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).
(B) The severely reduced survival rate of the mir-71(lf) mutant was suppressed by a null allele of unc-31(e928). The effect observed in ain-1(lf) mutants is likely the consequence of the combined effects of attenuating functions of these individual miRNAs. Previous studies revery play login indicate that the InsR pathway plays a dominant role in regulating L1 starvation survival and that reducing the activity of the insulin receptor daf-2, the PI3Kinase age-1, or the upstream regulator unc-31 results in increased L1 starvation survival rate (2, 3). (B) Survival rate of single and double mutants to indicate the functional relationship between ain-1 and age-1.
The two ain-1 loss-of-function alleles displayed significant reductions in L1 starvation survival rate. We further found that this survival rate reduction of ain-1 mutants was overcome by ectopic expression of the AIN-2 protein in the intestine but not in the muscle (Fig. 1A and Fig. S1A). We found that ain-1 but not ain-2 mutants displayed a significant reduction in L1 starvation survival rate compared with that of wild type (Fig. 1 A and D). 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).
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- (B) The severely reduced survival rate of the mir-71(lf) mutant was suppressed by a null allele of unc-31(e928).
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- The nematode Caenorhabditis elegans responds to starvation by entering developmental arrest at multiple stages of its life cycle (1).
- To test whether the activity of the InsR pathway was down-regulated by miR-71, we first examined the endogenous expression of components of the InsR pathway in mir-71(lf).
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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). (D) Bar graph showing that the delayed VPC timing defect of mir-71(lf) worms was enhanced by daf-16(lf) after 1 or 3 d of L1 starvation. (B) Bar graph showing the correlation between the severity of the retarded vulval precursor cell (VPC) timing defect of mir-71(lf) mutants and the duration of L1 starvation.
RRF Regulation
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. L1 starvation assay was adapted from a previously described protocol (3). Worms strains were grown and maintained at 20 °C as described (29).
Elegans Genetic Center (reference 257) and an N2 strain from the laboratory stock, respectively. Wild-type strains A and B are an N2 strain recently obtained from the C. (A) Survival rate curves of wild-type and mutant strains, as indicated. This is consistent with the previous reports that AIN-1 and AIN-2 are functional homologs with overlapping biochemical roles (16, 17). The roles of InsRs have also been implicated in arresting the cell cycle in germ cells and a portion of somatic cells during L1 diapause (2, 4). Contributed new reagents/analytic tools; X.Z., R.Z., and M.H.
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.
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- (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.
- These results suggest that a significant portion of the miR-71 activities in L1 diapause survival may be devoted to regulating the activities of UNC-31–mediated InsR/PI3K signaling and that the rest of miR-71 activity may regulate UNC-31–independent pathways.
- The effect observed in ain-1(lf) mutants is likely the consequence of the combined effects of attenuating functions of these individual miRNAs.
- 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).
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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. Furthermore, miR-71 plays a prominent role in developmental recovery from L1 diapause partly through repressing the expression of certain heterochronic genes. When you restore a backup that contains third-party account information you must enter the recovery password to decrypt the backup. If you opt-in to third-party account backup and restore, and have set an account recovery password, then the app backups to Google Drive (Android) or iCloud (iOS) do include the private key information for your third-party accounts.
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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. If this were true, the starved mir-71(lf); daf-16(lf) double-mutant worms should show a slow growth phenotype similar to that of daf-16(lf) worms, but no specific VPC timing defect. (H) Fluorescence and DIC images showing that a lin-42 3′UTR reporter was repressed in mir-71(+) worms (2/2 transgenic lines) and prominently derepressed in mir-71(−) worms (2/2 transgenic lines). We thus asked whether miR-71 was required for the reinitiation of developmental programs during the recovery phase after L1 starvation. These results suggest that miR-71 regulates the expression of unc-31 and age-1 through their 3′UTRs. Note that there are extra GFP-positive cells (red arrows) in mir-71(lf) mutants.
MiR-71 regulates vulval cell division during recovery of starved L1 worms. These results indicate that miR-71 is not essential for arresting seam cell or M-cell divisions during L1 diapause, suggesting that miR-71 function is distinct from DAF-16 function. DAF-16 (the FOXO homolog in C. elegans) has been shown to play an important role in cell cycle arrest and developmental progression partly by promoting cki-1 expression in some somatic cells during L1 arrest (2).
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If you haven’t enabled third-party account restore in Duo Mobile then app backups to Google account backup (Android) or iCloud (iOS) accounts DO NOT contain any private key or other sensitive data. Duo Mobile’s restore functionality lets you back up Duo-protected accounts and third-party OTP accounts (such as Google or Facebook) for recovery to the same device or to a new device. 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). Furthermore, the observed derepression of individual genes by mir-71(lf) seemed too weak to account for the phenotype, consistent with the idea that a prominent phenotype of an miRNA mutation is caused by the collective effect of changing expression in many genes, an important property of miRNA-mediated gene regulation. (F) Fluorescence and DIC images showing that an hbl-1 3′UTR reporter was repressed in mir-71(+) worms and slightly derepressed in mir-71(lf) mutants.
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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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(D) Fractions of worms that carry 3′UTR reporter transgene and show no GFP expression GFP(−), weak GFP expression GFP(+/−), and comparable GFP expression to mCherry GFP(+). We found that the mRNA level of UNC-31 was up-regulated by about 20% in mir-71(lf) (Fig. 3A). These results suggest that a significant portion of the miR-71 activities in L1 diapause survival may be devoted to regulating the activities of UNC-31–mediated InsR/PI3K signaling and that the rest of miR-71 activity may regulate UNC-31–independent pathways. We next examined the relationship between miR-71 and UNC-31, which functions upstream of AGE-1 during L1 diapause by regulating calcium-regulated dense-core vesicle fusion and the release of an insulin-like ligand (3). We identified 10 miRNA mutants that showed reduced survival rates with a stringent standard, as well as a few miRNA mutants with slightly increased survival rates (Table S1, Fig. 1D, and Fig. S1B).
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