Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes

Abstract

The RNA that is packaged into exosomes is termed as exosomal-shuttle RNA (esRNA); however, the players, which take this subset of RNA (esRNA) into exosomes, remain largely unknown. We hypothesized that RNA binding proteins (RBPs) could serve as key players in this mechanism, by making complexes with RNAs and transporting them into exosomes during the biosynthesis of exosomes. Here, we demonstrate the presence of 30 RBPs in exosomes that were shown to form RNA–RBP complexes with both cellular RNA and exosomal-RNA species. To assess the involvement of these RBPs in RNA-transfer into exosomes, the gene transcripts encoding six of the proteins identified in exosomes (HSP90AB1, XPO5, hnRNPH1, hnRNPM, hnRNPA2B1, and MVP) were silenced by siRNA and subsequent effect on esRNA was assessed. A significant reduction of total esRNA was observed by post-transcriptional silencing of MVP, compared to other RBPs. Furthermore, to confirm the binding of MVP with esRNA, a biotinylated-MVP was transiently expressed in HEK293F cells. Higher levels of esRNA were recovered from MVP that was eluted from exosomes of transfected cells, as compared to those of non-transfected cells. Our data indicate that these RBPs could end up in exosomes together with RNA molecules in the form of RNA–ribonucleoprotein complexes, which could be important for the transport of RNAs into exosomes and the maintenance of RNAs inside exosomes. This type of maintenance may favor the shuttling of RNAs from exosomes to recipient cells in the form of stable complexes.

Extracellular vesicle-mediated RNA release inHistoplasma capsulatum

“…In other systems, these proteins were also identified in association with EVs. For example, in EVs produced by human epithelial cells, 30 RBPs were identified (35), including heterogeneous nuclear ribonucleoproteins (hnRNPs). These proteins are responsible for directing pre-mRNAs in the maturation processes that culminate with transcriptional regulation, alternative splicing, transport, and localization (35).…”

“…For example, in EVs produced by human epithelial cells, 30 RBPs were identified (35), including heterogeneous nuclear ribonucleoproteins (hnRNPs). These proteins are responsible for directing pre-mRNAs in the maturation processes that culminate with transcriptional regulation, alternative splicing, transport, and localization (35). In addition, RBPs in EVs were identified in distinct models as hepatocytes, human embryonic kidney (HEK) cells, and mouse myoblast cells (35–37).…”

“…These proteins are responsible for directing pre-mRNAs in the maturation processes that culminate with transcriptional regulation, alternative splicing, transport, and localization (35). In addition, RBPs in EVs were identified in distinct models as hepatocytes, human embryonic kidney (HEK) cells, and mouse myoblast cells (35–37). Interestingly, one of the RBPs identified in EVs was SND1 (Staphylococcal nuclease domain-containing protein 1), which is a main component of RISC complex (RNA-induced silencing complex) that plays an important role in miRNA function (37).…”

Extracellular Vesicles in Non-Small-Cell Lung Cancer: Functional Role and Involvement in Resistance to Targeted Treatment and Immunotherapy

“…The miRNAs destined to be exported possess a conserved recognition motif, called the EXOmotif, which guides the binding to a series of specialized RBPs [44,45]. The selective binding of these RBPs is necessary for miRNA sorting into EVs, and coordinate the intracellular and extracellular miRNA distribution [46]. Hence, only a specific subpopulation of the total miRNAs are actively secreted outside the cell, and the relative abundance of miRNAs to be directed into the EVs is further controlled by dynamic post-transcriptional modifications [47].…”

“…In fact, some miRNAs, which can work as strong biomarkers to predict disease progression, are selectively loaded into the EVs of NSCLC patients [52]. The composition of EV-incorporated miRNAs distinguish a fraction of miRNAs that is different from the cytoplasm pool, and can have a specific role in cancer development [46]. Classical kinase pathways can also regulate miRNA sorting into EVs to sustain cell proliferation of lung cancer cells [46].…”