Components of the InsR pathway, including age-1, have recently been predicted to be targets of miR-71 in its role in aging (14). On the other hand, the role of a particular miRNA (miR-71) is executed by repressing the expression of many genes in multiple pathways. These results compelled us to examine specific interactions between individual miRNAs and their targets to gain mechanistic insights. This result is also consistent with the prediction from a miRISC immunoprecipitation analysis that hbl-1 is likely a target of one or more miRNAs, in addition to the let-7 family miRNAs, during early development (18). This result suggests that miR-71 likely functions upstream of, or in parallel to, HBL-1 in regulating VPC timing.
Knocking down lit-1 by RNAi in mir-71(lf); lin-42(lf) double mutants caused no significant suppression of the VPC timing defects of mir-71(lf) worms. To determine the functional relationship of miR-71 with LIN-42 and LIT-1, mir-71(lf); lin-42(lf) L1 worms were starved for 4 d and recovered on lit-1(RNAi) plates. 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. 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).
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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). 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. 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).
- As COVID-19 lockdowns unfolded around the world, and sales of games, puzzles, toys, and other playful artforms spiked, a friend asked me if I felt a sense of validation.
- The overall effect of miRNAs on L1 starvation survival is expected to be significantly stronger than that reflected by the data in Fig.
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(Right panels) The gonad of the same animals in the Left panels to indicate the similar developmental stage. The reporter construct, the control plasmid, and a transformation marker plasmid were coinjected into worms to generate the extrachromosomal arrays for analysis. We further examined the functional relationship between miR-71 and DAF-16, a FOXO transcription factor acting critically and negatively downstream of AGE-1/PI3K in the InsR pathway. Elegans Genetic Center (reference 257) and an N2 strain from the laboratory stock, respectively.
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{She tried to make herself into what others wanted her to be. She has a podcast called the courage to change. There are more recovery modalities than ever. Some features and devices may not be available in all languages or countries. Shared by Lindsay Broadwater from the Garrett County Local Management Board / Local Behavioral Health Authority Offices.}