Developing C. elegans as a model to understand tRNA-fragment biogenesis and function

Project Summary Small RNAs are ubiquitous regulators of eukaryotic gene expression in nearly all aspects of physiology from plants to humans. They function to regulate all facets of gene expression including transcription, RNA stability, and translation. An emerging class of small regulatory RNAs are tRNA-fragments (tRFs), produced from nucleolytic cleavage of tRNAs. tRFs have been implicated in cancer, neurodegenerative disease, viral infection, fertility, epigenetic inheritance, and aging. While the biogenesis of these RNAs is poorly understood, tRFs have been demonstrated to play roles in the regulation of transcription, posttranscriptional regulation of mRNA stability, and translation. Yet, the functions of tRFs throughout the different tissues of an organism in regulating cellular physiology are unknown. My lab is developing the roundworm C. elegans (worms) as a model to dissect the molecular mechanisms underlying the biogenesis tRF and cellular functions throughout the animal. The robust genetics, physiological assays, and molecular tools available in C. elegans provide a system ripe for the discovery of tRF biology. Importantly, we have developed small RNA-sequencing techniques to detect abundant tRFs in C. elegans, at levels much higher than previously published small RNA-seq datasets. Using these techniques, we will characterize tRFs in the different tissues of the fully developed adult using bulk and single-cell small RNA-seq. After determining the spatial and temporal expression of tRF species we will utilize both forward and reverse genetics to determine new factors required for their biogenesis. Further, we will employ biochemical enrichment strategies to determine factors that interact with and affect tRF function. Upon determining genes required for tRF biogenesis and function we will use mutant alleles of these factors to determine what roles these molecules have in regulating the physiology of the organism. Finally, we will use worm strains carrying loss-of-function mutations in RNA modifying enzymes to determine how RNA modifications affect tRF biogenesis, functions, and further regulate the physiology of the animal. The work on tRFs in C. elegans in my lab represents the first systematic dissections of all aspects of tRFs biology in a whole organism. What we learn about tRF biology in worms will be used to generate hypotheses about tRFs in other organisms. As tRFs have been implicated in many aspects of normal physiology in organisms ranging from yeast to humans, as well as in a wide range of diseases, comprehensively understanding how tRF are regulated and function represents a highly under-addressed aspect of biology.