Abstract
microRNAs (miRNAs) are increasingly recognized as important regulators of many biological processes in mosquitoes, vectors of numerous devastating infectious diseases. Identification of bona fide targets remains the bottleneck for functional studies of miRNAs. In this study, we used CLEAR-CLIP assays to systematically analyze miRNA-mRNA interactions in adult female Anopheles gambiae mosquitoes. Thousands of miRNA-target pairs were captured after direct ligation of the miRNA and its cognate target transcript in endogenous Argonaute–miRNA–mRNA complexes. Using two interactions detected in this manner, miR-309-SIX4 and let-7-kr-h1, we demonstrated the reliability of this experimental approach in identifying in vivo gene regulation by miRNAs. The miRNA-mRNA interaction dataset provided an invaluable opportunity to decipher targeting rules of mosquito miRNAs. Enriched motifs in the diverse targets of each miRNA indicated that the majority of mosquito miRNAs rely on seed-based canonical target recognition, while noncanonical miRNA binding sites are widespread and often contain motifs complementary to the central or 3’ ends of miRNAs. The time-lapse study of miRNA-target interactomes in adult female mosquitoes revealed dynamic miRNA regulation of gene expression in response to varying nutritional sources and physiological demands. Interestingly, some miRNAs exhibited flexibility to use distinct sequences at different stages for target recognition. Furthermore, many miRNA-mRNA interactions displayed stage-specific patterns, especially for those genes involved in metabolism, suggesting that miRNAs play critical roles in precise control of gene expression to cope with enormous physiological demands associated with egg production. The global mapping of miRNA-target interactions contributes to our understanding of miRNA targeting specificity in non-model organisms. It also provides a roadmap for additional studies focused on regulatory functions of miRNAs in Anopheles gambiae.
Highlights
Mosquito-borne infectious diseases, such as malaria, dengue, chikungunya, and Zika, pose an increasing threat to public health in many tropical and subtropical regions of the world [1]
Drastic changes in mRNA abundance have been previously reported when adult female mosquitoes attend to varying nutritional sources and physiological demands
The temporal patterns of miRNA-target interactions obtained in this study provide new insights into the roles of miRNAs in tightly controlled gene expression associated with blood-feeding and mosquito oogenesis
Summary
Mosquito-borne infectious diseases, such as malaria, dengue, chikungunya, and Zika, pose an increasing threat to public health in many tropical and subtropical regions of the world [1]. In the early hours after eclosion, adult mosquitoes rely on nutrient reserves (mainly glycogen and lipids) accumulated during the larval stage until they could find plant nectar or fruit juice [4]. During the remaining previtellogenic period, mosquitoes use carbohydrates of nectar to synthesize trehalose for immediate usage and store surplus nutrients in the form of glycogen and lipids [5]. By 72 hours post-eclosion (PE), female mosquitoes have completed previtellogenic maturation under the control of juvenile hormone (JH) and become competent for massive yolk protein synthesis and deposition [6, 7]. Teneral reserves and energy stored during the previtellogenic period are critically important for the fate of developing oocytes [8, 9]. Amino acids derived from blood proteins are used for the synthesis of yolk protein precursors and provide fuel to meet massive energy demand during vitellogenesis [10]
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