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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
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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 PPD(+) 4.4 billion PPD(-) CONFIDENTIAL
Landscape of TB latency 1.5 million deaths Active disease 16 million active infections Evolving lesions Bacterial replication Latency to Disease Caseouslesions Low numbers of bacteria 1.8 billion PPD(+) Calcified lesions Few viable bacteria 4.4 billion PPD(-) CONFIDENTIAL
Landscape of TB latency 1.5 million deaths Active disease 16 million active infections Evolving lesions Bacterial replication Latency to Disease Caseouslesions Low numbers of bacteria 1.8 billion PPD(+) Calcified lesions Few viable bacteria 4.4 billion PPD(-) GC-11 CONFIDENTIAL
Using gene expression to probe latent TB Infected tissue Gene regulatory network Concept – TB gene expression in vivo will reveal the conditions that it experiences. - Physiology - Drug targets
Using gene expression to probe latent TB Infected tissue Gene regulatory network Assumes TF binding = Tx initiation = mRNA abundance
mRNA abundance balance Transcription Rate Degradation Rate Shalem et al. 2008
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 0 5 10 15 20 30 60 Rifampicin Log Phase Cy dye label RNA Microarray Custom array design: 100,000 TB oligos 30,000 control oligos
mRNA decay by microarray T10 T5 T0 T0 T15 T20 T0 T0
Individual decay curve Rifampicin (rif)
Individual decay curve T1/2 Rifampicin (rif)
Data filtered for reproducibility and R2 • Inclusion criteria: • T1/2with R2>0.7 • Starting [RNA] > 4x background • Valid measures at > half replicates • 2139 genes met criteria
TB mRNA is very stable Average MTB T1/2 = 9.5 minutes
mRNA abundance and stability R2 = 0.8
mRNA abundance and stability • Inherent to the mRNA? • Inherent to abundance? • Test: • DosRregulon: ~48 genes induced by hypoxia, etc. • Place dosR under tet control. • Induce regulon in log phase
Induced transcripts degrade faster >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
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 0 10 20 30 120+ 60 Log Phase Rifampicin Hypoxia Cy dye label RNA Microarray
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! Bill Brabant Kyle Minch Tige Rustad Jessica Winkler Debbie Whitley Paul G Allen Family Foundation Bill and Melinda Gates Foundation NIAID contract # HHSN272200800059C
Rifampicinis stopping transcription • No rif induced genes • Lux assay to look for induction after rif treatment • Hypoxia sensitive promoter driving lux • Total degradation by array