ADARs employ a neural-specific mechanism to regulate PQM-1 expression and survival from hypoxia

Abstract

The ability to alter gene expression programs in response to changes in environmental conditions is central to the ability of an organism to thrive. For most organisms, the nervous system serves as the master regulator in communicating information about the animal's surroundings to other tissues. The information relay centers on signaling pathways that cue transcription factors in a given cell type to execute a specific gene expression program, but also provide a means to signal between tissues. The transcription factor PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as impacting survival from hypoxia. Herein, we reveal a novel mechanism for regulating PQM-1 expression specifically in neural cells of larval animals. Our studies reveal that the RNA binding protein, ADR-1, binds to pqm-1 mRNA in neural cells. This binding is regulated by the presence of a second RNA binding protein, ADR-2, which when absent leads to reduced expression of both pqm-1 and downstream PQM-1 activated genes. Interestingly, we find that neural pqm-1 expression is sufficient to impact gene expression throughout the animal and affect survival from hypoxia; phenotypes that we also observe in adr mutant animals. Together, these studies reveal an important post-transcriptional gene regulatory mechanism that allows the nervous system to sense and respond to environmental conditions to promote organismal survival from hypoxia.

Figure 1-. L1 animals lacking adr-2 have decreased expression of genes regulated by insulin signaling.

(A) Volcano plot depicting gene expression in adr-2(−) neural cells compared to wildtype (WT) neural cells. Dots represent individual genes that are upregulated (red; 186, p < 0.05, log2fold > 0.5), downregulated (blue; 502, p < 0.05, log2fold < −0.5) or not significantly different (grey, p ≥ 0.05) between three biological replicates of WT and adr-2(−) neural cells. (B-D) Expression of the indicated genes was determined relative to expression of the housekeeping gene gpd-3. Values were then normalized to wildtype neural cells (B) or wildtype L1 animals hatched in the absence of food (C, D) and the mean of three biological replicates was plotted. Error bars represent standard error of the mean (SEM). Statistical significance was calculated by two-way ANOVA test. **p < 0.005, ****p < 0.0001. ns indicates not significant (p > 0.05). For D, the indicated genotypes are of strains HAH30, HAH31, HAH32 and HAH33.

Figure 2-. Neural ADR-2 regulates insulin signaling cell non-autonomously in an editing independent manner.

(A, C) Gene expression of L1-arrested animals measured by qPCR. Expression of the indicated genes was determined relative to expression of the housekeeping gene gpd-3. Values were then normalized to WT and the mean of three (C) or four (A) biological replicates was plotted. Error bars represent standard error of the mean (SEM). Statistical significance was calculated by two-way ANOVA test. ****p < 0.0001, **p < 0.005, *p < 0.05 and ns indicates not significant (p > 0.05). For A, the indicated genotypes are of strains HAH23, HAH40 and HAH41. For C, the indicated genotypes are of strains N2, BB20 and HAH22 (B) A dashed line was used to outline the whole worm. For all the strains, the images are representative of 7–10 samples imaged in two biological replicates.

Figure 3-. Neural PQM-1 activity is sufficient to rescue expression of PQM-1 activated genes in adr-2(−) animals

(A-D) Gene expression of (A) neural cells and (B-D) L1-arrested animals measured by qPCR. Expression of indicated genes was determined relative to expression of the housekeeping gene gpd-3. Values were then normalized to WT and the mean of three biological replicates was plotted. Error bars represent standard error of the mean (SEM). Statistical significance was calculated by (A) multiple unpaired t tests and (B-D) two-way ANOVA test. ****p < 0.0001 and ns indicates not significant (p > 0.05). For A and B, the indicated genotypes are of strains HAH45 and HAH46. For C, the indicated genotypes are of strains HAH35, HAH37, HAH38 and HAH39. For D, the indicated genotypes are of strains HAH24, HAH41, HAH42, HAH43 and HAH44.

Figure 4-. Neural ADR-1 binding of pqm-1 affects expression of PQM-1 activated genes.

(A,C) Gene expression of L1-arrested animals measured by qPCR. Expression of the indicated genes was determined relative to expression of the housekeeping gene gpd-3. Values were then normalized to WT and the mean of three biological replicates was plotted. Error bars represent standard error of the mean (SEM). Statistical significance was calculated by two-way ANOVA test. ***p < 0.0005, **p < 0.01, *p <0.05. For A, the indicated genotypes are of strains N2, BB19, BB20 and BB21. For C, the indicated genotypes are of strains HAH48, HAH49 and HAH50. (B) Western blot depicting immunoprecipitation of neural ADR-1 from the indicated strains. Bar graph represents the fold enrichment determined by dividing IP/Input value from qPCR for the indicated strains divided by that of negative control. Values were then normalized to negative control and the mean of three biological replicates was plotted. Error bars represent SEM. Statistical significance was calculated by multiple unpaired t tests. **p < 0.005.

Figure 5-. Survival of hatched L1 animals after CoCl₂ exposure.

(A-B) Survival of transgenic (A) and non-transgenic (B) hatched L1 animals under hypoxic conditions induced by CoCl₂ exposure. Data plotted is average of three biological replicates. Error bars represent standard error of the mean (SEM).