1. Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash

2. 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

3. 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.

4. 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.

6. Components of Intercellular Communication Transfer of message Receive & Interpret the message Recipient Cell Type1 Response 1 State Dependent Encoding of Message Source Cell Receive & Interpret the message Recipient Cell Type 2 Response 2

7. HOW IS THE MESSAGE ENCODED?

8. Purification of Exosomes

9. The diversity in the isolation methods of secreted vesicles is a major source of heterogeneity in the results obtained from different research groups.

10. Correlation between replicates Cell TypeCorrelation Coefficient A5490.9797 BJ0.92 HELA0.8896 HMEC0.958 HUVEC0.974 IMR900.992 K5620.776 MCF70.947 U2OS0.867

12. Exosome Vs Whole Cell Y-axis : Mean log10(rpm) X-axis : log2(Fold Change) A549A549 H EL A K562K562 M CF 7 U2OSU2OS BJ H ME C HU VE C IM R9 0 Ge ne s A549 HELA K562 MCF7 U2OS BJ HMEC HUVEC IMR90 Genes Exosome Cell

13. Cell Type Specificity of exoRNA

14. exoRNA signature in Body Fluids Genes

15. Read length Distribution by families of RNA

16. miRNA in exosomes BJ CELL BJ Exosome

17. tRNA in exosomes CELL Exosome

18. IS THE MESSAGE DYNAMIC?

19. Dynamicity of exoRNA

20. Dynamicity of Exosomal RNA 707 – genes increased > 4 fold & 697 genes down >4 fold GeneFold Change miR-188356.030895G1/S transition supress - CDK miR-577279.406146G1/S transition supress - TSGA10 miR-369170.68972cells accumulate in G1/S - TNFalpha miR-580134.989963decreases EMT - target TWIST1 miR-561112.994978monitor efficacy to radiotherapy miR-33a110.649977increased metastasis - target Pim-1 LOC101929566104.000307 miR-500093.414501increased in side population - MDR miR-312892.770047 miR-472286.249888 miR-313679.085275 miR-22272.719991decreases cell proliferation - ILB4 miR-37372.304543increases invaiveness - CD4 GeneFold Change tRNA302.612218 RP11-713P17.3160.975108 Y-RNA151.189947 RNASEH2A111.922491 LINC0153878.193645 Y-RNA71.023952

21. TRANSFER OF EXOSOMAL RNA?

22. Sub-cellular localization of exosomes in recipient cells by lipid labeling of exosomal membrane. 1. Exosomes only 2. Mitotracker-Red 3. ER –Tracker Red 4. Lysotracker -Red

23. Detection of Intercellular transfer of exosomal RNA by metabolic labeling(EU) of RNA 3T3 cells + K562(+EU) exosomes 3T3 cells + Direct EU 3T3 cells + ActD + Direct EU 3T3 cells + ActD+ K562 exosomes(+EU)

24. Intercellular transfer of Y5 RNA(RNA-Seq) Recipient cells= HB4 cells(Mouse), Donor cells= K562(Human).

25. ELICIT A RESPONSE?

26. Molecular response BJ Fold change Time Point

27. Cell type specific response HUVEC Fold change BJ Fold change

28. miRNA targets mir99b accounts for almost 40% of the miRNA in K562 exosomes BJ Log2 (Fold change) HUVEC Log2 (Fold change)

29. Response of BJ to Serum Deprived K562 exosomes vs Mitomycin treated K562 exosomes

30. Transfer of message Receive & Interpret the message Recipient Cell Type1 Response 1 State Dependent Encoding of Message Source Cell Receive & Interpret the message Recipient Cell Type 2 Response 2 Summary Response 3

31. Acknowledgements Thomas Gingeras Sudipto Chakrabortty Alexandra Scavelli Jorg Drenkov Other lab members