Characterization, functional exploration, and evolutionary analysis of mirtronic microRNAs reveal their origin in the invasive vector mosquito, <i>Aedes albopictus</i>

The mirtron pathway represents a distinct category of noncanonical microRNA (miRNA) biogenesis mechanisms. Current studies suggest that the mirtron pathway may be widely prevalent across various taxa, including animals and plants, but investigation of this pathway has focused mainly on mammals, particularly humans, and the biological functions and emerging roles of several mirtrons in human diseases have been elucidated. In the context of insects, mirtrons have only been comprehensively characterized and preliminarily functionally analyzed in Drosophila. The Asian tiger mosquito, Aedes albopictus, is a highly invasive species and an important vector of arbovirus transmission to humans. Although canonical miRNA function has been studied in depth in mosquitoes, the role of mirtrons in this species remains to be revealed. In this study, we identified and validated 2 novel conventional mirtrons in Ae. albopictus that are precursors of miR-11900 and miR-11893. Mirtronic miRNA biogenesis depends on the splicing of introns and cleavage by Dicer but does not necessarily correlate with intron location in host genes. The molecular evolution of mirtrons was analyzed using methods based on host genes and their exon‒intron architecture; the results indicate that mirtronic miRNAs are relatively young and that they may have appeared in Culicinae after the Anophelinae and Culicinae diverged. According to small RNA sequencing (RNA-seq) and RNA-seq data on post-mirtronic miRNA overexpression, mosquito mirtronic miRNAs are present in low abundance, and the absence of typical target genes in Ae. albopictus suggests they are not involved in post-transcriptional gene regulation. Overall, our results indicate that the emergence of 2 mirtrons in Ae. albopictus is likely due to the formation of Dicer-recognized secondary structures during the evolution of the intron sequence; these structures are similar to byproducts processed by Dicer, and their abundance is controlled by an alternative adventitious mirtron emergence-dependent mechanism. Our study identifies for the 1st time mirtrons in insect species distinct from Drosophila melanogaster, provides new insights into mirtron evolution, and provides a reference for the functional analysis of mirtrons.