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

Landscape of TB latency

CONFIDENTIAL

4.4 billionPPD(-)

1.8 billionPPD(+)

16 millionactive

infections

1.5 milliondeaths

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

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

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

Using gene expression to probe latent TB

Infected tissue Gene regulatory network

Assumes TF binding = Tx initiation = mRNA abundance

mRNA abundance balance

Shalem et al. 2008

Transcription RateDegradation Rate

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

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

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

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]

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

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

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

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

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

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

Physical characteristics and mRNA stability

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

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

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

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

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

mRNA stability in hypoxia

RifampicinLogPhase

0 10 6020 30

Hypoxia

120+

RNA Cy dye label Microarray

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

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.

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

Thank you!Kyle Minch

Tige Rustad

Debbie Whitley

Bill Brabant

JessicaWinkler

Paul G Allen Family FoundationBill and Melinda Gates Foundation NIAID contract # HHSN272200800059C

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