Grant: Discovery Grants Program - Individual

Background: The plasma membrane erects a barrier to the spread of cytoplasmic materials. This enables cellular specialization that is essential for complex multicellular organisms. In this model cells should NOT exchange cytoplasmic or nuclear materials, as this would compromise the significant efforts devoted to establishing and maintaining cellular identity. Cells have evolved methods to communicate amongst themselves to allow the development and function of complex organs molecules on their surface or released into the extracellular space. Surprisingly, in some circumstances extracellular vesicles released by cells and called exosomes can overcome the plasma membrane barrier and deliver cytoplasmic contents produced in one cell into the cytoplasm of another1-4. This raises fundamental questions about how the physiological impact of intercellular communication of RNA by exosomes is controlled to ensure the maintenance of cellular identity and function. One principal mechanism that cells use to control exosome-mediated delivery of RNAs is by selectively packaging large amounts of only specific RNAs into exosomes (many other RNAs may be detectable in exosomes, but are often not abundant enough to be physiologically relevant). Understanding the mechanisms which package RNAs into exosomes can generate fundamental biological insights into RNA biology and physiological functions of exosomes. Over-Arching Aims and Rationale: Recent publications demonstrated that hundreds of novel RNA-binding proteins can be systematically identified by covalently cross-linking RNA to proteins to isolate RNA-protein complexes in stringent conditions31,32. We have established similar methods to purify all RNA-protein complexes from exosomes, and RNA-binding proteins bound to a pre-miR-451 which our preliminary data demonstrates is highly enriched in exosomes. We propose to generate a catalogue of new RNA binding proteins in exosomes and dissect their roles in packaging specific RNAs into exosomes. Finally, we will test the biological impact of new RNA-binding proteins and RNAs in exosomes in cell or mouse models. Hypothesis: Proteins bind specific RNA sequences and structures to recruit these into exosomes Specific Aims 1. Purify pre-miR-451 complexes from cells and exosomes to identify and characterize the impact of proteins on packaging pre-miR-451 into exosomes. 2. Purify all RNA-protein complexes from exosomes of multiple cell types and identify RNA packaged into exosomes by a subset of these. 3. Test the biological impact in mice of new RNA-binding proteins and RNAs in exosomes. Significance: mRNAs represent a tiny fraction of the RNAs in exosomes. The most abundant RNAs in exosomes are poorly understood non-coding RNAs. Using unbiased approaches this research will define how RNA of several little studied types is selectively packaged into exosomes and aims to understand the impact of intercellular transfer of these RNAs by exosomes.