Molecular factors governing the biogenesis and release of short immunostimulatory RNAs - Project Summary and Abstract Host defense against microbial pathogens involves a complex interplay of cellular and molecular mechanisms, including those mediated by short non-coding RNAs (sncRNAs). For over two decades, work in this area has focused largely on microRNAs (miRNAs) and small interfering RNAs (siRNAs), yet recent advances in RNA biology have uncovered additional, often more abundant classes of functional sncRNAs derived from transfer RNAs (tRNAs). The contributions of these non-miRNA-sncRNAs to immune responses remain poorly defined, in part because standard RNA-seq fails to capture their full repertoire. In particular, 5′-tRNA halves, the most abundant class of tRNA-derived sncRNAs, bear a 2′,3′-cyclic phosphate (cP) at their 3′-end, which prevents ligation to 3′-adaptors during conventional library preparation and renders them largely invisible to standard sequencing. To overcome this barrier, we developed cP-RNA-seq, a specialized sequencing method that selectively captures 5′-tRNA halves and other cP-containing sncRNAs, together with tRNA half-specific TaqMan RT-qPCR assays for sensitive, quantitative detection. Using these tools, we recently demonstrated that mycobacterial infection and surface Toll-like receptor (TLR) activation markedly upregulate 5′-tRNA halves in human monocyte-derived macrophages (HMDMs) and their secreted extracellular vesicles (EVs). Moreover, specific 5′-tRNA halves act as potent immunostimulatory ligands by activating the endosomal sncRNA receptor TLR7 and inducing cytokine production when delivered into recipient HMDM endosomes via EVs. Despite these advances, the mechanistic steps that generate immunostimulatory 5′-tRNA halves remain largely unexplored. A key unresolved question is what molecular events follow tRNA anticodon-loop cleavage to yield distinct tRNA halves that can be selectively exported. In HMDMs, only 5′-tRNA halves, and not their 3′-counterparts, are efficiently packaged into EVs, implying that “nicked-tRNAs” must be actively unwound and then selectively sorted. Recent evidence implicates a subset of DExD/H-box RNA helicases in unwinding nicked-tRNAs in diverse cell types, suggesting that these enzymes could function as “release factors” for 5′-tRNA halves. Our work further points to a dedicated EV cargo-selecting RNA-binding protein that specifically recognizes 5′-tRNA halves, supporting a model in which helicase-driven release and sequence-specific recognition act together to enable their selective packaging into EVs and subsequent engagement of TLR7. In this study, we will use integrated biochemical, sequencing, and functional approaches to establish mechanistic proof-of-concept that a helicase- RNA-binding protein axis governs the biogenesis, recognition, and EV export of immunostimulatory 5′-tRNA halves in macrophages. The results will uncover a previously underappreciated sncRNA pathway in innate immune regulation and lay a foundation for exploiting immunostimulatory tRNA halves and their upstream regulators as biomarkers and therapeutic entry points in infection and inflammation.
