Melanoma cells release extracellular vesicles which contain RNA-binding proteins able to bind the mRNA encoding histone H1°
- Authors: Schiera, G.; Di Liegro, C.; Puleo, V.; Colletta, O.; Di Liegro, I.
- Publication year: 2015
- Type: eedings
- Key words: Extracellular vesicles (EVs), G26/24 oligodendroglioma cells, xtracellular matrix remodelling proteases, A375 melanoma cells, H1° histone, RNA-protein complexes, myelin expression factor-2 (MYEF2)
- OA Link: http://hdl.handle.net/10447/249070
Abstract
Extracellular vesicles (EVs) are produced by most prokaryotic and eukaryotic cells; tumour cells, however, release much higher amounts of EVs, which contain cancer-specific proteins and RNAs. Molecules carried by EVs are captured by surrounding cells, which then undergo profound phenotypic modifications. G26/24 oligodendroglioma cells release, for example, EVs containing FasL and TRAIL, which induce apoptosis in rat cortical neurons and astrocytes in culture. By metabolic labelling of cells, EV-mediated horizontal transfer of radioactive proteins was clearly demonstrated. Among the proteins present in EVs produced by oligodendroglioma cells, extracellular matrix remodelling proteases, and the linker histone H1°, a differentiation-specific histone, were identified. We also found that A375 melanoma cells release EVs which, like the once produced by oligodendroglioma cells, contain H1°. Interestingly, H1° histone sorted to vesicles has a molecular mass higher than expected, and is probably sumoylated. More recently, by a T1 RNase-protection assay, done by mixing an in-vitro transcribed, H1°- encoding RNA, and EVs, three main RNA-protein complexes were evidenced, the most abundant of which had an apparent molecular mass of about 65 kDa. We then synthesized a biotinylated H1° RNA, with the aim to fish, by affinity chromatography, the evidenced proteins. The RNA-bound fraction was finally analysed by mass spectrometry. The most abundant protein identified was the myelin expression factor-2 (MYEF2), which has indeed a molecular mass of about 60 kDa. The presence of MYEF2 in EVs released from A375 melanoma cells was finally confirmed by Western blot analysis.