Investigating small non-coding RNA as mediators of extracellular vesicle communication

Once thought to be little more than a way for cells to offload waste, extracellular vesicles (EVs) are now recognised to be a deliberate way for a cell to secrete cargos of RNA, DNA and proteins to reshape tissues and act as signal carriers. These somewhat overlooked organelles may hold the key to understanding how tissues and systems in our bodies communicate, particularly within complex diseases. The James lab lead in the study of EV mediated communication via RNAs and this project will expand upon our work in this area. Based on pilot data, the project will seek to determine the different categories of non-coding RNA loaded into vesicles and the mechanisms that regulate this selective loading process. The above aims will be achieved though the following, utilising two disease models systems, cancer (prostate) and osteoarthritis to produce EV materials to study alongside non-disease controls: 1. Use existing datasets from the group and collaborators to document the RNA types identified in EVs and their abundance in disease and non-disease contexts. This work can be carried out away from the labs and during the current period of home-working. We have data sets in house and have already identified publicly available data which we can work with. 2. Validate our pilot data by characterising the different non-coding RNA types present in EVs from both non-disease and disease models. Focused quantitative PCR approach, the student selected for the project has already completed elements of this aim as part of their rotation). 3. Determine the protein complexes associated with key classes of EV RNA e.g. where ribosomal RNAs are present is the full complement of ribosomal proteins also present. Affinity capture techniques will be teamed with proteomics approaches (either targeted or non-biased global). 4. Identify if manipulation of these protein complexes alters RNA loading to determine a potential targetable means of controlling EV cargos. Over-expression and depletion of key proteins within the complexes (and/or proteins associated with the complex) will be used to determine the impact on the abundance of different classes of RNA within EVs. We are looking to identify effects on entire classes of RNA e.g. ribosomal RNA or SnoRNAs rather than an individual RNA, so will be looking to target major proteins/pathways. Within the group we have experience and proof of principle data by depleting members of the miRNA interaction complexes to disrupt miRNA loading into EVs.