Transcriptomic analysis of RNA Granules and Exosomes: Unmasking the Intra- and Intercellular organelle overlap in Huntington's Disease
Introduction:Repetitive RNA sequences like CAG repeats that trigger aberrant RNA-protein interactions cause several neurodegenerative diseases including Huntington's disease (HD). Importantly, these aberrant RNA-protein complexes also seed the formation of cytoplasmic liquid-like granules, such as the stress granules. Emerging evidence demonstrates that granules formed via liquid-liquid phase separation can mature into gel-like inclusions that persist within the cell and may act as precursor to aggregates that occur in patients' tissue. Thus, deregulation of RNA granule biology is an important component of neurodegeneration. Interestingly, the formation of intracellular membrane-less organelles like stress granules increases along with the secretion of exosomes. Exosomes are small membrane-bound vesicles that are secreted generally by all cell types. They may participate in the spreading of misfolded proteins and aberrant RNA-protein complexes across the central nervous system in neurodegenerative diseases like HD. Moreover, due to their capability to cross the blood-brain barrier, exosomes hold great potential as sources of biomarkers available from the periphery, e.g., from blood. In this study, we performed a comparative transcriptomic analysis of exosomes and RNA granules in an HD model.Methods: Exosomes and RNA granules were isolated from an HD cell model, stably expressing HTT exon 1 with 83 CAG repeats under an inducible promoter. Both exosomes and RNA granules were isolated from induced (HD) and non-induced (control) cells and analysed by RNA sequencing.Results: Comparative analysis between the transcriptomics data of HD RNA granules and exosomes showed that: (I) intracellular RNA granules and extracellular RNA vesicles share content, (II) several non-coding RNAs translocate to RNA granules, (III) the composition of RNA granules and exosomes is affected by expression of mutant HTT.Discussion: Our data show that intracellular RNA granules and exosomes share content, suggesting that formation of RNA granules and exosome loading may be related. Moreover, our data suggest that both intracellular RNA granules and extracellular RNA vesicles may serve as a source for diagnostic strategies. For example, disease-specific RNA-signatures of exosomes can serve as biomarker of central nervous system diseases. However, the cell-type specific signature of EV-secreted RNAs may make it difficult to detect these biomarkers in blood. Overall design: To explore and compare the composition of RNA content in exosomes and RNA granules under WT conditions and in Huntington, exosomes and RNA granules were separately extracted from control or HD-induced HEK293 cells and total RNA was sequenced. Five biological replicates were perfomed for both sample types and both conditions.