microRNAs play critical roles in the survival and recovery of Caenorhabditis elegans from starvation-induced L1 diapause

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We further examined worms recovering from 4 d of L1 starvation and found that around 90% of the mir-71(lf) mutants displayed retarded vulval precursor cell (VPC) division, compared with less than 5% in wild type (Fig. 4A). We found that the 3′UTRs of several genes of the InsR pathway, including unc-31, age-1, pdk-1, akt-2, and sgk-1, contain predicted miR-71 targeting sites (as predicted by TargetScan and mirWIP). (H and I) Fluorescence images (H) and statistical data (I) showing that the M cell diveded in fed animals but remained undivided in 4-, 7-, or 11-d–starved L1 wild-type and mir-71(lf) worms. (E) Fluorescence and DIC images showing that the unc-31 3′UTR reporter was repressed in mir-71(+)worms (2/2 transgenic lines) but not in mir-71(lf) worms (4/4 transgenic lines). We found that the poor survival rate of daf-16(mu86)(lf) was further decreased by mir-71(lf) (Fig. 2C), consistent with the notion that a portion of miR-71 activities regulate genes that act in parallel to UNC-31–mediated InsR/PI3K signaling for long-term survival during L1 diapause. Mutating miR-71 drastically reduces the survival rate of animals in L1 diapause, and the effect can be suppressed by mutations of insulin receptor pathway genes age-1 and unc-31.
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Among short-lived miRNA mutants, a mir-71 deletion mutant, mir-71(n4115) (referred to as mir-71(lf) hereafter), displayed a severe reduction in L1 starvation survival rate (Table S1 and Fig. 2A). We found that the reduced survival rate of ain-1 was suppressed by either reduction of age-1 function or loss of unc-31 function (Fig. 1 B and C), suggesting that a significant portion of the overall miRNA functions in L1 diapause is upstream of, or in parallel to, the InsR pathway. In this study, we addressed the questions of whether and how miRNAs impact developmental arrest and the long-term survival of early L1 stage worms in response to food starvation.

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1A because the ain-1 mutations reduce, but do not eliminate, miRISC functions. The overall effect of miRNAs on L1 starvation survival is expected to be significantly stronger than that reflected by the data in Fig. 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).
(E) DIC images showing that hbl-1(RNAi) caused precocious VPC divisions in late L2/early L3 in both wild-type and mir-71(lf) worms recovered from 4 d of L1 starvation. Note that the daf-16(lf) worms recovering from 3 d of L1 starvation displayed a ∼12-h delay in overall development and that the mir-71(lf); daf-16(lf) double mutants displayed an ∼24-h delay. (C) Bar graph showing that the delayed VPC timing defects of mir-71(lf) worms was suppressed by an unc-31(lf) mutation and partially suppressed by an age-1(rf) mutation. In worms that recovered from 4 d of L1 starvation, we also found that a significant portion of the mir-71(lf) mutants displayed egg-laying defects and overproliferating or precociously reflexed gonads.

Intestinal miRNAs Play Critical Roles in L1 Starvation Survival.

To test the hypothesis that these developmental timing genes mediate the regulatory role of miR-71 in larval development during recovery from starvation-induced L1 diapause, we examined whether knocking down HBL-1 function can suppress the retarded VPC timing defect of mir-71(lf). Reduction-of-function mutation (rf) in the age-1/PI3 kinase gene, age-1(hx546), made worms long-lived in the L1 starvation assay and was able to suppress the reduced L1 survival rate of mir-71(lf); the rate of the double mutants was comparable to that of wild type (Fig. 2A). Our genetic analysis indicated that for both L1 diapause survival and developmental recovery functions, miR-71 regulates expressions of genes in both the insulin receptor-dependent and -independent pathways.

  • Our genetic analysis indicated that for both L1 diapause survival and developmental recovery functions, miR-71 regulates expressions of genes in both the insulin receptor-dependent and -independent pathways.
  • We found that the 3′UTRs of several genes of the InsR pathway, including unc-31, age-1, pdk-1, akt-2, and sgk-1, contain predicted miR-71 targeting sites (as predicted by TargetScan and mirWIP).
  • To identify individual miRNAs that play prominent roles in L1 diapause, we screened 72 available mutant strains of individual miRNAs and miRNA families (87 miRNAs in total) using the L1 starvation assay.
  • Biomass recovery was similar across growth strategies, suggesting that growth-related differences play a minimal role in short-term recovery; however, early regrowth was characterised by contrasting trait shifts.
  • 1A because the ain-1 mutations reduce, but do not eliminate, miRISC functions.
  • Among short-lived miRNA mutants, a mir-71 deletion mutant, mir-71(n4115) (referred to as mir-71(lf) hereafter), displayed a severe reduction in L1 starvation survival rate (Table S1 and Fig. 2A).

miR-71 Likely Directly Represses the Expression of age-1 and unc-31 by Acting on Their 3′ Untranslated Regions.

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To investigate the roles of miRNAs in animal survival during starvation-induced L1 diapause, we impaired the overall miRISC function with loss-of-function (lf) mutants of ain-1 (ku322, ku425, and tm3681) and ain-2(tm2432) and examined their L1 starvation survival rate (Materials and Methods). The strong suppression of the mir-71(lf) defect by hbl-1(RNAi), and the relatively weak effect of miR-71 on hbl-1 expression, are consistent with the idea that miR-71 exerts its role by modulating activities of multiple genes related to hbl-1 function in developmental timing. In contrast, the nuclear-localized GFP expression under the control of the 3′UTR of age-1(Fig. 3 C and D) or unc-31 (Fig. 3 E and F) was strongly repressed in the control worms, but prominently derepressed in mir-71(lf) mutant worms. If the 3′UTR of age-1 or unc-31 is repressed by miR-71, the GFP expression will be repressed in tissues where miR-71 is expressed in wild-type worms, but derepressed in the same tissues of mir-71(lf) worms. (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.