microRNA gene of a classic evolutionary hotspot locus
Antónia Monteiro lab: He mainly focuses on the accumulation mechanism of related pigment molecules in organisms
Background (Why this study is very important?)
A region known as a “hot spot” locus has been repeatedly implicated in the generation of similar phenotypic variation during evolution. Specifically, this locus is associated with polymorphism in melanin wing color, but it was previously unknown that the major effector gene in this locus is not the previously thought protein-coding gene cortex, but a microrna (miRNA) called mir-193. So, it provides more clues about how organisms operate during evolution
How does the author address the proposed problem, and what is the author’s novel model or design? And the results obtained.
First, the researchers explored the relationship to pigment accumulation by studying the genetic level of mir-193. Briefly, in FIG1, the process of miRNA from primary miRNA (pri-miRNA) to mature miRNA, including the processing sites of Drosha and Dicer, is shown in FIG1. And how mirnas guide gene silencing by binding to target mrnas. How to use CRISPR-Cas9 technology to disrupt Drosha or Dicer processing sites, Thus, the biosynthesis of mature miRNA Bicyclus is inhibited Two highly conserved miRNAs (mir-193 and mir-2788) around the cortex locus in anynana (a butterfly model species) as well as the mutant lines of four candidate protein-coding genes mir-193 and mir-2788 and their corresponding genotype mir-193 and mir-2788 fingers The expression levels of guide strand in the corresponding mutant lines and the wild type, together with the statistically significant markers, show the position of the three model butterfly species in the Lepidopteran phylogenetic tree, These species are all related to the cortex locus showing images of mKOs of mir-193 in Pieris canidia and Papilio polytes, which were flipped horizontally when necessary.
Then, briefly in Fig2, using RNA-seq data analysis, the investigators identified a approximately 740-bp transcript in the mir-193 mutant, which was likely derived from a large primary transcript spanning a 370-kb chromosomal region. This newly identified long non-coding RNA (lncRNA), named ivory, has the same transcription direction as mir-193 and is the only transcript that overlaps with mir-193. The expression pattern of ivory, mir-193 and mir-2788 is very similar, and its expression in the larva wing is very low. Expression was high in pupal wings. In particular, the peak expression of ivory occurs earlier than the two mature mirnas, consistent with the general pattern of miRNA processing in Drosophila. By blocking Drosha processing, the researchers found that ivory expression was slightly increased in the mir-193 mutant and significantly increased in the mir-2788 mutant, providing moderate evidence that the two mirnas are derived from ivory. The researchers examined the spatial expression of pri-mir-193, pri-mir-2788, and ivory and found that their expression signals matched the black/brown regions of the Bicyclus anynana wing pattern. The researchers found that ivory TSS mutants exhibit phenotypes similar to mir-193 mutants in several butterflies, suggesting that ivory acts as pri-mir-193/2788 and that mir-193 is a functional product of ivory. By comparing transcriptome data between mir-193 mutants and wild type, mir-193 was found to trigger numerous transcriptomic changes, mainly in the late pupal developmental stages. They also identified direct target genes of mir-193-3p, including ebony (e), Esp1, and yellow-e3 (ELECTR-E3), which have known or potential roles in color regulation. Overall, further evidence is provided that mir-193 is derived from lncRNA ivory, and detailed information on how mir-193 regulates butterfly wing color by directly targeting multiple pigment genes.
At the same time, detailed information on the molecular mechanism of how mir-193 regulates butterfly wing color by directly targeting multiple pigment genes was further investigated. In short, in Fig.3. Differences in gene expression in wing tissues at different developmental stages of mir-193 mutants compared with wild type are demonstrated. In particular, genes associated with pigmentation or potential color regulation in butterflies are highlighted. The number of genes differentially expressed at different developmental stages (larval, pupal day 1, and pupal day 6) in mir-193 m4 mutant compared with wild type, and the expression changes of these genes. By analyzing the up-regulated genes in the mutants and looking for complementary binding sites with mir-193-3p, the researchers predicted 49 genes that might be directly targeted by mir-193-3p. Three candidate target genes –Ebony (e), Esp1, and yellow-e3 (ELECTR-E3) -with known or potential roles in color regulation were selected. Graphs show the predicted miR-193-3p binding sites in these genes. The direct silencing effect of miR-193-3p on four predicted target sites in these three candidate genes was verified in vitro using a dual luciferase reporter assay. In particular, miR-193-3p showed a dose-dependent silencing effect on two target sites of the ebony gene. The effect of miR-193-3p mimic concentration gradient on reporter gene activity, thus verifying the direct regulation of miR-193-3p on candidate genes.
Finally, the genomic location and functional study of mir-193 in Drosophila, as well as its conservation across different species, supported the role of mir-193 as an ancient melanin regulator and highlighted the importance of non-coding Rnas in animal evolution and phenotypic diversity. Briefly, in FIG4, the genomic location of mir-193 and its relationship with surrounding protein-coding genes in Drosophila melanogaster are shown. In Drosophila, mir-193 is located in a different genomic region, independent of the long noncoding RNA ivory, and between two protein-coding genes with the same transcription orientation. By using the pannier Gal4 driver (pnr-Gal4), Investigators expressed mir-193 sponges (a series of binding sites complementary to the seed region of Mir-193-3p for mir-193 inhibition) or additional miR-193 precursors to enhance mir-193 function. The results showed that the wings of flies with mir-193 sponge expression became brighter, while the wings of flies with mir-193 overexpression became darker. Conservation of mir-193 in different animal phyla, and its role in promoting melanogenesis. This suggests that the role of mir-193 in Drosophila and Lepidopteran insects is conserved and not affected by the genomic context. By comparing the functions of mir-193 in different species, the investigators concluded that mir-193 has an ancestral role in regulating melanin formation and that this function is conserved across species.
Why is this article significant, and how does it contribute to our research?
This study not only reveals the molecular basis of butterfly wing color variation, but also highlights the importance of non-coding Rnas in evolution and provides a new perspective on understanding genome complexity and species diversity. It also provides information for more efficient and accurate biological mechanism research strategies.