Comparative transcriptomic analysis of human and Drosophila extracellular vesicles reveals extensive conservation. Comparative transcriptomic analysis of human and Drosophila extracellular vesicles reveals extensive conservation

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that Drosophila, like human cells release exosome-like EVs with diameter ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNA pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these extensive similarities suggest that EVs could also enable RNA-mediated intercellular communication in Drosophila. Overall design: We performed RNA-seq on extracellular vesicles purified from of human and Drosophila cell line cultures. S2R+ and D17 Drosophila EVs were analyzed, along with human A431 and HepG2 EVs. No ribosomal RNA depletion or polyA selection was performed on EV samples. For comparative analyses, we also analyzed total cellular RNA from Drosophila D17 and human HepG2. Ribodepletion was performed on cellular samples.

细胞外囊泡（Extracellular vesicles, EVs）是一类膜包被的纳米颗粒，携带有特定组成的遗传物质。在哺乳动物中，EVs可介导多种生物载荷与信号分子的横向转移，其中尤以微小RNA（microRNA, miRNA）和信使RNA（messenger RNA, mRNA）类分子最为典型。这种细胞间通讯形式是否广泛存在于其他后生动物中，目前仍不明确。本研究首次对果蝇（Drosophila）与人类细胞模型来源的EVs开展了平行比较形态学与转录组学表征。电子显微镜（Electron microscopy）观察显示，与人类细胞类似，果蝇细胞也会释放直径介于30至200纳米的外泌体样EVs，其内部含有复杂的转录本群体。RNA测序（RNA sequencing, RNA-seq）结果表明，人类与果蝇EVs中均存在丰富的核糖体RNA假基因与逆转录转座子。人类样本中穹窿RNA（Vault RNAs）与Y RNA（Y RNAs）含量丰富，而果蝇EVs中参与假尿苷酸化的小核仁RNA（small nucleolar RNAs, snoRNAs）最为普遍。研究还鉴定出大量mRNA，其序列主要由外显子区域构成且呈现全长读段覆盖特征，功能富集分析显示这些mRNA主要富集于翻译过程与电子传递链相关功能。相较于人类系统，这些广泛的相似性提示，果蝇中EVs同样可能介导RNA依赖的细胞间通讯。

实验整体设计：我们对人类与果蝇细胞系培养物中纯化得到的细胞外囊泡开展了RNA测序。分析样本包括果蝇S2R+、D17细胞来源的EVs，以及人类A431、HepG2细胞来源的EVs。所有EV样本均未进行核糖体RNA去除或polyA尾选择富集。为开展比较分析，我们同时提取了果蝇D17细胞与人类HepG2细胞的总细胞RNA，并对这些细胞样本进行了核糖体RNA去除处理。