The functional roles of ANG, RNASE1, and AGO2 in the biogenesis and stablization regualtion of cellular and extracellular tDRs

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that tRNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, the RNA modifications on tDRs have been obstacles for accurately identifying tDRs with conventional RNA sequencing. Here, we use AlkB-facilitated methylation sequencing (ARM-seq) to uncover a comprehensive landscape of cellular and extracellular tDR expression in various cells during different stress responses. We find that extracellular tDRs have a distinct fragmentation signature from intracellular tDRs and these tDR signatures are better discriminators of different stress responses than miRNAs. The distinct extracellular tDR fragmentation pattern and signatures are also noted in plasma from patients on cardiopulmonary bypass. Most importantly, we demonstrate that ANG and RNASE1 critically contribute to the stress-modulated cellular and extracellular tDR signatures and we identify a sub population of extracellular tDRs that are either transported or stabilized by AGO2. Together, our findings further the knowledge about tDR biogenesis in both cells and extracellular environments in response to different stressors and position extracellular tDRs as reliable candidates for novel biomarkers in human diseases. Overall design: Cellular and extracellular tDRs expression profiles of ANG knockout, RNASE1 knockout, and AGO2 knockout HEK cells with different stress treatments (3 independent replicates for each condition)