Volume 21, Issue 6, Pages 1434-1441 (November 2017) MicroRNA Regulation of nAChR Expression and Nicotine-Dependent Behavior in C. elegans  Manish Rauthan, Jianke Gong, Jinzhi Liu, Zhaoyu Li, Seth A. Wescott, Jianfeng Liu, X.Z. Shawn Xu  Cell Reports  Volume 21, Issue 6, Pages 1434-1441 (November 2017) DOI: 10.1016/j.celrep.2017.10.043 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 1434-1441DOI: (10.1016/j.celrep.2017.10.043) Copyright © 2017 The Authors Terms and Conditions

Figure 1 The acr-19 Gene Is Essential for Nicotine Withdrawal but Not Acute Response, and Its Transcript Is Upregulated by Chronic Nicotine Treatment (A) Relative mRNA level of acr-15 and acr-19 is upregulated by chronic nicotine treatment (16 hr). ∗∗p < 0.001 (t test). (B) Withdrawal response of wild-type (WT), acr-15, and acr-19 mutant worms after chronic nicotine treatment. n = 10. ∗∗p < 0.001 (t test). Error bars, SEM. See the rescue data of acr-19(ok967) worms in Figure 4B. (C) acr-19 but not acr-15 mutant worms show normal nicotine acute response. n = 10. ∗∗p < 0.001 (t test). (D) Nicotine-induced upregulation of acr-19 mRNA level is suppressed in acr-15 mutant worms. ∗∗p < 0.001 (t test). All qPCR experiments in this study were run in triplicates (biological replicates) for each genotype. Each experiment was repeated at least three times. All error bars represent SEM. Cell Reports 2017 21, 1434-1441DOI: (10.1016/j.celrep.2017.10.043) Copyright © 2017 The Authors Terms and Conditions

Figure 2 The MicroRNA Machinery Is Required for acr-19-Mediated Nicotine Withdrawal Response (A) alg-1 but not rde-1 mutant worms show no nicotine withdrawal response. n = 10. ∗∗p < 0.001, ∗∗∗p < 0.0001 (t test). (B) alg-1 mutant worms exhibit normal nicotine acute response. n = 10. ∗∗p < 0.001, ∗∗∗p < 0.0001 (t test). (C) The upregulation of acr-19 mRNA after prolonged nicotine treatment is suppressed in alg-1 mutant worms. ∗p < 0.01 (t test). (D) alg-1 mRNA level is significantly downregulated after prolonged nicotine treatment in WT but not in acr-15 mutant worms. ∗∗p < 0.001 (t test). All error bars represent SEM. See also Figure S1. Cell Reports 2017 21, 1434-1441DOI: (10.1016/j.celrep.2017.10.043) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Nicotine-Mediated Upregulation of acr-19 Gene Requires the MicroRNA miR-238 (A) Predicted binding site of miR-238 in the 3′ UTR of acr-19 gene. (B) Nicotine-mediated upregulation of acr-19 mRNA after prolonged treatment is suppressed in mir-238 mutant worms. ∗∗p < 0.001 (t test). (C and D) mir-238(n4112) mutant worms show no nicotine withdrawal response (C) but exhibit normal nicotine acute response (D). n = 10. ∗∗p < 0.001 (t test). (E) Transgenic rescue of the nicotine withdrawal phenotype in mir-238, alg-1, and acr-15 mutants by directing the expression of their wild-type gene in acr-19-expressing cells. acr-19 promoter was used to drive the expression of the transgenes. n = 10. ∗∗p < 0.001 (t test). All error bars represent SEM. See also Figures S2 and S3. Cell Reports 2017 21, 1434-1441DOI: (10.1016/j.celrep.2017.10.043) Copyright © 2017 The Authors Terms and Conditions

Figure 4 The 3′ UTR of acr-19 Gene Is Essential for Nicotine Withdrawal Response and Nicotine-Induced Upregulation of acr-19 (A) Schematic of different constructs used to rescue nicotine withdrawal response in acr-19 mutants worms. The rescuing plasmid was generated by amplifying acr-19 along with a 3.5-kb promoter and 600 bp of 3′ UTR. The miR-238 binding site in the 3′ UTR of acr-19 was mutated from AGTACAAA to AAAGCTTA to introduce a HindIII site. (B) acr-19 transgene with its native 3′ UTR, but not the unc-54 3′ UTR, rescues the nicotine withdrawal phenotype in acr-19 mutant worms. A transgene containing acr-19 gene with its 3′ UTR harboring a mutation in the miR-238 binding site is unable to fully rescue the nicotine withdrawal phenotype. n = 10. ∗∗∗p < 0.0001 (t test). (C) Nicotine treatment can upregulate the mRNA level of acr-19 transgene with its native 3′ UTR, but not the unc-54 3′ UTR or the acr-19 3′ UTR harboring a mutation in the miR-238 binding site in acr-19(ok967) mutant worms. ∗∗∗p < 0.0001 (t test). (D) Schematic of GFP-expressing constructs with distinct 3′ UTR. (E and F) Chronic nicotine treatment upregulates the protein level of GFP transgene attached with the 3′ UTR of acr-19, but not the 3′ UTR of unc-54, or the 3′ UTR of acr-19 harboring a mutation in the miR-238 binding site (repeated at least 3 times). (E) Western blot. (F) Bar graph. Band intensity was normalized to mock-treated sample and was also calibrated based on the β-tubulin level of each sample. Nic, nicotine. ∗∗p < 0.001 (ANOVA with Dunnett’s test). (G) A genetic model. In addition to miR-238, some unknown microRNAs (denoted by the question mark) may also regulate acr-19 expression. All error bars represent SEM. Cell Reports 2017 21, 1434-1441DOI: (10.1016/j.celrep.2017.10.043) Copyright © 2017 The Authors Terms and Conditions