Institute of Biochemistry, Justus Liebig University of Gießen, 35392 Gießen, Germany.
Institute for Tumor Immunology, Center for Tumor Biology and Immunology (ZTI), Philipps University of Marburg, 35043 Marburg, Germany.
Cells. 2021 Oct 6;10(10):2674. doi: 10.3390/cells10102674.
Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.
细胞外囊泡 (EVs) 在细胞间通讯中起着重要作用，是生物物质（如各种编码和非编码 RNA 类）的载体，其中一些被证明可以选择性地靶向囊泡。然而，对于促成这种特异性的蛋白质因子、机制和序列元件，目前仍知之甚少。在这里，我们使用一种报告系统，该系统会产生不同类型的修饰转录本，以解析释放到 EV 中的 RNA 的特异性决定因素。首先，我们发现小 RNA 比大 RNA 更有效地被包裹到 EV 中，其次，我们确定了几种内源性 RNA 转录物在 EV 中的绝对数量（U6 snRNA、U1 snRNA、Y1 RNA 和 GAPDH mRNA）。我们发现与 pol II 衍生的转录物相比，RNA 聚合酶 III (pol III) 转录物更有效地分泌到 EV 中。令人惊讶的是，我们的定量分析显示，基于过表达报告转录本和内源性 RNA，囊泡中 RNA 的积累相对于细胞总水平没有增加。RNA 似乎仅以低拷贝数与 EV 相关，每个 EV 中存在 0.02 到 1 个分子。这种 RNA 相关性可能反映了囊泡内部的包裹或在囊泡表面的结合状态较不紧密。
