Abstract
Some RNA species, especially microRNAs, are non-randomly sorted into exosomes, but how selectivity of RNA exosomal sorting is achieved is unknown. We found that all three variants of RNA-binding ubiquitin E3 ligase (MEX3C)–MEX3C-1, MEX3C-2, and MEX3C-3 –interact with adaptor-related protein complex 2 (AP-2), a cargo adaptor in clathrin-mediated endocytosis. MEX3C’s C-terminal RING finger domain and the hnRNP K homology (KH) domain shared by the three MEX3C variants are both necessary for MEX3C/AP-2 interaction. MEX3C associates with the endolysosomal compartment through an endocytosis-like process. siRNA-mediated inhibition of the MEX3C or AP-2 complex substantially decreased exosomal but not cellular microRNA miR-451a expression. Exosomal sorting is ceramide-dependent but not ESCRT-dependent in microRNA miR-451a. That RNA-binding protein associates with membrane trafficking machinery, and that its involvement in exosomal microRNA expression, suggest the existence of a mechanism for specific recruiting of RNA molecules to endosomes for subsequent exosomal sorting.
Highlights
Extracellular vesicles (EVs) contain RNA species such as microRNAs, and may mediate intercellular RNA transfer [1]
Since mRNAs coding for MEX3C-2 is most widely expressed [28], we transiently expressed C-terminal Flag-tagged MEX3C-2 in HEK293 cells, and used anti-Flag antibody-conjugated agarose beads to pull down MEX3C-2 and its associated proteins
All three MEX3C variants (MEX3C-1, MEX3C-2, and MEX3C-3) can associate with adaptor-related protein complex 2 (AP-2), and the interaction is RNA independent: 1) RNase treatment eliminated MEX3C/THOC4 interaction but not MEX3C/AP-2 interaction; 2) The C-terminal truncated MEX3C mutant (MEX3C-1ΔC) preserved RNA binding and nuclear export activity [28] but lost the ability to interact with AP-2; 3) MEX3C-1mKH mutant lost RNA binding activity [25,26] yet still interacted with AP-2
Summary
Extracellular vesicles (EVs) contain RNA species such as microRNAs (miRNA), and may mediate intercellular RNA transfer [1]. Consistent with the observation that ceramide triggers budding of exosome vesicles into multivesicular endosomes [3], miRNAs can be sorted to the exosomes through a ceramide-dependent mechanism [4]. The Endosomal Complexes Required for Transport (ESCRT) complexes are involved in exosome biogenesis [5], whether they play a role in miRNA exosomal sorting is unclear. Some miRNAs contain sequence motifs to guide their EV sorting [6,7]. RNA-binding proteins, such as SYNCRIP, hnRNPA2B1, and Y-box protein 1, mediate the exosomal sorting of miRNAs [8,9,10]. The link between ribonucleoprotein particles and membrane vesicles is unknown
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