Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes

Abstract

Telomeric repeat-containing RNA (TERRA) has been identified as a telomere-associated regulator of chromosome end protection. Here, we report that TERRA can also be found in extracellular fractions that stimulate innate immune signaling. We identified extracellular forms of TERRA in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. Cell-free TERRA (cfTERRA) could be isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. cfTERRA is a shorter form (∼200 nt) of cellular TERRA and copurifies with CD63-and CD83-positive exosome vesicles that could be visualized by cyro-electron microscopy. These fractions were also enriched for histone proteins that physically associate with TERRA in extracellular ChIP assays. Incubation of cfTERRAcontaining exosomes with peripheral blood mononuclear cells stimulated transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10) Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. These findings imply a previously unidentified extrinsic function for TERRA and a mechanism of communication between telomeres and innate immune signals in tissue and tumor microenvironments.TERRA | telomere | exosome | innate immunity | cytokine

The crosstalk of telomere dysfunction and inflammation through cell-free TERRA containing exosomes

“…Based on these data and our recently published study, 20 we propose that telomere-induced stress leads to the upregulation of TERRA that is processed into smaller fragments and secreted in exosome-like vesicles. These TERRA containing exosomes are relatively stable and circulate in extracellular spaces, including blood and plasma.…”

“…10 We previously demonstrated that exosomal TERRA is smaller than cellular TERRA, and is associated with a small percentage of telomeric DNA and histone protein components. 20 Using identical extraction methods described previously, we measured exosome-associated TERRA by dot blot (Fig. 1C).…”

“…We also observed that cfTERRA is co-purified with extracellular telomeric DNA (G and C-rich strands), and is associated with histone, as detected by extracellular ChIP assays with histone H3 specific antibodies. 20 Others have reported that nucleosomes and telomeric DNA can be found in cell-free fractions, and are considered contaminants of exosomes. We addressed this concern by demonstrating the exosome-associated cfTERRA is highly resistant to RNAse, while the majority of chromatin DNA can be eliminated by DNase.…”

“…20 These cfTERRA-containing exosomes were strong inducers of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs). We also demonstrated that synthetic TERRA was able to stimulate inflammatory cytokines when delivered by liposomes.…”

Role of exosomes in the protection of cellular homeostasis

“…55 Telomeric repeat-containing RNA (TERRA) is release into exosomes by cells that present telomere dysfunction. 56,57 Although several works have reported the presence of DNA in exosomes, the function of exosomes in the regulation of DNA homeostasis is far from been elucidated.…”