| Autor: |
Scheepbouwer C; Department of Neurosurgery, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Cancer Biology, Amsterdam, Netherlands.; Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands., Aparicio-Puerta E; Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY, USA., Gómez-Martin C; Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands., van Eijndhoven MAJ; Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands., Drees EEE; Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands.; Department of Hematology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands., Bosch L; Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands., de Jong D; Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands., Wurdinger T; Department of Neurosurgery, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Cancer Biology, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands., Zijlstra JM; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands.; Department of Hematology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands., Hackenberg M; Bioinformatics Laboratory, Biomedical Research Centre (CIBM), Biotechnology Institute, PTS, Avda. del Conocimiento s/n, 18100 Granada, Spain.; Genetics Department, Faculty of Science, Universidad de Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain.; Excellence Research Unit 'Modeling Nature' (MNat), University of Granada, Spain.; Instituto de Investigación Biosanitaria ibs. Granada, University Hospitals of Granada-University of Granada, Spain; Conocimiento s/n 18100, Granada. Spain., Gerber A; Department of Neurosurgery, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Cancer Biology, Amsterdam, Netherlands., Pegtel DM; Department of Pathology, Cancer Center Amsterdam, Amsterdam University Medical Center, VU University, Amsterdam, Netherlands.; Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, Netherlands. |
| Abstrakt: |
Small extracellular vesicles (sEVs) are heterogenous lipid membrane particles typically less than 200 nm in size and secreted by most cell types either constitutively or upon activation signals. sEVs isolated from biofluids contain RNAs, including small non-coding RNAs (ncRNAs), that can be either encapsulated within the EV lumen or bound to the EV surface. EV-associated microRNAs (miRNAs) are, despite a relatively low abundance, extensively investigated for their selective incorporation and their role in cell-cell communication. In contrast, the sorting of highly-structured ncRNA species is understudied, mainly due to technical limitations of traditional small RNA sequencing protocols. Here, we adapted ALL-tRNAseq to profile the relative abundance of highly structured and potentially methylated small ncRNA species, including transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and Y RNAs in bulk EV preparations. We determined that full-length tRNAs, typically 75 to 90 nucleotides in length, were the dominant small ncRNA species (>60% of all reads in the 18-120 nucleotides size-range) in all cell culture-derived EVs, as well as in human plasma-derived EV samples, vastly outnumbering 21 nucleotides-long miRNAs. Nearly all EV-associated tRNAs were protected from external RNAse treatment, indicating a location within the EV lumen. Strikingly, the vast majority of luminal-sorted, full-length, nucleobase modification-containing EV-tRNA sequences, harbored a dysfunctional 3' CCA tail, 1 to 3 nucleotides truncated, rendering them incompetent for amino acid loading. In contrast, in non-EV associated extracellular particle fractions (NVEPs), tRNAs appeared almost exclusively fragmented or 'nicked' into tRNA-derived small RNAs (tsRNAs) with lengths between 18 to 35 nucleotides. We propose that in mammalian cells, tRNAs that lack a functional 3' CCA tail are selectively sorted into EVs and shuttled out of the producing cell, offering a new perspective into the physiological role of secreted EVs and luminal cargo-selection. |
