Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Author:

Tosar Juan Pablo1234ORCID,Segovia Mercedes5,Castellano Mauricio12,Gámbaro Fabiana26,Akiyama Yasutoshi34,Fagúndez Pablo12,Olivera Álvaro7,Costa Bruno12ORCID,Possi Tania2,Hill Marcelo5,Ivanov Pavel348ORCID,Cayota Alfonso29

Affiliation:

1. Analytical Biochemistry Unit. Nuclear Research Center. Faculty of Science. Universidad de la República, Uruguay

2. Functional Genomics Unit, Institut Pasteur de Montevideo, Uruguay

3. Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Boston, MA, USA

4. Department of Medicine, Harvard Medical School, Boston, MA, USA

5. Laboratory of Immunoregulation and Inflammation, Institut Pasteur de Montevideo, Uruguay. Immunobiology Department, Faculty of Medicine, Universidad de la República, Uruguay

6. Molecular Virology Laboratory, Nuclear Research Center. Faculty of Science. Universidad de la República, Uruguay

7. Centro Universitario Regional Este, Universidad de la República, Uruguay

8. The Broad Institute of Harvard and M.I.T., Cambridge, MA, USA

9. Department of Medicine, University Hospital, Universidad de la República, Uruguay

Abstract

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.

Funder

Agencia Nacional de Investigación e Innovación

Comisión Sectorial de Investigación Científica

National Institutes of Health

Publisher

Oxford University Press (OUP)

Subject

Genetics

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