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.
Subject(s)
Extracellular Vesicles/genetics , RNA, Transfer/genetics , RNA/genetics , Ribosomes/genetics , Animals , Culture Media, Conditioned/pharmacology , Enzyme Inhibitors/pharmacology , High-Throughput Nucleotide Sequencing , Humans , Ribonucleases/antagonists & inhibitors , Ribonucleases/geneticsABSTRACT
Rat brain ribonucleases (RNases) were studied. Three types of RNases with maximum activities at pH 5.0, 7.2 and 9.5 were found. The activity of the pH 7.2 enzyme can be detected only by avoiding the interference of a very active inhibitor with p-chlor-mercuri-benzoic acid (PCMB). The effect of bivalent cations (Ca2+, Mg2+), Na+ and ethylenediamine tetraacetic acid (EDTA) was investigated. The activities studied showed a different subcellular distribution. Changes in RNase activity during postnatal rat brain development were studied. The pH 7.2 and 9.5 enzymes have a similar behavior increasing up to the 15th-20th day and remaining constant thereafter. The pH 5.0 enzyme remains constant from the 5th to the 20th day, decreasing thereafter.
Subject(s)
Brain/enzymology , Ribonucleases/metabolism , Age Factors , Animals , Brain/drug effects , Cations, Divalent/pharmacology , Chloromercuribenzoates/pharmacology , Detergents/pharmacology , Edetic Acid/pharmacology , Female , Hydrogen-Ion Concentration , Male , Rats , Ribonucleases/antagonists & inhibitorsABSTRACT
Measurement of ribonucleases activity in rat diaphragm muscle formed the central part of the work described in this thesis. Two main forms of ribonucleases activity at physiological pH were demonstrated, as characterised by cation dependence and inhibition by ribonuclease inhibitor. These two forms are believed to be alkaline ribonuclease I and alkaline ribonuclease IV previously characterised in liver. Rat diaphragm contained much less alkaline ribonuclease IV activity than liver and kidney. No effect on alkaline ribonuclease IV activity by insulin, fasting, actinomycin D, phrenic denervation, or cycloheximide was observed. Insulin did not have any effect on alkaline ribonuclease I activity, nor on ribonuclease inhibitor activity. Partially purified inhibitor from liver and skeletal muscle stimulated the incorporation of 14C-leucine by heaptic ribosomes. However, this stimulation in amino acid incorporation by the inhibitor seems to be unrelated to the stimulation of incorporation of amino acids caused by insulin (AU)