ABSTRACT

The transfer of microRNAs (miRs) via extracellular vesicles (EVs) is a functionally relevant mechanism of intercellular communication that regulates both organ homoeostasis and disease development. Little is known about the packaging of miRs into EVs. Previous studies have shown that certain miRs are exported by RNA-binding proteins into small EVs, while for other miRs and for large EVs, in general, the export mechanisms remain unclear. Therefore, a proteomic analysis of endothelial cell-derived large EVs was performed, which revealed that heterogeneous nuclear ribonucleoprotein U (hnRNPU) is abundantly present in EVs. EVs were characterized by electron microscopy, immunoblotting and nanoparticle tracking analysis. Taqman microRNA array and single qPCR experiments identified specific miR patterns to be exported into EVs in an hnRNPU-dependent way. The specific role of hnRNPU for vesicular miR-sorting was confirmed independently by gain- and loss-of-function experiments. In our study, miR-30c-5p was the miR whose export was most significantly regulated by hnRNPU. Mechanistically, in silico binding analysis showed that the export of miRs into EVs depends on the binding efficiency of the respective miRs to hnRNPU. Among the exported miRs, a significant enrichment of the sequence motif AAMRUGCU was detected as a potential sorting signal. Experimentally, binding of miR-30c-5p to hnRNPU was confirmed independently by RNA-immunoprecipitation, electrophoretic mobility shift assay and reciprocally by miR-pulldown. Nuclear binding of miR-30c-5p to hnRNPU and subsequent stabilization was associated with a lower cytoplasmatic abundance and consequently reduced availability for vesicular export. hnRNPU-dependent miR-30c-5p export reduced cellular migration as well as pro-angiogenic gene expression in EV-recipient cells. In summary, hnRNPU retains miR-30c-5p and other miRs and thereby prevents their export into large EVs. The data presented provide a novel and functionally relevant mechanism of vesicular miR export.

摘要

microRNA（miRs）通过细胞外囊泡（EVs）的转移是功能上相关的细胞间通讯机制，可调节器官均位和疾病发展。关于将miR包装到EV的知之甚少。先前的研究表明，某些miR通过RNA结合蛋白输出到小型EV中，而对于其他miR和大型EV，总的来说，其输出机制尚不清楚。因此，对源自内皮细胞的大型电动汽车进行了蛋白质组学分析，发现电动汽车中大量存在异质核糖核蛋白U（hnRNPU）。电动汽车通过电子显微镜，免疫印迹和纳米颗粒跟踪分析进行表征。Taqman microRNA阵列和单次qPCR实验确定了特定的miR模式，将以hnRNPU依赖的方式输出到EV中。hnRNPU在水泡miR分选中的特定作用已通过功能获得和丧失实验独立确认。在我们的研究中，miR-30c-5p是受RNRNPU最显着调控的miR。机械上，在计算机上结合分析表明，miR向电动汽车的出口取决于各自miR与hnRNPU的结合效率。在输出的miRs中，序列基序AAMRUGCU的大量富集被检测为潜在的分类信号。实验上，miR-30c-5p与hnRNPU的结合通过RNA免疫沉淀，电泳迁移率移动分析独立确定，而通过miR-pulldown相互确认。miR-30c-5p与hnRNPU的核结合以及随后的稳定与较低的细胞质丰度相关，因此减少了水泡输出的可用性。hnRNPU依赖的miR-30c-5p输出减少了EV受体细胞中的细胞迁移以及促血管生成基因的表达。总之，hnRNPU保留了miR-30c-5p和其他miR，从而阻止了它们出口到大型电动汽车中。