G, mir-H, mir-I and mir-K was confirmed by TaqMan qRT-PCR in wild-type (N2) C. elegans. Consistent with their classification as miRNAs, their expression was significantly reduced in alg-1(gk214) mutant animals. The expression of the miRNA let-7 is shown as a positive control. The expression of a non-miRNA small RNA species (snoRNA sn2429) does not change in alg-1(gk214) background (DNS). *, p < 0.05, alg-1(gk215) compared to wild-type (N2) (Student's t-test).

Figure 2. Spatio-temporal expression changes in miRNA expression during aging

The expression of GFP in C. elegans reporter strains for 4 aging-associated miRNAs was surveyed during adulthood. Consistent with deep-sequencing and qRT-PCR results, the expression of GFP driven by the promoters of mir-71, mir-238, mir-239 and mir-246 increased during aging. miR-71 and miR-238 are expressed ubiquitously and miR-246 is expressed in gonadal sheath cells, as reported before [13]. During aging, pmir-71::GFP expression increases in the intestine and in the pharynx, while pmir-239::GFP expression is seen primarily in intestine and in neurons, tissues previously implicated in regulation of lifespan in C. elegans [1]. Representative images showing changes in GFP intensity are shown (warmer colors indicate higher GFP expression).

Figure 3. Deletion or over-expression of aging-associated miRNAs affects lifespan

(A) Mutations in differentially expressed miRNAs affect lifespan. Four miRNA genes that are among the most over-expressed in aged animals - mir-71, mir-238, mir-239, and mir-246 - exhibit significant changes in lifespan when mutated. C. elegans mutants that contain deletions of miR-71, miR-238 and miR-246 - mir-71(n4115) (red), mir-238(n4112) (blue), and mir-246(n4636) (green) exhibit significantly reduced lifespan compared to wild-type (N2) animals (black), whereas deletion of miR-239 – mir-239(nDf62) – extends lifespan. (B–C) miR-71 and miR-246 overexpression extends life. Three C. elegans lines that overexpress (o/e) miR-71 or miR-246 exhibit longer lifespans compared to wild-type N2 animals. (D) miR-239 overexpression reduces lifespan. Three lines overexpressing (o/e) miR-239 (red, blue and green) exhibit significantly shorter lifespan as compared to wild-type N2 animals (black). All lifespan assays conducted at 20°C. Statistics are shown in Table 3.

Figure 4. Stress resistance in miRNA mutants correlates with their lifespan phenotypes

(A) In response to heat shock (12h at 35°C), the short-lived miRNA mutants mir-71(n4115) and mir-246(n4636) exhibit increased thermo-sensitivity while the long-lived mir-239(nDf62) mutant exhibited significantly increased thermo-resistance as compared to wild-type animals (N2). (B) Similarly, in response to oxidative stress (Paraquat, 6 hours), the short-lived mutants mir-71(n4115) and mir-238(n4112) exhibited decreased survival while the long-lived mir-239(nDf62) mutant exhibited significantly increased survival compared to wild-type animals. Each strain (n>30) was examined in triplicate. Survival (%) and error bars represent the average survival and standard deviation between triplicate samples, respectively. p-values were calculated using Student's t-test: p=0.42 for mir-238(n4112) (heat stress); p=0.99 for mir-246(n4636) (paraquot); *, p < 0.05, compared to wild-type (N2).

Figure 5. miR-239 and miR-71 function through the IGF-1/insulin-like and the DNA Damage Checkpoint pathways

(A) The long lifespan of mir-239(nDf62) is suppressed by daf-16(RNAi) but is unaffected by control (empty vector) RNAi. mir-239(nDf62) does not further enhance the lifespan on daf-2(RNAi), suggesting that they function in the same pathway. (B–C) The long lifespan of daf-2(RNAi) is partially suppressed by mir-71(n4115) and daf-16(RNAi) does not further decrease the lifespan of mir-71(n4115), suggesting miR-71 functions in the same pathway as DAF-2 and DAF-16. (D) miR-71 additionally functions in DNA damage checkpoint pathway. mir-71(n4115) fully suppresses the longevity induced by RNAi to CDC-25.1, a factor of the DNA damage checkpoint pathway. All lifespan assays conducted at 20°C. Statistics are shown in Table S5.

Figure 6. Targets of miR-71 in IIS and DNA Damage Checkpoint pathways

(A–B) Schematic representation and secondary structures of miR-71 binding sites on the 3’UTR of PDK-1, a component of IIS pathway, and on the 3’UTR of CDC-25.1, a component of the DNA Damage Checkpoint pathway. (C) In a miR-71 deletion genetic background (mir-71(n4115)), the mRNA levels of PDK-1 and CDC-25.1 are significantly up-regulated specifically in older adult animals (Day 10) as compared to wild-type N2 animals, as measured by qRT-PCR, and after normalization by endogenous control ACT-1. (D) Model for molecular and genetic interactions of age-related miRNAs with known aging pathways. Arrows denote positive interaction, -| denotes negative interactions.