Posttranscriptional regulation plays a very important role in animal oocytes and embryos. Maternally synthesized mRNAs and proteins control early animal development up until the maternal-to-zygotic transition (MZT). This is the point when the zygotic genome takes control. The maternally deposited mRNAs are posttranscriptionally regulated right from the time they are produced during oogenesis, through egg activation, and in the embryo. microRNAs (miRNAs) are posttranscriptional regulators that have been shown to play a role in both RNA stability and translation. I examined miRNA abundance in Drosophila stage 14 oocytes, activated unfertilized eggs, and embryos and have grouped all the then known Drosophila miRNAs into four distinct temporal classes. Class I and III appear to be maternally deposited, while Class II appears to be both maternally and zygotically transcribed, and Class IV appears to be strictly zygotically transcribed. Follow-up experiments validated three of the four classes.

Posttranscriptional regulation plays a very important role in animal oocytes and embryos. Maternally synthesized mRNAs and proteins control early animal development up until the maternal-to-zygotic transition (MZT). This is the point when the zygotic genome takes control. The maternally deposited mRNAs are posttranscriptionally regulated right from the time they are produced during oogenesis, through egg activation, and in the embryo. microRNAs (miRNAs) are posttranscriptional regulators that have been shown to play a role in both RNA stability and translation. I examined miRNA abundance in Drosophila stage 14 oocytes, activated unfertilized eggs, and embryos and have grouped all the then known Drosophila miRNAs into four distinct temporal classes. Class I and III appear to be maternally deposited, while Class II appears to be both maternally and zygotically transcribed, and Class IV appears to be strictly zygotically transcribed. Follow-up experiments validated three of the four classes.

Female adults of many mosquito species possess distinct physiological features adapting to blood feeding for successful reproduction. The disease pathogens that are transmitted by mosquitoes have evolved to take advantages of the indispensable blood feedings to complete their transmission cycles and to survive attacks from the mosquito's innate immune system. Normal egg development and mosquito immunity are tightly controlled by tissue- and stage-specific gene expression and coordinated by many signal molecules in the mosquito. Understanding gene regulation affecting mosquito reproduction and malaria parasites infection is of paramount importance for developing novel malaria control strategies. A growing body of evidence indicates that microRNAs (miRNAs) are involved in egg maturation and immune reactions against invading pathogens in mosquitoes. However, the molecular mechanisms by which specific miRNAs selectively modulate reproduction and the survival of pathogens are largely unknown. The miRNA-induced gene-silencing pathway in mosquitoes was mostly extrapolated from the studies of flies. To explore the dynamics of miRNAs in reproduction, I used small RNAs sequencing to monitor miRNAs expression and their association with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) in the malaria mosquito Anopheles gambiae (An. gambiae) during the 72-h period immediately after blood feeding. I found the abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. I confirmed that miR-309 is essential for normal egg development by depletion of endogenous miR-309 with a specific antagomir. In addition, my results showed that the Ago association of some miRNAs was not proportional to their cellular abundance implying additional regulation at miRNA integration. To investigate the functional roles of miRNAs and define context-dependent miRNA-mRNA interactions during the reproductive process, I have applied an innovative experimental approach to study miRNA-mRNA interactome. CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP can generate miRNA-mRNA chimeras from UV-irradiation stabilized Ago-miRNA-mRNA complex. My results have defined tens of thousands of miRNA-mRNA interactions in mosquitoes, including novel targets for mosquito-specific miRNAs. Verification of the predicted interactions using mRNA-seq, ribosome-profiling, and luciferase reporter assay revealed a reliable miRNA-mRNA interaction network. Based on the detected interactions, I refined the paring rules for mosquito miRNAs and illustrated the dynamic pairing between different regions of miRNAs with their targets in vivo. The miRNA-mRNA interactions were compared using this approach at multiple time points before and after blood feeding. Importantly, this study showed that the interactions were dynamic and enriched in genes that are involved in metabolisms, supporting the proposed functions of miRNAs in coordinating the gene regulation in mosquito reproduction. Plasmodium falciparum (P. falciparum) is a major human malaria parasite. To understand the functions of miRNAs in the mosquito resistance to Plasmodium infection, we analyzed the miRNA-mRNA interactions after female mosquitoes taking a P. falciparum-infected blood meal or an uninfected blood meal. Comparison of the interactions revealed enhanced miRNA-mRNA interactions after P. falciparum infection involving a group of immunity-related genes. In summary, this study has provided a systematic view and significantly advanced our understanding of the miRNA functions in mosquito reproduction and P. falciparum infection. / PHD / Female mosquito is able to transmit lots of disease to the human when it bites for blood. The blood meal provides necessary nutrient for mosquito reproduction and spread the pathogens such as malaria and Zika at the same time. Thus understanding the molecular mechanism behind this process would be greatly helpful to develop novel vector control strategy. Here, we found a distinct class of RNAs contributing to the regulation of mosquito blood meal and parasite infection. We used a novel biochemical method to decoding the special role of these kinds of RNAs in these processes. We found them regulating mosquito metabolism and immunity. This study significantly deepened our knowledge about the process of mosquito reproduction and transmitting diseases.