Skip to main content
. 2019 Dec 4;8:e49930. doi: 10.7554/eLife.49930

Figure 1. mir-1 protects against proteotoxic stress.

(A) Alignment of mature miR-1 sequences indicates deep conservation. The seed sequence of each miR-1 family member is highlighted in blue and the conservation of C. elegans mir-1 is highlighted in gray. hsa = Homo sapiens, mmu = Mus musculus, gga = Gallus gallus, xtr = Xenopus tropicalis, dre = Danio rerio, dme = Drosophila melanogaster, cel = Caenorhabditis elegans. (B–C) Visualization of Q40::YFP aggregates (green foci) in (B) wild-type and (C) mir-1(gk276) animals. Scale bar, 50 μm. (D) Quantification of Q40::YFP aggregation in wild-type, mir-1(gk276), mir-1(n4102) and mir-80(nDf53) animals. (E) Quantification of Q40::YFP aggregates in wild-type, mir-1(gk276) and mir-1(gk276) animals transgenically-expressing the mir-1 hairpin in body wall muscle (myo-3 promoter), pharynx (myo-2 promoter) or intestine (ges-1 promoter). Mutation of the mir-1 seed sequence (Muscle mir-1*) abrogates rescue from body wall muscle. Mature mir-1 sequences (wild-type mir-1 or mutated mir-1*) used for rescue experiments are shown (box). Red nucleotides indicate the mutations in the seed sequence used in mir-1* rescue experiments, which are predicted to hinder interactions with mir-1 targets. (F) Body bends in wild-type, mir-1(gk276) and mir-1(n4102) mutant animals expressing α-synuclein::YFP. (G) Survival of wild-type, mir-1(gk276) and mir-1(n4102) animals after exposure to 4 hr of 35°C heat stress. Transgenic expression of wild-type mir-1 hairpin, but not mutated mir-1*, in body wall muscle rescues mir-1(gk276) heat stress sensitivity. All experiments were performed in triplicate and at least 10 animals were scored per experiment. Error bars show standard error of the mean (SEM). ****p<0.0001, n.s. not significant to the control (one-way ANOVA analysis, followed by Dunnett’s multiple comparison test).

Figure 1.

Figure 1—figure supplement 1. Quantification of Q40::YFP Expression.

Figure 1—figure supplement 1.

(A) Location of mir-1(T09B4.11) on chromosome I, reverse strand of assembly; http://www.wormbase.org, WS258, showing the two deletion strains used in this study, gk276 and n4102 (orange bars). (B–C) WB analysis (B) and quantification (C) of Q40::YFP protein lysates from wild-type, mir-1(gk276) and mir-1(n4102) animals for YFP expression using an α-GFP antibody and α-tubulin antibody as a loading control (n = 3). n.s. not significant (one-way ANOVA analysis, followed by Dunnett’s multiple comparison test).

Figure 1—figure supplement 2. Motility Analysis.

Figure 1—figure supplement 2.

(A–C) Quantification of body bends in wild-type and mir-1(gk276) mutant animals without a transgene (A), expressing the Q0::YFP transgene (B) or expressing the Q40::YFP transgene (C). All experiments were performed in triplicate (number of animals scored are shown in each bar). ± SEM. *p<0.05, **p<0.01, ****p<0.0001, n.s. not significant (one-way ANOVA analysis, followed by Dunnett’s multiple comparison test).

Figure 1—figure supplement 3. mir-1 prevents the formation of α-synuclein inclusions.

Figure 1—figure supplement 3.

(A–B) Visualization of α-synuclein::YFP inclusions (yellow arrowheads) in (A) wild-type and (B) mir-1(gk276) animals in the first day of adulthood. Scale bar, 50 μm. (C) Quantification of α-synuclein::YFP inclusions in wild-type and mir-1(gk276) animals. n > 25. ± SEM ****p<0.0001 (Welch's t-test).

Figure 1—figure supplement 4. mir-1 Lifespan Analysis.

Figure 1—figure supplement 4.

(A) Lifespan analysis of wild-type (n = 121), mir-1(gk276) (n = 131) and mir-1(n4102) (n = 125) animals. Log-rank (Mantel-Cox) test - n.s. not significant.