UC Berkeley

Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown.

In the work described herein I used biochemical and genetic tools to fractionate distinct EV sub-populations and dissect their mechanisms of miRNA sorting. I report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. I then characterized their RNA content and observed that the EV sub-types use different mechanisms of miRNA sorting (selective and non-selective). Using a cell-free reaction I identified the Lupus La protein as the mediator of miR-122 in vitro packaging. I next showed that the La protein is required for the secretion of selectively sorted miRNAs and 5’ TOP mRNAs in vivo. Finally by using proximity labeling I sought to understand how the La protein is recognized for sorting in the endosomal membrane. I found that the La protein is recognized for sorting by the sequestosome-1 protein. Its secretion is dependent on the presence of LC3-II in the endosomal compartment. In sum, this work provides a mechanistic understanding of miRNA sorting into EVs derived from cancer cells. Moreover I provide preliminary data on how RNA binding proteins might be recognized for secretion in endosomal compartments.