Deciphering Regulation of MicroRNA Expression in Disease Vector, Aedes aegypti

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in post-transcriptional gene expression regulation in eukaryotic organisms. miRNA function is driven by base-pair binding to a complementary mRNA strand which in turn results in mRNA silencing. Here, we employed a novel co-immunoprecipitation technique (CLIP-seq) to identify miRNA-mRNA interactions during the post-eclosion (PE) and blood meal stages of Aedes aegypti, and validated miRNA binding using a dual luciferase assay. Next, we investigated the transcriptional factors that govern miRNA expression regulation. We used a double-stranded RNA (dsRNA) knockdown approach to silence drosha to enrich for pri-miRNAs in the total RNA pool. Three pri-miRNAs (miR-276, miR-2940 and miR-252) were identified and their respective promoter regions were selected for transcription factor binding analysis using a luciferase based assay. Only one transcription factor, E75, induced luciferase activity and we further explored E75 as part of the juvenile hormone (JH) hierarchical network. E75 transcript levels could be induced by topical application of JH, and reduced through the blockage of JH pathway by RNAi targeting of the key receptor Met. Using small-RNA sequencing, we found that iMet and iE75 treatments both had a global negative impact on miRNA expression. These findings established the role of E75 within the JH hierarchical network in miRNA activation during the PE phase. We next examined the functional roles of E75 regulated miRNAs, miRNA-276, miRNA-2940 and miR-252, during the gonotrophic cycle. Antagomir treatment with Ant-2940-3p resulted in reduced follicle size and number of eggs laid. We performed computational mRNA-miRNA target predictions to elucidate the mRNA gene(s) affected by miR-2940 knockdown and confirmed by RT-qPCR that the putative target Clumsy (AAEL002518) had elevated gene levels from Ant-2940-3p treated fatbody tissues. This mRNA-miRNA interaction was confirmed using an in vitro dual luciferase assay in Drosophila S2 and in native Ae. aegypti Aag2 cell lines. Finally, we performed a phenotypic rescue experiment to demonstrate that the elevated expression of Clumsy was responsible for the disruption we noted in egg development upon Ant-2940-3p treatment. Collectively, these results confirm the importance of miR-2940-3p in the fatbody tissue during the PE developmental phase of the gonotrophic cycle