Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Abstract

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities

“…This method, called RI-SEC-seq (for: size-exclusion chromatography [SEC] fractionation of RNase inhibitor [RI]-treated cell-conditioned medium), enabled us to study RNAs in the form they were effectively released from cells. Following this procedure, we discovered the presence of extracellular ribosomes and tRNAs, and the extracellular biogenesis of extracellular tRNA-derived fragments (Tosar et al, 2020). The latter was independently validated by others, using cell lines devoid of RNase I (Nechooshtan et al, 2020).…”

“…Analyze data as described in Tosar et al (2020). Briefly, identify and clip 3' adapter sequences, map clipped reads to the human genome and count features corresponding to microRNAs, tRNAs, www.bio-protocol.org/e3918 Bio-protocol 11(04): e3918.…”

“…Perform single-end sequencing in an Illumina platform. In our study (Tosar et al, 2020) we sequenced for 200 cycles in order to be able to detect some full-length RNAs (e.g., 5.8S rRNA) and not only their fragments, based on gel electrophoresis results (Figure 2). However, doing so has a major drawback.…”

How does an RNA selfie work? EV‐associated RNA in innate immunity as self or danger

“…To be sure, EV association may not be needed to trigger responses. In addition to being loaded into EVs [17], non‐coding RNA species from MCF‐7 cells, mainly dimers of tRNA‐Gly‐GCC halves and ribosomal RNA, are also released in non‐EV fractions [18]. In this form, they can activate primary bone‐marrow‐derived dendritic cells and induce the release of IL‐1β.…”

Exogenous RNA as a Regulatory Signal during a Plant’s Interaction with the Biotic Environment: An Evolutionary Perspective and Future Applications in Agriculture

“…While similar mechanisms have not yet been found to be involved in RNA uptake in plants, the clathrin-mediated endocytosis pathway has been shown to be responsible for uptake of exogenous dsRNA in the white mold phytopathogen Sclerotinia sclerotiorum [ 42 ]. Recent findings detailing new types of extracellular RNAs and mechanisms of RNA secretion in non-plant organisms [ 43 , 44 ] also suggest that we cannot exclude the possibility of discovering new mechanisms of naked regRNA or ribonucleoprotein complexes transfer, both by secretion and uptake, in plant cells in addition to the reported extracellular vesicles-mediated transfer of sRNAs.…”