Transcript Exosomal RNA mediated non-cell autonomous regulation of gene

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