The RNA-binding protein hnRNPU regulates the sorting of microRNA-30c-5p into large extracellular vesicles

Andreas Zietzer; Mohammed Rabiul Hosen; Han Wang; Philip Roger Goody; Marc Sylvester; Eicke Latz; Georg Nickenig; Nikos Werner; Felix Jansen

doi:10.1080/20013078.2020.1786967

The RNA-binding protein hnRNPU regulates the sorting of microRNA-30c-5p into large extracellular vesicles

J Extracell Vesicles. 2020 Jul 2;9(1):1786967. doi: 10.1080/20013078.2020.1786967.

Authors

Andreas Zietzer¹, Mohammed Rabiul Hosen¹, Han Wang¹, Philip Roger Goody¹, Marc Sylvester², Eicke Latz³, Georg Nickenig¹, Nikos Werner^{1

4}, Felix Jansen¹

Affiliations

¹ Heart Center Bonn, Medical Department II, University Hospital Bonn, Venusberg-Campus 1, Bonn, Germany.
² Core Facility Mass Spectrometry, Institute of Biochemistry and Molecular Biology,Medical Faculty, University of Bonn, Bonn, Germany.
³ Institute of Innate Immunity, University Hospital Bonn, University of Bonn, Bonn, Germany.
⁴ Internal Medicine III, Krankenhaus Der Barmherzigen Brüder Trier, Trier, Germany.

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.

Keywords: Extracellular vesicles; RNA-binding protein; SAF-A; endothelial cell; export; hnRNPU; microRNA; microRNA-30c-5p; microvesicles; migration; transfer.