S1A indicated a dominant role of intestinal miRNAs in regulating L1 starvation survival. We used a dual-color 3′UTR reporter system (18) to test the computational, prediction-based hypothesis that the 3′UTRs of age-1 and unc-31 are directly regulated by miR-71 (Fig. 3B and Materials and Methods). Among these potential miRNA targets, the predicted miR-71–targeting sites in the 3′UTRs of age-1 and unc-31 are conserved between C.
These results suggest that miRNAs act in the intestine, and possibly in other tissues, to promote L1 starvation survival. MicroRNAs (miRNAs) are well known for their functions in controlling developmental timing in the nematode (5, 6). Upon entering L1 diapause, RNA polymerase II quickly accumulates and pauses at promoter regions, and this accumulation was speculated to stop transcription and facilitate the immediate reinitiation of gene expression when food becomes available (2).
miR-71 Regulates the Timing of Vulval Cell Division in Animals Recovering from L1 Diapause.
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Furthermore, a recent study suggests that the expression of certain miRNAs is differentially regulated by starvation-induced dauer diapause (15). Consistent with these ideas, several recent lines of evidence suggest that miRNA let-7 and the heterochronic genes lin-42 and hbl-1 are required to regulate the starvation-induced dauer diapause (10–12) and that a number of miRNAs including lin-4 and mir-71 are involved in regulating life span (13, 14). Furthermore, worms that are long-lived due to dietary restriction or decreased mitochondrial respiratory rates are short-lived during L1 diapause, suggesting that the mechanisms controlling L1 starvation survival are different at least in some aspects from those controlling aging (3).
- Upon entering L1 diapause, RNA polymerase II quickly accumulates and pauses at promoter regions, and this accumulation was speculated to stop transcription and facilitate the immediate reinitiation of gene expression when food becomes available (2).
- When late, first larval stage (L1) worms sense unfavorable conditions, they enter an alternative and long-lived larval stage called dauer larvae (or dauer diapause).
- We speculate that the expression of heterochronic genes controlling the L2/L3 programs, including that of hbl-1 and lin-42, are increased during L1 diapause to arrest the developmental progression, and miR-71 is probably required to suppress these “excess” signals during the recovery phase (Fig. S5).
- Consistent with the observation described above, the 4-d–starved mir-71(lf) mutants recovering on the RNAi control plates displayed the highly penetrant retarded defect in VPC division.
- Unlike dauer diapause, L1 diapause is not accompanied by life cycle changes and has not been shown to require certain signaling pathways that control the formation of dauer diapause such as TGF-β signaling (daf-1, daf-7) and nuclear hormone receptor (daf-12) (2, 3).
- Consistent with these ideas, several recent lines of evidence suggest that miRNA let-7 and the heterochronic genes lin-42 and hbl-1 are required to regulate the starvation-induced dauer diapause (10–12) and that a number of miRNAs including lin-4 and mir-71 are involved in regulating life span (13, 14).
To determine viability, 20-μL aliquots (60–100 worms) were placed every 3 d onto two 6-cm nematode growth medium (NGM) plates seeded with OP50, and the numbers of L1 worms were recorded as number of plated worms (Np). A total of 16–24 h later, the density of newly hatched L1 worms was adjusted to three to five worms per microliter S-basal. The eggs were transferred to plates seeded with HB101 and bleached again 3 d later. Briefly, worms were well fed for at least two generations, and gravid adults were bleached with hypochlorite and sodium hydroxide.
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- We provide evidence that miRNA miR-71 is not required for the animals’ entry into L1 diapause, but plays a critical role in long-term survival by repressing the expression of insulin receptor/PI3K pathway genes and genes acting downstream or in parallel to the pathway.
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- This result is consistent with the observation that miR-71 is specifically required for the starvation-induced stress response (Fig. S5).
- We then compared the expression of a hbl-1 3′UTR reporter (18) in the mir-71(lf) mutants with that in wild type and found that the expression of this reporter was slightly derepressed at L3 in the mir-71 mutant (Fig. 4 F and G).
- The presented results indicate that interactions between multiple miRNAs and likely a large number of their mRNA targets in multiple pathways regulate the response to starvation-induced L1 diapause.
On the other hand, the role of a particular miRNA (miR-71) is executed by repressing the expression of many genes in multiple pathways. On one hand, we showed that deletions of a good number of miRNAs have varying impacts on the L1 diapause survival rate, although they may effect the rate through different mechanisms. Instead, many specific physiological functions, such as the starvation-induced stress response, are regulated by a miRNA-target network, often involving multiple miRNAs and a large number of their targets. We found that the known developmental timing genes, hbl-1, lin-42, and lit-1, were at the top of the list (TargetScan). To understand how miR-71 affects VPC division, we searched its predicted targets for potential genes involved in regulating developmental timing. These results indicate that miR-71 plays a significant role in larval development of animals recovering from L1 diapause and likely does so by regulating the expression of components of the insulin receptor/DAF-16 pathway, as well as factors acting downstream, or in parallel to, DAF-16.
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(A) The mir-71(n4115, lf) mutant displayed severe reduction in L1 starvation survival rate, and the reduced survival rate of mir-71(lf) was suppressed by a reduction-of-function allele of age-1(hx546). (C) The reduced L1 starvation survival rate of ain-1(lf) mutants was significantly suppressed by a null allele of unc-31. Compromising overall miRNA function dramatically reduces the survival rate of L1 worms in starvation-induced diapause, and the effect can be significantly suppressed by an age-1/PI3K mutation.
