Diverse Long RNAs Are Differentially Sorted into Extracellular Vesicles Secreted by Colorectal Cancer Cells

Abstract

SUMMARY The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/ Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA.

Long noncoding RNA expression profiling in cancer: Challenges and opportunities

“…Particular interest is given to the profiling of IncRNAs contained in extracellular vesicles (EVs) . Specific long coding and noncoding RNAs have been shown to be enriched in EVs compared with matched cognate cells, suggesting that mechanisms are in place to control their export . These RNAs are also protected from ribonuclease activity, and some of them have been shown to be present as full‐length transcripts .…”

“…[47][48][49] Specific long coding and noncoding RNAs have been shown to be enriched in EVs compared with matched cognate cells, suggesting that mechanisms are in place to control their export. 50 These RNAs are also protected from ribonuclease activity, 51 and some of them have been shown to be present as full-length transcripts. 50,51 Moreover, RNAs selectively exported in tumor-derived EVs are more likely to serve as useful biomarkers of disease.…”

“…50,51 Moreover, RNAs selectively exported in tumor-derived EVs are more likely to serve as useful biomarkers of disease. 49,50 RNA content of bio-fluids or EVs is generally much lower compared to tissue samples, making it even more challenging to accurately quantify lncRNA expression. RNA-capture sequencing workflows, dedicated lncRNA probesets, and efficient total RNA-seq methods can overcome these challenges, enabling sensitive and accurate expression analysis of lncRNAs in human body fluids.…”

Extracellular Vesicles: The New Frontier of Stem Cell Regenerative Medicine?

“…The composition of EVs depend mainly on their cell origin and on the stimuli they receive, under both physiological and pathological conditions. Indeed, it is well recognized that targeting of bioactive molecules within EVs is a selective process, being specific molecules included or excluded [109][110][111][112]. After their release into the extracellular environment many EVs rapidly break down liberating their cargo in the surrounding microenvironment, otherwise EVs can deliver their cargo to target cells via different pathways: direct interaction, resulting in EV fusion with the target cell membrane or in their endocytosis, and interaction mediated by ligand-receptor binding [113][114][115] (Figure 2).…”

Sexual dimorphic expression and release of transcription factors in bovine embryos exposed to oxidative stress

“…The role of EVs is not only in cell-cell communication (Saeed-Zidane et al, 2017) but also in the removal of undesirable cellular molecules (Takahashi et al, 2017). Contrary to cellular expression, the NOTCH1 was increased in EVs derived from male embryos exposed to OS compared with female counterparts, which may indicate the selectivity of cargo molecules to be exported by EVs (Bhome et al, 2018;Hinger et al, 2018) to facilitate cell to cell communication or maintain of cellular homeostasis. Herein, we can summarize that exposing preimplantation bovine embryos to OS compromised the transformation of blastocysts irrespective of embryo sex, due to increasing intracellular ROS accumulation, which in turn leads to increase apoptosis and delay of differentiation in response to dysregulating of the TFs related to stress response, development, differentiation, and apoptosis.…”