Functional Genomics Unit, Institut Pasteur de Montevideo , Montevideo, Uruguay.
Molecular Virology Laboratory, Nuclear Research Center, Faculty of Science, Universidad de la República , Montevideo, Uruguay.
RNA Biol. 2020 Aug;17(8):1168-1182. doi: 10.1080/15476286.2019.1708548. Epub 2019 Dec 29.
Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5' tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5' tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.
细胞外囊泡 (EVs) 是细胞衍生的纳米颗粒,可在细胞间充当核酸的天然载体。它们作为治疗性核酸(如小 RNA)的递送载体具有优势。一旦纯化,就可以通过电穿孔或转染将所需的核酸加载到 EV 中。一种有吸引力的替代方法是用所需的小 RNA 转染细胞,并利用细胞机制将 RNA 分选到 EV 中。这种可能性尚未得到充分探索,因为人们认为细胞仅分泌特定的 RNA。然而,我们假设,即使存在选择性分泌,浓度驱动的 RNA 分选到 EVs 仍然是可行的。为了证明这一点,我们用甘氨酸 5' tRNA 片段转染细胞,我们之前已经证明这些片段可以更好地抵抗核糖核酸酶。然后,我们测量了它们在 EV 和受体细胞中的水平,发现与随机序列的不稳定 RNA 相反,这些 tRNA 片段以与用于转染的 RNA 浓度成正比的量存在于囊泡和受体细胞中。具有随机序列的其他稳定寡核苷酸也获得了相似的效率。我们的结果表明,RNA 稳定性是维持高细胞内浓度所必需的关键因素,这是高效非选择性 RNA 分选到 EV 并递送到细胞的前提。鉴于甘氨酸 5' tRNA 片段属于应激诱导的 tRNA 片段组,这些片段经常在细胞外空间和生物液中检测到,我们提出细胞外 tRNA 片段的上调是细胞应激的结果,可能参与细胞间信号传递。