transcriptional profiling and mrna stability – don’t shoot the messenger
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Transcriptional profiling and mRNA stability – don’t shoot the messenger. David R. Sherman Seattle Biomedical Research Institute Grand Challenge of Latent TB Mtg. Cape Town February 25, 2012. Landscape of TB latency. 1.5 million deaths. 16 million active infections. 1.8 billion - PowerPoint PPT PresentationTRANSCRIPT
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Transcriptional profiling and mRNA stability – don’t shoot the messenger
David R. ShermanSeattle Biomedical Research Institute
Grand Challenge of Latent TB Mtg.Cape Town
February 25, 2012
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Landscape of TB latency
CONFIDENTIAL
4.4 billionPPD(-)
1.8 billionPPD(+)
16 millionactive
infections
1.5 milliondeaths
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Landscape of TB latency
CONFIDENTIAL
4.4 billionPPD(-)
1.8 billionPPD(+)
16 millionactive
infections
1.5 milliondeaths
Calcified lesionsFew viable bacteria
Caseous lesionsLow numbers of bacteria
Evolving lesionsBacterial replication
Late
ncy
to D
isea
se
Active disease
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Landscape of TB latency
CONFIDENTIAL
4.4 billionPPD(-)
1.8 billionPPD(+)
16 millionactive
infections
1.5 milliondeaths
Calcified lesionsFew viable bacteria
Caseous lesionsLow numbers of bacteria
Evolving lesionsBacterial replication
Late
ncy
to D
isea
se
Active disease
GC-11
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Using gene expression to probe latent TB
Concept – TB gene expression in vivo will reveal the conditions that it experiences.
- Physiology- Drug targets
Infected tissue Gene regulatory network
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Using gene expression to probe latent TB
Infected tissue Gene regulatory network
Assumes TF binding = Tx initiation = mRNA abundance
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mRNA abundance balance
Shalem et al. 2008
Transcription RateDegradation Rate
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Talk outline
• Characterize MTB mRNA decay– Global mRNA half life (T1/2)– Most stable/labile messages– Factors affecting stability
• mRNA decay in stress response– Mild cold shock– Hypoxia
• Summary
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Measuring mRNA degradation
RifampicinLog Phase
0 10 60
RNA Cy dye label Microarray
20 305 15
Custom array design:100,000 TB oligos30,000 control oligos
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8 9 10 11 12 13 14 15 16
8
9
10
11
12
13
14
15
16
T0
T5
8 9 10 11 12 13 14 15 16
8
9
10
11
12
13
14
15
16
T0
T10
8 9 10 11 12 13 14 15 16
8
9
10
11
12
13
14
15
16
T0
T15
7 8 9 10 11 12 13 14 15 16
7
8
9
10
11
12
13
14
15
16
T0
T20
T0
T0 T0
T0
T20
T5 T10
T15
mRNA decay by microarray
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Individual decay curve
Rifampicin (rif)
0 5 10 15 20 25 30 350
1
2
3
4
5
6
7
8
Rv0011c (example)
Rv0011c
Time (min)
Log(
2) [R
NA]
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Individual decay curve
Rifampicin (rif)1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.000
5
10
15
20
25
30
35
f(x) = − 6.02087620439056 x + 40.0112985990332R² = 0.98814512464103
Rv0011c
Rv0011cLinear (Rv0011c)
Log(2) [RNA]
Tim
e (m
in)
T1/2
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Data filtered for reproducibility and R2
• Inclusion criteria:– T1/2 with R2>0.7– Starting [RNA] > 4x background– Valid measures at > half replicates
• 2139 genes met criteria
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TB mRNA is very stable
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16More0
5
10
15
20
25
MTB
1/2 life (minutes)
% T
rans
crip
ts
Average MTB T1/2 = 9.5 minutes
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TB mRNA is very stable
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16More0
5
10
15
20
25
MTBE. coliB. subtilis
1/2 life (minutes)
% T
rans
crip
ts
0 10 20 30 40 50 60 700.0001
0.001
0.01
0.1
1
MTBE. coli
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Functional CategoryMean half-life (minutes)
information pathways 8.7*
virulence, detoxification, and adaptation 9.3
lipid metabolism 9.4cell wall and cell processes 9.4metabolism and respiration 9.5hypothetical protein 9.6Regulatory proteins 9.6insertion seqs and phages 10.4**
PE/PPE 11.1**
*=significantly lower**=significantly higher
mRNA T1/2 by functional category
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Functional subcategory Specified Gene %
Whole Genome %
P Value
Functional Enrichment for the genes with the shortest 1/2 lifePosttranslational modification, protein turnover, chaperones 12.8 2.3 0Translation, ribosomal structure and biogenesis 21.4 3.0 0Energy production and conversion 14.2 5.2 0.002Intracellular trafficking, secretion, and vesicular transport 2.8 0.3 0.025
Functional Enrichment for the genes with the longest 1/2 lifeReplication, recombination and repair 12.8 4.8 0.004Amino acid transport and metabolism 11.4 4.5 0.01PE/PPE 2.8 0.2 0.014Energy production and conversion 11.4 5.2 0.019
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Physical characteristics and mRNA stability
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4 6 8 10 12 14 16 184
5
6
7
8
9
10
1/2 Life (min)
Star
ting
inte
nsity
(log
2)
mRNA abundance and stability
R2 = 0.8
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mRNA abundance and stability
• Inherent to the mRNA?
• Inherent to abundance?
Test:• DosR regulon: ~48 genes induced by hypoxia, etc.• Place dosR under tet control.• Induce regulon in log phase
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Induced transcripts degrade faster
0 5 10 15 20 25 300123456789
10
25 genes of the DosR regulon
DosRInducedUninduced
Half-life (min)
Initi
al tr
ansc
ript a
bund
ance
(log
2)
>2000 transcripts did not change stability
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Modified mRNA decay in response to stress conditions
• Are specific transcripts (de)stabilized?• Does the global mRNA ½ life change?
• Is mRNA decay regulated to change transcript abundance?
Temperature Hypoxia
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7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16
7.5
8
8.5
9
9.5
10
10.5
11
11.5
12
12.5
13
13.5
14
14.5
15
15.5
16
T0
T5 H
ours
aft
er r
ifam
pin
(20C
)
mRNA degradation at 20C
After 5 hrs:Only 55 genes decayed 2x or more.
Degradation not measurable.
T1/2 very sensitive to temp.
T = 5 hrs
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mRNA stability in hypoxia
RifampicinLogPhase
0 10 6020 30
Hypoxia
120+
RNA Cy dye label Microarray
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0 10 20 30 40 50 60 70
1%
10%
100%
Reaeration
Time post rifampin (min)
% In
itial
mRN
A0 10 20 30 40 50 60 70
1%
10%
100%
1 hour hypoxiaReaeration
Time post rifampin (min)
% In
itial
mRN
A0 10 20 30 40 50 60 70
1%
10%
100%
1 hour hypoxia5 days hypoxiaReaeration
Time post rifampin (min)
% In
itial
mRN
A
0 10 20 30 40 50 60 70
1%
10%
100%
1 hour hypoxia5 days hypoxiaStarvationReaeration
Time post rifampin (min)
% In
itial
mRN
A
mRNA stability in altered O2
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MTB mRNA decay characterization
• Reliable mRNA half lives for >2000 genes.• Average half life ~9.5 minutes.• Half life inversely correlated with transcript
concentration.• Transcripts stabilized by cold, hypooxia.
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Questions to explore
• Why is TB mRNA very stable?– RNases or mRNA secondary structure?
• Hypoxia and low temp: Transcription decreased to balance decay? Mechanism?
• How to repress genes?• What are the consequences?– Systems modeling
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Thank you!Kyle Minch
Tige Rustad
Debbie Whitley
Bill Brabant
JessicaWinkler
Paul G Allen Family FoundationBill and Melinda Gates Foundation NIAID contract # HHSN272200800059C
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Rifampicin is stopping transcription
• No rif induced genes• Lux assay to look for induction after rif treatment– Hypoxia sensitive promoter driving lux
• Total degradation by array
0 Rif 1h Hypoxia 50 Rif 1h Hypoxia No Hypoxia0
4000
8000
12000
16000
Lux induction in response to hypoxia