Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Abstract

A major proportion of extracellular RNAs (exRNAs) do not co-isolate 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 RI-SEC-seq: 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 can be sensed by dendritic cells. Extracellular ribosomes and/or tRNAs could therefore be decoded as damage-associated molecular patterns.

Extracellular tRNAs and tRNA-derived fragments

“…To test this hypothesis, we added recombinant ribonuclease inhibitor (RI) directly to the cell-conditioned medium of different mammalian cell lines. To our surprise, addition of RI dramatically changed extracellular RNA profiles [119]. Without RI, most exRNAs outside EVs were 30-31 nt 5ʹ tRNA halves derived from acceptors of glycine and glutamic acid, as has been described in previous sections of this review.…”

“…However, the authors of this study purified EVs by ultracentrifugation, which results in less pure preparations compared to the density gradient method used in [72]. We have combined density gradient separation with non-radioactive digoxigenin-based Northern blot, and found that, indeed, EVs from nonstressed cells contain mostly full-length tRNAs instead of tRNA-derived fragments [119]. We will resume this discussion in chapter six.…”

“…These tRNA halves were originated in the extracellular space, by enzymatic fragmentation of fulllength tRNAs. Thus, in samples with high RNase activity such as serum, full-length tRNAs are only found inside EVs, while the most stable tRNA halves can accumulate in the outside [119].…”

“…Based on the use of a non-retroviral thermostable reverse transcriptase and sequencing [70,72], electrophoretic profiles [114,115] or Northern blot [95,119] it seems clear that EVs contain more full-length tRNAs than tRNA-derived fragments. However, the mechanisms used by cells to encapsulate tRNAs in EVs are still not completely understood.…”

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], noncoding 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.…”