Mechanisms of RNA sorting into exosomes
- Author(s): Shurtleff, Matthew James
- Advisor(s): Schekman, Randy W
- et al.
Exosomes are vesicles that are released by cells into the extracellular environment and populate all bodily fluids. These vesicles contain molecular cargo, including RNA, proteins and lipids and therefore may serve as vehicles for intercellular communication by transferring unconventional signals between cells. Despite widespread scientific interest in the physiological role of exosomes in health and disease, little is currently known about how molecules are selectively sorted into exosomes.
In the work described herein, I used biochemical approaches to purify exosomes from cells grown in culture and identify microRNAs that are selectively sorted into exosomes. I then developed a cell-free reaction that reconstitutes the selective sorting of microRNA into exosomes in vitro. The reaction was then utilized to identify an RNA binding protein, Y-box Protein I (YBX1), that is required for sorting an exosomal microRNA. Next, I used the cell-free reaction as the basis for a selection strategy (termed Exo-SELEX) to identify primary RNA sequence motifs that act as positive and negative sorting signals in vitro. Finally, I used a high throughput RNA sequencing approach (TGIRT-seq) that allowed for a comprehensive transcriptome analysis (including highly structured or modified transcripts) of purified exosomes from normal and YBX1 knockout cells. TGIRT-seq analysis revealed that the most abundant transcript biotypes in exosomes are tRNA followed by other small non-coding RNA species. The abundant small non-coding RNA (tRNA, Y-RNA and Vault RNA) were strongly depleted in YBX1 knockout exosomes while sequences representing long non-coding RNA and protein coding genes were unaffected, indicating that these are sorted through a separate mechanism and allowing for broad classification of exosomal RNA based on YBX1-dependence or independence. In sum, this work provides a preliminary mechanistic understanding of the process of RNA sorting into exosomes and establishes multiple tools for the continued dissection of these pathways.