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 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.

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

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

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 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

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

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

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

TRANSFER OF EXOSOMAL RNA?

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

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)

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).

ELICIT A RESPONSE?

Molecular responseBJ

Fol

d ch

ange

Time Point

Cell type specific responseH

UVE

C Fo

ld c

hang

e

BJ Fold change

miRNA targets

mir99b accounts for almost 40% of the miRNA in K562 exosomes

BJ Log2 (Fold change)

HU

VEC

Log2

(Fol

d ch

ange

)

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

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

Acknowledgements

Thomas GingerasSudipto Chakrabortty

Alexandra ScavelliJorg Drenkov

Other lab members