Melanoma cells release extracellular vesicles which contain H1° RNA and RNA-binding proteins

Abstract

G26/24 oligodendroglioma cells produce EVs that contain pro-apoptotic proteins, such as FasL and TRAIL, able to induce neuronal- [1] and astrocytic- [2] death. Cancer cells release EVs [3] through which transferring proteins, such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. By releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used A375 melanoma cells. EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait [7]. Melanoma cells were found to synthesize H1° and secrete it via EVs. Moreover, EVs also contain H1° mRNA. Interestingly, H1° histone sorted to vesicles seems to be sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot. [1] D’Agostino et al. 2006, Int J Oncol 29:1075-85. [2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7 [3] Di Liegro et al. 2015, Biomed Res Int, 2015, Article ID 152926. [4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33 [5] Schiera G et al. 2013, Int J Oncol 43:1771-6 [6] Scaturro et al. 1998, J Biol Chem 273:22788-91 [7] Scaturro et al. 2003, Int J Mol Med 11:509-511