Background/Purpose: Extracellular vesicles (EVs) are small structures that enclose a variety of nucleic acids. While much of the published work on EV RNA has focused on the role of mRNAs and microRNAs in intercellular communication, these RNAs are a small fraction of the RNA cargoes of EVs (1). RNA surveillance pathways eliminate defective RNAs and are also implicated in maintaining immunological tolerance to host RNA (2). We hypothesized that EVs package defective noncoding RNAs (ncRNAs) as part of a novel RNA surveillance pathway, which may prevent these RNAs from being recognized as foreign and activating an interferon response.

Methods: We generated mouse macrophage RAW 264.7 cell lines that stably express doxycycline-inducible shRNAs against Dis3l2 as well as control nonsilencing shRNAs. Dis3l2 is a ribonuclease that degrades aberrant structured ncRNAs in cytosol. We purified EVs from these cell lines and used a thermostable group II intron reverse transcriptase to make cDNA libraries and sequence RNA from whole cells and EVs, which allowed us to characterize highly structured RNAs that are missed by conventional RNA-Seq. We also performed conventional RNA-Seq on Dis3l2-depleted cells. To determine effects of preventing exosome packaging, we treated RAW 264.7 cells with pharmacological EV inhibitors and performed Northern blots and qPCR on cellular RNA after drug treatment.

Results: Across multiple classes of RNAs, including small ncRNAs, repeat RNAs, mRNAs, and microRNAs, EVs are enriched for aberrant transcripts. Many aberrant transcripts are further enriched in EVs purified from cells depleted of Dis3l2. We confirmed our sequencing results with Northern blots. Transcriptomic analysis of Dis3l2-depleted RAW 264.7 cells show a striking Type I interferon signature. Treating cells with an EV inhibitor was associated with cellular accumulation of some of the aberrant RNAs found in EVs, as well as upregulation of interferon-stimulated genes.

Conclusion: Our data implicate EVs in a novel RNA surveillance pathway that competes with other surveillance pathways to purge cells of RNAs that may otherwise activate an interferon response. Thus, EVs may be involved in protecting cells from aberrant activation of innate immune sensors. Such pathways may be important for diseases like systemic lupus erythematosus, where aberrant nucleic acid sensing and an interferon response are implicated in pathogenesis. 1. Shurtleff MJ, Yao J, Qin Y, Nottingham RM, Temoche-Diaz MM, Schekman R, et al. Broad role for YBX1 in defining the small noncoding RNA composition of exosomes. Proc Natl Acad Sci U S A. 2017;114(43):E8987-E95.2. Wolin SL, Maquat LE. Cellular RNA surveillance in health and disease. Science. 2019;366(6467):822-7.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/aberrant-noncoding-rnas-are-enriched-in-extracellular-vesicles-and-implicated-in-interferon-activation/