Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

Juan Pablo Tosar,Tania Possi,Mauricio Castellano,Álvaro Olivera,Fabiana Gámbaro,Marcelo Hill,Bruno Costa,Mercedes Segovia,Pavel Ivanov,Pablo Fagúndez,Alfonso Cayota,Yasutoshi Akiyama

doi:10.1093/nar/gkaa674

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.

Highlights

Extracellular RNA profiling in biofluids such as urine, plasma or serum is a promising approach for early disease detection and monitoring in minimally invasive liquid biopsies 1
Because RNA is less stable than DNA in biofluids such as plasma due to the high RNase activity of these samples 8, Extracellular RNA (exRNA) are typically studied in the context of lipid membrane-containing extracellular vesicles (EVs) 7,9,10 or lipoprotein particles (LPPs) extracellular stability by their association with proteins[13,14]
A substantial fraction of exRNAs is not encapsulated inside EVs, yet the extracellular nonvesicular

Summary

Introduction

Extracellular RNA (exRNA) profiling in biofluids such as urine, plasma or serum is a promising approach for early disease detection and monitoring in minimally invasive liquid biopsies 1. Plasma cellfree DNA analysis has proven powerful to detect cancer-associated mutations or altered DNA methylation events , exRNA analysis has the potential to inform about transcript expression, posttranscriptional modifications and splicing variants 4. Because RNA is less stable than DNA in biofluids such as plasma due to the high RNase activity of these samples 8, exRNAs are typically studied in the context of lipid membrane-containing extracellular vesicles (EVs) 7,9,10 or lipoprotein particles (LPPs) extracellular stability by their association with proteins[13,14]. Extracellular soluble ribonucleoproteins remain the least studied exRNA carriers 15, with most attention far placed at the level of EVs. A new extracellular nanoparticle was recently discovered

Methods

Results

Conclusion