The "hidden" proteins in Aedes aegypti

Abstract Aedes aegypti is a major vector of dengue, chikungunya, yellow fever, and Zika viruses. The global incidence of dengue increased significantly in recent decades, directly impacting the US and US territories. No specific treatment for dengue exists and vaccine options are limited. Current prevention depends mainly on effective vector control, which is hindered by increasing insecticide-resistance. Novel strategies to control these devastating diseases, informed by a better understanding of mosquito biology, are being actively explored. Omics-enabled research and effective application of modern genetic methods have significantly improved our understanding of mosquito biology, potentially leading to new targets and/or novel approaches for controlling mosquito-borne infectious diseases. However, an important and prevalent component of the proteome known as the microproteins have been largely "hidden" from genome annotations and thus ignored. The advent of Ribo-seq, a technique that sequences ribosome protected fragments, resulted in the discovery of thousands of small open reading frames (sORFs) engaged with actively translating ribosomes. These sORF-encoded microproteins represent a rapidly growing treasure of the proteome that plays significant roles in various aspects of biology including development, physiology, and metabolism. A significant portion of the sORF-encoded microproteins represent de novo inventions which may be adaptive and confer lineage-specific functions. Long non- coding RNAs (LncRNAs) are a major source of these "hidden" microproteins. We have recently identified LncRNAs in Ae. aegypti that show strong evidence of protein-coding based on ribosome protected fragments and/or evolutionary signatures. We will pursue two specific aims: 1) Discover LncRNA- encoded microproteins in Ae. aegypti using systematic and complementary approaches; 2) Determine the function of selected microproteins in Ae. aegypti male reproduction. The proposed research is significant to basic biology as it has the potential to uncover >1000 "hidden" proteins in Ae. aegypti. These "hidden" proteins can lead to new targets for the control of mosquito-borne diseases as they are often restricted to certain mosquito lineages and the study of microprotein function in male reproduction is relevant to genetic control programs. The proposed research will have a broad impact beyond Ae. aegypti, as a list of well-curated and experimentally supported sORFs and the tools and methods developed in this project will help identify and characterize microproteins in other mosquito lineages or other arthropod vectors including ticks. The proposed research will also open doors to research into other "hidden" non-canonical ORFs and provide valuable resources for future investigations of translation regulation, potentially bridging a gap between transcriptomics and proteomics.