Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells

Abstract

Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrPC, and the abnormal infectious form, PrPSc, are found associated with exosomes, which are small 50–130 nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, non-coding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease.

141 Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells - evidence of unique microRNA cargos

“…1A), which is similar to previously published data. 22 These results show that our protocol for specifically exosome isolation results in no contamination of nuclear, mitochondrial, or endoplasmic reticulum membranes components, whereas the other two EV subsets seem to contain such markers. The presence of different types of vesicles in the different EV isolates was also confirmed by electron microscopy (Fig.…”

“…22,23 Interestingly, we found enrichment of multiple types of non-coding RNAs in all EV subsets. Despite our focus on short RNA molecules up to 200 nucleotides, fragmented mRNA can also be detected in the vesicular RNA, and are captured by the cDNA library preparation (data not shown).…”

“…Our data of vtRNA and Y-RNA are in accordance with previous study on T-cell/dendritic cell exosomes, also describing enrichment of Y-RNA and vault RNA in exosomes. 22,23 Together, these data show that the different subsets of EVs contain different types of small non-coding RNAs, which putatively could be used as a surrogate signature to distinguish the EV subsets.…”

“…41 Most studies describing RNA species in exosomes and other extracellular vesicles have applied array-based methods, 14 and only few have utilized deep sequencing to illustrate the RNA content in detail. 22,23 Until today, no study has described the RNA content in different vesicle subsets secreted from a single cell type, using any methodology. This is thus the first study that utilizes RNA sequencing to profile small RNAs concomitantly in apoptotic bodies, microvesicles and exosomes released by any single cell type under any condition.…”

“…[17][18][19][20][21] Importantly, many small RNAs are released in the extracellular environment as EV cargo, which has been specifically documented for the exosomes. [22][23][24][25][26][27] Importantly, many cellular RNAs, including miRNAs are detected in the extracellular space, especially in extracellular vesicles. 28,29 Currently, extracellular RNA communication is also considered to be putatively important in animals, with important implications for biology, disease and medicine.…”