RNY1 partitions into extracellular vesicles and ribonucleoprotein particles and regulates macrophage programming by airway fluids during inflammation.

YRNAs are small noncoding RNAs that are abundant in the extracellular space. Prior research has shown that YRNAs are present in most biofluids and that their levels fluctuate during disease. Yet our understanding of YRNA dynamics in the context of biofluid extracellular particles during disease processes is limited. In this study, we found that RNY1, one of two murine YRNAs, increases in airway fluid during allergen-induced lung inflammation and correlates with neutrophil infiltration. Using RNase sensitivity assays and size exclusion chromatography, we determined that airway fluid RNY1 is protected by both EVs and protein-containing structures in distinct extracellular compartments. In contrast the other murine YRNA, RNY3, is only enriched in BALF EVs, suggesting that RNY1 is uniquely partitioned in heterogenous extracellular particles. Both EV-enriched and protein-enriched RNY1-containing extracellular compartments were able program robust pro-inflammatory transcriptional responses in target macrophages. RNY1 contributed to this programming, as macrophages treated with a protein-enriched compartment isolated from RNY1-/- mice demonstrated lower induction of interferon-associated genes. Together, this study demonstrates that YRNAs are heterogeneously packaged in complex biofluids and contribute to intercellular signaling axes during inflammation.