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

La

ten

cy

to D

ise

as

e 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

La

ten

cy

to D

ise

as

e 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 0

Energy 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 0.004

Amino acid transport and metabolism 11.4 4.5 0.01

PE/PPE 2.8 0.2 0.014

Energy 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

nsit

y (l

og2)

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 25 300

1

2

3

4

5

6

7

8

9

10

25 genes of the DosR regulon

DosRInducedUninduced

Half-life (min)

Initi

al tr

ansc

ript

abu

ndan

ce (l

og2)

>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

Hou

rs a

fter

rifa

mp

in (

20

C)

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