A microRNA is the effector gene of a classic evolutionary hotspot locus

Abstract

In Lepidoptera (butterflies and moths), the genomic region around the gene cortex is a "hotspot" locus, repeatedly implicated in generating intraspecific melanic wing color polymorphisms across 100 million years of evolution. However, the identity of the effector gene regulating melanic wing color within this locus remains unknown. We show that none of the four candidate protein-coding genes within this locus, including cortex, serve as major effectors. Instead, a microRNA (miRNA), mir-193, serves as the major effector across three deeply diverged lineages of butterflies, and its role is conserved in Drosophila. In Lepidoptera, mir-193 is derived from a gigantic primary long noncoding RNA, ivory, and it functions by directly repressing multiple pigmentation genes. We show that a miRNA can drive repeated instances of adaptive evolution in animals.

Fig. 1.. mir-193 is the major melanic color regulator in the cortex locus.

(A) Biogenesis and silencing machinery of miRNAs. (B) Disrupting Drosha or Dicer processing sites using CRISPR-Cas9 inhibits the biogenesis of mature miRNAs. (C) The core cortex locus in B. anynana showing two highly conserved miRNAs and four candidate protein-coding genes. (D) Homozygous mutant lines of mir-193 and mir-2788 in B. anynana and their corresponding genotypes. A dotted line denotes the cutting site. (E) Expression levels of the guide strand of mir-193, miR-193-3p, and the two mature strands of mir-2788, across the corresponding mutant lines and wild type. n = 3 to 4 replicates; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Expression levels with the same letter are not significantly different from each other. Error bar, SEM. (F) Phylogenetic placement of the three model butterfly species in the tree of lepidopterans previously mapped to the cortex locus. Phylogeny is from (40). Mosaic knockouts (mKOs) of mir-193 in (G) Pieris canidia and (H) Papilio polytes. Images were horizontally flipped when necessary.

Fig. 2.. The lncRNA ivory functions as primary mir-193.

(A) A gigantic lncRNA, ivory, was found in the RNA-seq data of three mir-193 mutants, with a deeply conserved TSS whose chromatin accessibility increased during the larval-pupal transition (shaded), and an undefined 3′ terminus. No open chromatin was found around the miRNA region (shaded). (B) Time series expression of miRNA mature strands, miR-193-3p, miR-2788-5p, and ivory. (C) Expression levels of ivory across the miRNA mutants and wild type. (D) HCR probes were designed in the first exonic region for ivory, and in a 1-kb intronic region flanking each miRNA precursor for pri-miRNAs. (E) Spatial expression (HCR) of pri-mir-193 (i), pri-mir-2788 (ii), ivory (iii), and merged panels (iv) in the B. anynana “eyespot” wing color pattern and (F) their expression signals within individual nuclei. DAPI staining is in blue. (G) ivory TSS mKO phenotypes in B. anynana, P. canidia, and P. polytes. (H) A homozygous ivory TSS mutant line in B. anynana and its corresponding genotype. A dotted line denotes the cutting site. (I) Expression levels of ivory and the guide strands of mir-193 and mir-2788 across ivory TSS mutants and WT. Images were horizontally flipped when necessary. For qPCR, n = 3 to 4 replicates; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Error bar, SEM.

Fig. 3.. mir-193 directly targets multiple pigmentation genes.

(A) Differentially expressed genes (padj<0.01) across sib-paired female mir-193 m4 mutant and WT wing tissues across wing development. Genes highlighted were previously associated with butterfly pigmentation or potential color regulators. Candidates for in vitro validation are in bold (B) Four putative binding sites of miR-193-3p, the guide strand of mir-193, were found in CDS and/or 3′ UTR regions of three candidate genes, ebony (e), Esp1, and yellow-e3 (yel-e3) that are up-regulated in day 6 mutant wings. Full sequence complementarity (allowing G:U wobble base pairing) between the seed region (nucleotides 2 to 8 from the 5′ terminus of the miRNA guide strand) and the miRNA targets are highlighted. (C) Dual luciferase reporter assay was used to validate the direct miRNA target silencing across the four predicted binding sites in vitro, with a concentration gradient of miR-193-3p mimic. n = 6 replicates; *P < 0.05; **P < 0.01; ***P < 0.001. Error bar, SEM.

Fig. 4.. mir-193 is an ancestral melanic color regulator.

(A) Genomic context of D. melanogaster mir-193. (B) Phenotypes of pnr-Gal4 transgenic lines expressing either a mir-193 sponge with a sequence of seed-complementary binding sites for the guide strand miR-193-3p (reduced mir-193), or extra mir-193 precursors (increased mir-193), or a mir-193 sponge with scrambled miR-193-3p binding sites (control). (C) Functional conservation of mir-193 across a broader animal phylogeny where mir-193 is deeply conserved. Phylogeny is from (40).