Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.
-
Upload
dorthy-jenkins -
Category
Documents
-
view
237 -
download
0
Transcript of Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.
![Page 1: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/1.jpg)
Exosomal RNA mediated non-cell autonomous regulation of gene expression
Ashwin Prakash
![Page 2: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/2.jpg)
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
![Page 3: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/3.jpg)
Extracellular VesiclesExosomes 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.
![Page 4: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/4.jpg)
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.
![Page 5: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/5.jpg)
![Page 6: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/6.jpg)
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
![Page 7: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/7.jpg)
HOW IS THE MESSAGE ENCODED?
![Page 8: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/8.jpg)
Purification of Exosomes
![Page 9: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/9.jpg)
The diversity in the isolation methods of secreted vesicles is a major source of heterogeneity in the results obtained from different research groups.
![Page 10: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/10.jpg)
Correlation between replicatesCell Type Correlation
CoefficientA549 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
![Page 11: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/11.jpg)
![Page 12: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/12.jpg)
Exosome Vs Whole Cell
Y-axis : Mean log10(rpm)X-axis : log2(Fold Change)
A549
HEL
A
K562
MCF
7
U2O
S B JH
MEC
HU
VEC
IMR9
0
Gen
es
Exosome Cell
![Page 13: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/13.jpg)
Cell Type Specificity of exoRNA
![Page 14: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/14.jpg)
exoRNA signature in Body Fluids
Genes
![Page 15: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/15.jpg)
Read length Distribution by families of RNA
![Page 16: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/16.jpg)
miRNA in exosomes
BJ CELL
BJ Exosome
![Page 17: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/17.jpg)
tRNA in exosomes
CELL
Exosome
![Page 18: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/18.jpg)
IS THE MESSAGE DYNAMIC?
![Page 19: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/19.jpg)
Dynamicity of exoRNA
![Page 20: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/20.jpg)
Dynamicity of Exosomal RNA
707 – genes increased > 4 fold & 697 genes down >4 fold
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
![Page 21: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/21.jpg)
TRANSFER OF EXOSOMAL RNA?
![Page 22: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/22.jpg)
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
![Page 23: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/23.jpg)
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)
![Page 24: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/24.jpg)
Intercellular transfer of Y5 RNA(RNA-Seq)
RNY5 detection in receipient cells
01020304050
60708090
100
0 6 12 24 48 Neg.control
Time (hours)
Y5 r
pm
Recipient cells= HB4 cells(Mouse), Donor cells= K562(Human).
![Page 25: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/25.jpg)
ELICIT A RESPONSE?
![Page 26: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/26.jpg)
Molecular responseBJ
Fol
d ch
ange
Time Point
![Page 27: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/27.jpg)
Cell type specific responseH
UVE
C Fo
ld c
hang
e
BJ Fold change
![Page 28: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/28.jpg)
miRNA targets
mir99b accounts for almost 40% of the miRNA in K562 exosomes
BJ Log2 (Fold change)
HU
VEC
Log2
(Fol
d ch
ange
)
![Page 29: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/29.jpg)
Response of BJ to Serum Deprived K562 exosomes vs Mitomycin treated K562 exosomes
![Page 30: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/30.jpg)
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
![Page 31: Exosomal RNA mediated non-cell autonomous regulation of gene expression Ashwin Prakash.](https://reader035.fdocuments.net/reader035/viewer/2022081506/5697bf911a28abf838c8e5a8/html5/thumbnails/31.jpg)
Acknowledgements
Thomas GingerasSudipto Chakrabortty
Alexandra ScavelliJorg Drenkov
Other lab members