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. Author manuscript; available in PMC: 2019 Sep 18.
Published in final edited form as: Nat Metab. 2019 Feb 18;1(3):350–359. doi: 10.1038/s42255-019-0033-z

Figure 2. Negative regulation of tir-1 by mir-71 important for proteostasis and longevity.

Figure 2

a, mRNA level of predicted mir-71 targets (TargetScanWorm Release 6.2). Color-coded transcriptional responses of mRNAs expressed in AWC neurons under multiple proteotoxic stress conditions or in mir-71(n4115) deletion mutant. Expression levels are relative to untreated wild-type (WT) worms and were determined via RNA sequencing (upper panel) (n=3 biological replicates) or microarray (lower panel) (n=4 biological replicates), n.s.=not significant. b, h, Representative western blots from day 1 adult worms of indicated genotypes show UbV-GFP and tubulin (TUB) level. Data derived from at least 3 independent experiments with similar results. Molecular weights are shown in kDa. b, The tir-1(ok2859) deletion mutant suppresses mir-71(n4115)-induced proteolytic defects. c, Proteotoxic stress increases TIR-1 levels in AWC neurons. Confocal microscopy images showing tir-1::GFP in green and AWC neurons in red. Scale bar: 15 µm. d, Quantification of TIR-1::GFP fluorescence intensity shown in (c). DMSO was used as a solvent control but did not show a significant difference compared to the control condition (1322.24 ± 96.05, data not shown). Bars show mean values ± SEM obtained from n=12 individual animals (represented by dots). Statistics were determined by one-way ANOVA with post-hoc test. e, Heat stress sensitivity of mir-71(n4115) mutant is suppressed by tir-1(ok2859) deletion. The hsf-1(sy441) mutant served as control. Bars show mean values ± SEM obtained from n=3 biological replicates using at least 50 worms (mean values represented by dots); statistics were determined by one-way ANOVA with post-hoc test. f, tir-1(ok2859) deletion extends the lifespan of mir-71(n4115) worms. For statistics details see Supplementary Table 1. g, mir-71 negatively regulates tir-1 transcript level. Relative tir-1 mRNA levels measured by qRT-PCR in day 1 adult worms. Data normalized to wild-type (WT). Bars show mean values ± SEM obtained from n=3 biological replicates with 3 technical replicates each (mean values represented by dots); statistics were determined by one-way ANOVA with post-hoc test; ns=not significant). h, AWC-selective expression of mir-71 does not suppress proteostasis defects caused by the tir-1 3’UTR* mutation. i, The tir-1 3’UTR* mutation shortens lifespan. For statistics details see Supplementary Table 1.