Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash Regulation of gene expression • Endogenous – cell cycle, growth, senescence, differentiation, development etc. • Exogenous – Extracellular conditions – temperature, pH, electrolytes etc. – Secreted factors • • • • Neurotransmitters Proteins Hormones Metabolites Extracellular Vesicles Exosomes were discovered by Johnstone and colleagues in 1987, Extracellular vesicles are membrane bound structures released by cells under physiological and pathological conditions. Exosomes are homogenous in shape, they range between 50 and 100 nm in diameter. Microvesicles range between 100 and 1000 nm in diameter. Apoptotic bodies which are formed through membrane blebbing of dying cells, these vesicles are over 1000 nm but much more heterogeneous in shape. Mechanism of Biogenesis and Secretion • Internalized into early endosomes • Inward vesiculation of the limiting membrane to form multivesicular bodies (MVBs) • The formation of MVBs is dependent on Tsg101, while hepatocyte growth factor receptor substrate (Hrs) is needed for the accumulation of vesicles within these MVBs. • Directed to the plasma membrane resulting in the release of the intraluminal vesicles. Components of Intercellular Communication Recipient Cell Type1 Receive & Interpret the message Source Cell State Dependent Encoding of Message Transfer of message Response 1 Recipient Cell Type 2 Receive & Interpret the message Response 2 HOW IS THE MESSAGE ENCODED? Purification of Exosomes The diversity in the isolation methods of secreted vesicles is a major source of heterogeneity in the results obtained from different research groups. Correlation between replicates Cell Type Correlation Coefficient A549 0.9797 BJ 0.92 HELA 0.8896 HMEC 0.958 HUVEC 0.974 IMR90 0.992 K562 0.776 MCF7 0.947 U2OS 0.867 IMR90 HMEC BJ U2OS MCF7 K562 HUVEC Y-axis : Mean log10(rpm) X-axis : log2(Fold Change) HELA Cell Exosome A549 Genes Exosome Vs Whole Cell Cell Type Specificity of exoRNA exoRNA signature in Body Fluids Genes Read length Distribution by families of RNA miRNA in exosomes BJ CELL BJ Exosome tRNA in exosomes CELL Exosome IS THE MESSAGE DYNAMIC? Dynamicity of exoRNA Dynamicity of Exosomal RNA Gene Fold Change miR-188 356.030895 G1/S transition supress - CDK miR-577 279.406146 G1/S transition supress - TSGA10 miR-369 170.68972 cells accumulate in G1/S - TNFalpha miR-580 134.989963 decreases EMT - target TWIST1 miR-561 112.994978 monitor efficacy to radiotherapy miR-33a 110.649977 increased metastasis - target Pim-1 LOC101929566 104.000307 miR-5000 93.414501 increased in side population - MDR miR-3128 92.770047 miR-4722 86.249888 miR-3136 79.085275 miR-222 72.719991 decreases cell proliferation - ILB4 miR-373 72.304543 increases invaiveness - CD4 Gene Fold Change tRNA 302.612218 RP11-713P17.3 160.975108 Y-RNA 151.189947 RNASEH2A 111.922491 LINC01538 78.193645 Y-RNA 71.023952 707 – genes increased > 4 fold & 697 genes down >4 fold TRANSFER OF EXOSOMAL RNA? Sub-cellular localization of exosomes in recipient cells by lipid labeling of exosomal membrane. 1. Exosomes only 3. ER –Tracker Red 2. Mitotracker-Red 4. Lysotracker -Red Detection of Intercellular transfer of exosomal RNA by metabolic labeling(EU) of RNA 3T3 cells + K562(+EU) exosomes 3T3 cells + ActD + Direct EU 3T3 cells + Direct EU 3T3 cells + ActD+ K562 exosomes(+EU) Intercellular transfer of Y5 RNA(RNA-Seq) Y5 rpm RNY5 de te ction in re ce ipie nt ce lls 100 90 80 70 60 50 40 30 20 10 0 0 6 12 24 48 Neg. control Tim e (hours ) Recipient cells= HB4 cells(Mouse), Donor cells= K562(Human). ELICIT A RESPONSE? BJ Fold change Molecular response Time Point HUVEC Fold change Cell type specific response BJ Fold change HUVEC Log2 (Fold change) miRNA targets mir99b accounts for almost 40% of the miRNA in K562 exosomes BJ Log2 (Fold change) Response of BJ to Serum Deprived K562 exosomes vs Mitomycin treated K562 exosomes Summary Recipient Cell Type1 Receive & Interpret the message Source Cell State Dependent Encoding of Message Transfer of message Response 1 Recipient Cell Type 2 Receive & Interpret the message Response 3 Response 2 Acknowledgements Thomas Gingeras Sudipto Chakrabortty Alexandra Scavelli Jorg Drenkov Other lab members