Circular RNAs In EVs – A Novel Class Of Released Non-coding RNAs

Abstract
Non-coding RNAs (ncRNAs) were established in the last decade as a new valuable biomarker class for human diseases. Specifically, circular RNAs (circRNAs), which represent the newest class of ncRNAs, turned into research focus within the last decade. Although single examples of these particular RNA class had been known for more than forty years, circRNAs were established as a large RNA class only a few years ago, based on the advent of tailored RNA-Seq technologies and Bioinformatics. CircRNAs were identified in all eukaryotic cells investigated so far, are cell-type specifically expressed, and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form, resulting in a metabolically more stable form of RNA compared to their linear counterparts. These biological properties and the correlation with various human diseases, such as cardiovascular diseases and cancer, immediately suggesting their potential use as novel attractive RNA-based biomarkers. We have focused on the analysis of circRNAs and the corresponding linear splice isoforms from human blood cells, in particular platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles (EVs) from purified and in vitro activated human platelets, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. Furthermore, to unravel mechanisms that contribute to the specific packaging of RNAs into EVs, we developed a novel approach to determine specific sequence motifs required for selective loading of (circ)RNA into EVs. This unbiased method should contribute to our understanding of how RNAs are specifically packaged into EVs. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and harbor great biomarker potential. In addition, we would also like to stress the strong need for standardization and for setting up minimal requirements for validating this class of ncRNA.