van Hout, F. A. H.; Himanen, S. V.; Vihervaara, A.; Miesen, P.

The Aedes aegypti mosquito transmits arboviruses such as dengue, Zika, and chikungunya virus, posing a substantial threat to global health. The mosquito immune response determines virus transmission, yet, insight into the transcriptional regulation of mosquito immunity remains limited. In this study, we optimized the nascent RNA-sequencing method Precision Run-On sequencing (PRO-seq) for Aedes aegypti Aag2 cells. PRO-seq enabled profiling the distribution of active RNA polymerases across the mosquito genome at nucleotide precision and identified the exact transcription start nucleotides (TSN) of expressed genes. Based on exact positioning of the TSN, we uncovered core promoter elements, including the Initiator and Downstream Promoter Elements. Notably, RNA polymerase accumulates at the promoter-proximal region of genes, but transcribes into the divergent region to a lesser extent than in vertebrates. To investigate rapid and dynamic immune responses, Aag2 cells were immune-stimulated with heat-inactivated E. coli for 1 and 4 hours. Differential gene expression analysis revealed different groups of genes to be induced over time. While Clip domain serine proteases and antimicrobial peptides were induced promptly and sustained, a delayed stress response consisting of heat shock-related genes was only seen at 4 hours after stimulation. Strikingly, gene sets with different temporal expression profiles were associated with distinct transcription factor binding motifs. Altogether, our study provides valuable insights into the functional genomics of Aedes aegypti and indicates that even within a rapid response, different dynamics emerge, potentially regulated by distinct transcription factors. These insights are crucial to gain a better understanding of the mosquito immune response and its regulation.